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1.  Mammalian Adaptation in the PB2 Gene of Avian H5N1 Influenza Virus 
Journal of Virology  2013;87(19):10884-10888.
The substitution of glutamic acid (E) for lysine (K) at position 627 of the PB2 protein of avian H5N1 viruses has been identified as a virulence and host range determinant for infection of mammals. Here, we report that the E-to-K host-adaptive mutation in the PB2 gene appeared from day 4 and 5 along the respiratory tracts of mice and was complete by day 6 postinoculation. This mutation correlated with efficient replication of the virus in mice.
doi:10.1128/JVI.01016-13
PMCID: PMC3807384  PMID: 23864613
2.  A Single Dose of Any of Four Different Live Attenuated Tetravalent Dengue Vaccines Is Safe and Immunogenic in Flavivirus-naive Adults: A Randomized, Double-blind Clinical Trial 
The Journal of Infectious Diseases  2013;207(6):957-965.
Background. Dengue virus (DENV) causes hundreds of millions of infections annually. Four dengue serotypes exist, and previous infection with one serotype increases the likelihood of severe disease with a second, heterotypic DENV infection.
Methods. In a randomized, placebo-controlled study, the safety and immunogenicity of 4 different admixtures of a live attenuated tetravalent (LATV) dengue vaccine were evaluated in 113 flavivirus-naive adults. Serum neutralizing antibody levels to all 4 dengue viruses were measured on days 0, 28, 42, and 180.
Results. A single dose of each LATV admixture induced a trivalent or better neutralizing antibody response in 75%–90% of vaccinees. There was no significant difference in the incidence of adverse events between vaccinees and placebo-recipients other than rash. A trivalent or better response correlated with rash and with non-black race (P < .0001). Black race was significantly associated with a reduced incidence of vaccine viremia.
Conclusions. TV003 induced a trivalent or greater antibody response in 90% of flavivirus-naive vaccinees and is a promising candidate for the prevention of dengue. Race was identified as a factor influencing the infectivity of the LATV viruses, reflecting observations of the effect of race on disease severity in natural dengue infection.
Clinical Trials Registration NCT01072786.
doi:10.1093/infdis/jis936
PMCID: PMC3571448  PMID: 23329850
dengue vaccine; live attenuated tetravalent; clinical trial
3.  Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm 
PLoS Pathogens  2014;10(3):e1003971.
Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.
Author Summary
Influenza A viruses, containing eight single stranded RNA segments, cause seasonal epidemics and occasional pandemics. Reassortment of the influenza viral genome in co-infected cells confers an evolutionary advantage for the virus, and can result in viruses with pandemic potential like the 2009 pandemic H1N1 and 2013 H7N9 virus. Replication of the viral genome occurs in the nucleus of the host cell and the progeny viral RNA (vRNA) segments must be transported to the plasma membrane for budding. The dynamics of vRNA assembly into progeny virions remains unknown. We used novel techniques to visualize the 3D-localization of four distinct vRNA segments in an infected cell and a fluorescent virus to visualize vRNA transport during a productive infection to determine where, when and how assembly occurs. Our data suggest that vRNA segments are exported from the nucleus as subcomplexes that undergo additional assembly en route to the plasma membrane through dynamic fusion events of vRNA-containing cytoplasmic foci. These observations have broad implications for understanding the intracellular requirements behind reassortment of influenza viruses and may lead to the development of new antiviral targets.
doi:10.1371/journal.ppat.1003971
PMCID: PMC3946384  PMID: 24603687
5.  Intranasal Vaccination Promotes Detrimental Th17-Mediated Immunity against Influenza Infection 
PLoS Pathogens  2014;10(1):e1003875.
Influenza disease is a global health issue that causes significant morbidity and mortality through seasonal epidemics. Currently, inactivated influenza virus vaccines given intramuscularly or live attenuated influenza virus vaccines administered intranasally are the only approved options for vaccination against influenza virus in humans. We evaluated the efficacy of a synthetic toll-like receptor 4 agonist CRX-601 as an adjuvant for enhancing vaccine-induced protection against influenza infection. Intranasal administration of CRX-601 adjuvant combined with detergent split-influenza antigen (A/Uruguay/716/2007 (H3N2)) generated strong local and systemic immunity against co-administered influenza antigens while exhibiting high efficacy against two heterotypic influenza challenges. Intranasal vaccination with CRX-601 adjuvanted vaccines promoted antigen-specific IgG and IgA antibody responses and the generation of polyfunctional antigen-specific Th17 cells (CD4+IL-17A+TNFα+). Following challenge with influenza virus, vaccinated mice transiently exhibited increased weight loss and morbidity during early stages of disease but eventually controlled infection. This disease exacerbation following influenza infection in vaccinated mice was dependent on both the route of vaccination and the addition of the adjuvant. Neutralization of IL-17A confirmed a detrimental role for this cytokine during influenza infection. The expansion of vaccine-primed Th17 cells during influenza infection was also accompanied by an augmented lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it highlights the importance of both route of vaccination and adjuvant selection in vaccine development
Author Summary
Influenza virus remains a global health risk causing significant morbidity and mortality each year, with the elderly (>65 years) and the very young particularly prone to severe respiratory disease. Scientists are working to develop highly efficacious vaccines capable of eliciting broad cross-clade protection from influenza infection. Adjuvants as well as the route of immunization are known to modulate the type, quality and breadth of immune responses to vaccines. In this study, we demonstrated intranasal vaccination with influenza antigens, and a novel synthetic TLR4-based adjuvant system provided protection against a lethal heterologous viral challenge. Immunization stimulated mucosal influenza-specific IgA antibody responses together with systemic IgG antibodies. While intranasal immunization stimulated the production of protective antibodies, vaccination via this route also promoted the generation of influenza-specific Th17 CD4+ T cells. These vaccine-induced Th17 cells increased inflammation and morbidity without contributing to viral clearance following challenge. Antibody neutralization of IL-17A during influenza infection significantly reduced the enhanced lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it demonstrates the importance of both route of immunization and adjuvant selection in vaccine development.
doi:10.1371/journal.ppat.1003875
PMCID: PMC3900655  PMID: 24465206
6.  Heterovariant Cross-Reactive B-Cell Responses Induced by the 2009 Pandemic Influenza Virus A Subtype H1N1 Vaccine 
The Journal of Infectious Diseases  2012;207(2):288-296.
Background. The generation of heterovariant immunity is a highly desirable feature of influenza vaccines. The goal of this study was to compare the heterovariant B-cell response induced by the monovalent inactivated 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) vaccine with that induced by the 2009 seasonal trivalent influenza vaccine (sTIV) containing a seasonal influenza A virus subtype H1N1 (A[H1N1]) component in young and elderly adults.
Methods. Plasmablast-derived polyclonal antibodies (PPAb) from young and elderly recipients of A(H1N1)pdm09 vaccine or sTIV were tested for binding activity to various influenza antigens.
Results. In A(H1N1)pdm09 recipients, the PPAb titers against homotypic A(H1N1)pdm09 vaccine were similar to those against the heterovariant seasonal A(H1N1) vaccine and were similar between young and elderly subjects. The PPAb avidity was higher among elderly individuals, compared with young individuals. In contrast, the young sTIV recipients had 10-fold lower heterovariant PPAb titers against the A(H1N1)pdm09 vaccine than against the homotypic seasonal A(H1N1) vaccine. In binding assays with recombinant head and stalk domains of hemagglutinin, PPAb from the A(H1N1)pdm09 recipients but not PPAb from the sTIV recipients bound to the conserved stalk domain.
Conclusion. The A(H1N1)pdm09 vaccine induced production of PPAb with heterovariant reactivity, including antibodies targeting the conserved hemagglutinin stalk domain.
doi:10.1093/infdis/jis664
PMCID: PMC3532823  PMID: 23107783
influenza; vaccine; antibody; cross-reactivity
7.  An Open Label Phase I Trial of a Live Attenuated H2N2 Influenza Virus Vaccine in Healthy Adults 
Background
Live attenuated influenza vaccines (LAIV) against a variety of strains of pandemic potential are being developed and tested. We describe the results of an open label Phase I trial of a live attenuated H2N2 virus vaccine.
Objectives
To evaluate the safety, infectivity and immunogenicity of a live attenuated H2N2 influenza virus vaccine.
Participants/Methods
The A/Ann Arbor/6/60 (H2N2) virus used in this study is the attenuated, cold-adapted, temperature-sensitive strain that provides the genetic backbone of seasonal LAIV (MedImmune). We evaluated the safety, infectivity, and immunogenicity of two doses of 107TCID50 of this vaccine administered by nasal spray 4 weeks apart to normal healthy seronegative adults.
Results
Twenty-one participants received a first dose of the vaccine; 18 participants received a second dose. No serious adverse events occurred during the trial. The most common adverse events after vaccination were headache and musculoskeletal pain. The vaccine was restricted in replication: 24% and 17% had virus detectable by culture or rRT-PCR after the first and second dose, respectively. Antibody responses to the vaccine were also restricted: 24% of participants developed an antibody response as measured by either hemagglutination-inhibition assay (10%), or ELISA for H2 HA-specific serum IgG (24%) or IgA (16%) after either one or two doses. None of the participants had a neutralizing antibody response. Vaccine-specific IgG-secreting cells as measured by ELISPOT increased from a mean of 0.5 to 2.0/106 Peripheral Blood Mononuclear Cells (PBMCs); vaccine-specific IgA-secreting cells increased from 0.1 to 0.5/106PBMCs.
Conclusions
The live attenuated H2N2 1960 AA ca vaccine demonstrated a safety profile consistent with seasonal trivalent LAIV but was restricted in replication and minimally immunogenic in healthy seronegative adults.
doi:10.1111/j.1750-2659.2012.00350.x
PMCID: PMC3527634  PMID: 22417012
H2N2; human; influenza; influenza vaccine; LAIV; NAI
8.  Replication and Immunogenicity of Swine, Equine, and Avian H3 Subtype Influenza Viruses in Mice and Ferrets 
Journal of Virology  2013;87(12):6901-6910.
Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines.
doi:10.1128/JVI.03520-12
PMCID: PMC3676140  PMID: 23576512
9.  B Cell Response and Hemagglutinin Stalk-Reactive Antibody Production in Different Age Cohorts following 2009 H1N1 Influenza Virus Vaccination 
The 2009 pandemic H1N1 (pH1N1) influenza virus carried a swine-origin hemagglutinin (HA) that was closely related to the HAs of pre-1947 H1N1 viruses but highly divergent from the HAs of recently circulating H1N1 strains. Consequently, prior exposure to pH1N1-like viruses was mostly limited to individuals over the age of about 60 years. We related age and associated differences in immune history to the B cell response to an inactivated monovalent pH1N1 vaccine given intramuscularly to subjects in three age cohorts: 18 to 32 years, 60 to 69 years, and ≥70 years. The day 0 pH1N1-specific hemagglutination inhibition (HAI) and microneutralization (MN) titers were generally higher in the older cohorts, consistent with greater prevaccination exposure to pH1N1-like viruses. Most subjects in each cohort responded well to vaccination, with early formation of circulating virus-specific antibody (Ab)-secreting cells and ≥4-fold increases in HAI and MN titers. However, the response was strongest in the 18- to 32-year cohort. Circulating levels of HA stalk-reactive Abs were increased after vaccination, especially in the 18- to 32-year cohort, raising the possibility of elevated levels of cross-reactive neutralizing Abs. In the young cohort, an increase in MN activity against the seasonal influenza virus A/Brisbane/59/07 after vaccination was generally associated with an increase in the anti-Brisbane/59/07 HAI titer, suggesting an effect mediated primarily by HA head-reactive rather than stalk-reactive Abs. Our findings support recent proposals that immunization with a relatively novel HA favors the induction of Abs against conserved epitopes. They also emphasize the need to clarify how the level of circulating stalk-reactive Abs relates to resistance to influenza.
doi:10.1128/CVI.00735-12
PMCID: PMC3675965  PMID: 23576673
10.  Mode of Parainfluenza Virus Transmission Determines the Dynamics of Primary Infection and Protection from Reinfection 
PLoS Pathogens  2013;9(11):e1003786.
Little is known about how the mode of respiratory virus transmission determines the dynamics of primary infection and protection from reinfection. Using non-invasive imaging of murine parainfluenza virus 1 (Sendai virus) in living mice, we determined the frequency, timing, dynamics, and virulence of primary infection after contact and airborne transmission, as well as the tropism and magnitude of reinfection after subsequent challenge. Contact transmission of Sendai virus was 100% efficient, phenotypically uniform, initiated and grew to robust levels in the upper respiratory tract (URT), later spread to the lungs, grew to a lower level in the lungs than the URT, and protected from reinfection completely in the URT yet only partially in the lungs. Airborne transmission through 7.6-cm and 15.2-cm separations between donor and recipient mice was 86%–100% efficient. The dynamics of primary infection after airborne transmission varied between individual mice and included the following categories: (a) non-productive transmission, (b) tracheal dominant, (c) tracheal initiated yet respiratory disseminated, and (d) nasopharyngeal initiated yet respiratory disseminated. Any previous exposure to Sendai virus infection protected from mortality and severe morbidity after lethal challenge. Furthermore, a higher level of primary infection in a given respiratory tissue (nasopharynx, trachea, or lungs) was inversely correlated with the level of reinfection in that same tissue. Overall, the mode of transmission determined the dynamics and tropism of primary infection, which in turn governed the level of seroconversion and protection from reinfection. These data are the first description of the dynamics of respiratory virus infection and protection from reinfection throughout the respiratory tracts of living animals after airborne transmission. This work provides a basis for understanding parainfluenza virus transmission and protective immunity and for developing novel vaccines and non-pharmaceutical interventions.
Author Summary
Parainfluenza viruses are highly contagious, a leading cause of acute respiratory infection (ARI) in children, often reoccurring, and currently controlled by non-pharmaceutical interventions. We tracked infection and reinfection of a prototypic murine parainfluenza virus after contact or airborne transmission. Our studies reveal that the mode of transmission determines the dynamics of primary infection. Additionally, higher levels of protection from reinfection are induced in individual respiratory tissues by higher levels of primary infection in those same tissues. Natural infection after either contact or airborne transmission tends to initiate in the URT, but not the lungs. Complete protection from infection in the URT was afforded by URT-biased, non-pathogenic infection after low-dose intranasal vaccination. Overall, the data suggest that parainfluenza virus transmission may be effectively controlled by handwashing, disinfection of surfaces, and environmental control of short-range transmission, in addition to the development of live attenuated vaccines that target the URT.
doi:10.1371/journal.ppat.1003786
PMCID: PMC3836739  PMID: 24278024
12.  New World Bats Harbor Diverse Influenza A Viruses 
PLoS Pathogens  2013;9(10):e1003657.
Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.
Author Summary
Previous studies indicated that a novel influenza A virus (H17N10) was circulating in fruit bats from Guatemala (Central America). Herein, we investigated whether similar viruses are present in bat species from South America. Analysis of rectal swabs from bats sampled in the Amazon rainforest region of Peru identified another new influenza A virus from bats that is phylogenetically distinct from the one identified in Guatemala. The genes that encode the surface proteins of the new virus from the flat-faced fruit bat were designated as new subtype H18N11. Serologic testing of blood samples from several species of Peruvian bats indicated a high prevalence of antibodies to the surface proteins. Phylogenetic analyses demonstrate that bat populations from Central and South America maintain as much influenza virus genetic diversity in some gene segments as all other mammalian and avian species combined. The crystal structures of the hemagglutinin and neuraminidase proteins indicate that sialic acid is not a receptor for virus attachment nor a substrate for release, suggesting a novel mechanism of influenza A virus attachment and activation of membrane fusion for entry into host cells. In summary, our findings indicate that bats constitute a potentially important reservoir for influenza viruses.
doi:10.1371/journal.ppat.1003657
PMCID: PMC3794996  PMID: 24130481
13.  Evaluation of replication, immunogenicity and protective efficacy of a live attenuated cold-adapted pandemic H1N1 influenza virus vaccine in non-human primates 
Vaccine  2012;30(38):5603-5610.
We studied the replication of influenza A/California/07/09 (H1N1) wild type (CA09wt) virus in two non-human primate species and used one of these models to evaluate the immunogenicity and protective efficacy of a live attenuated cold-adapted vaccine, which contains the hemagglutinin and neuraminidase from the H1N1 wild type virus and six internal protein gene segments of the A/Ann Arbor/6/60 cold-adapted (ca) master donor virus. We infected African green monkeys (AGMs) and rhesus macaques with 2 × 106 TCID50 of CA09wt and CA09ca influenza viruses. The virus replicated in the upper respiratory tract of all animals but the titers in upper respiratory tract tissues of rhesus macaques were significant higher than in AGMs (mean peak titers 104.5 TCID50/g and 102.0 TCID50/g on days 4 and 2 post-infection, respectively; p<0.01). Virus replication was observed in the lungs of all rhesus macaques (102.0–105.4TCID50/g) whereas only 2 out of 4 AGMs had virus recovered from the lungs (102.5– 103.5 TCID50/g). The CA09ca vaccine virus was attenuated and highly restricted in replication in both AGMs and rhesus macaques. We evaluated the immunogenicity and protective efficacy of the CA09ca vaccine in rhesus macaques because CA09wt virus replicated more efficiently in this species. One or two doses of vaccine were administered intranasally and intratracheally to rhesus macaques. For the two-dose group, the vaccine was administered 4-weeks apart. Immunogenicity was assessed by measuring hemagglutination-inhibiting (HAI) antibodies in the serum and specific IgA antibodies to CA09wt virus in the nasal wash. One or two doses of the vaccine elicited a significant rise in HAI titers (range 40–320). Two doses of CA09ca elicited higher pH1N1-specific IgA titers than in the mock-immunized group (p<0.01). Vaccine efficacy was assessed by comparing titers of CA09wt challenge virus in the respiratory tract of mock immunized and CA09ca vaccinated monkeys. Significantly lower virus titers were observed in the lungs of vaccinated animals than mock-immunized animals (p ≤ 0.01). Our results demonstrate that AGMs and rhesus macaques support the replication of pandemic H1N1 influenza virus to different degrees and a cold-adapted pH1N1 vaccine elicits protective immunity against pH1N1 virus infection in rhesus macaques.
doi:10.1016/j.vaccine.2012.06.088
PMCID: PMC3412900  PMID: 22789506
Pandemic H1N1; Non-human primate; Influenza vaccine
14.  Engineering H5N1 avian influenza viruses to study human adaptation 
Nature  2012;486(7403):335-340.
Two studies of H5N1 avian influenza viruses that had been genetically engineered to render them transmissible between ferrets have proved highly controversial. Divergent opinions exist about the importance of these studies of influenza transmission and about potential ‘dual use’ research implications. No consensus has developed yet about how to balance these concerns. After not recommending immediate full publication of earlier, less complete versions of the studies, the United States National Science Advisory Board for Biosecurity subsequently recommended full publication of more complete manuscripts; however, controversy about this and similar research remains.
doi:10.1038/nature11170
PMCID: PMC3731044  PMID: 22722191
15.  Antigen-activated dendritic cells ameliorate influenza A infections 
The Journal of Clinical Investigation  2013;123(7):2850-2861.
Influenza A viruses cause significant morbidity and mortality worldwide. There is a need for alternative or adjunct therapies, as resistance to currently used antiviral drugs is emerging rapidly. We tested ligand epitope antigen presentation system (LEAPS) technology as a new immune-based treatment for influenza virus infection in a mouse model. Influenza-J-LEAPS peptides were synthesized by conjugating the binding ligand derived from the β2-microglobulin chain of the human MHC class I molecule (J-LEAPS) with 15 to 30 amino acid–long peptides derived from influenza virus NP, M, or HA proteins. DCs were stimulated with influenza-J-LEAPS peptides (influenza-J-LEAPS) and injected intravenously into infected mice. Antigen-specific LEAPS-stimulated DCs were effective in reducing influenza virus replication in the lungs and enhancing survival of infected animals. Additionally, they augmented influenza-specific T cell responses in the lungs and reduced the severity of disease by limiting excessive cytokine responses, which are known to contribute to morbidity and mortality following influenza virus infection. Our data demonstrate that influenza-J-LEAPS–pulsed DCs reduce virus replication in the lungs, enhance survival, and modulate the protective immune responses that eliminate the virus while preventing excessive cytokines that could injure the host. This approach shows promise as an adjunct to antiviral treatment of influenza virus infections.
doi:10.1172/JCI67550
PMCID: PMC3696566  PMID: 23934125
16.  Antigenic Drift of the Pandemic 2009 A(H1N1) Influenza Virus in a Ferret Model 
PLoS Pathogens  2013;9(5):e1003354.
Surveillance data indicate that most circulating A(H1N1)pdm09 influenza viruses have remained antigenically similar since they emerged in humans in 2009. However, antigenic drift is likely to occur in the future in response to increasing population immunity induced by infection or vaccination. In this study, sequential passaging of A(H1N1)pdm09 virus by contact transmission through two independent series of suboptimally vaccinated ferrets resulted in selection of variant viruses with an amino acid substitution (N156K, H1 numbering without signal peptide; N159K, H3 numbering without signal peptide; N173K, H1 numbering from first methionine) in a known antigenic site of the viral HA. The N156K HA variant replicated and transmitted efficiently between naïve ferrets and outgrew wildtype virus in vivo in ferrets in the presence and absence of immune pressure. In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties. The N156K escape mutant was antigenically distinct from wildtype virus as shown by binding of HA-specific antibodies. Glycan binding assays demonstrated the N156K escape mutant had altered receptor binding preferences compared to wildtype virus, which was supported by computational modeling predictions. The N156K substitution, and culture adaptations, have been detected in human A(H1N1)pdm09 viruses with N156K preferentially reported in sequences from original clinical samples rather than cultured isolates. This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection. Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance.
Author Summary
Infection with influenza virus leads to significant morbidity and mortality. Annual vaccination may prevent subsequent disease by inducing neutralizing antibodies to currently circulating strains in the human population. To escape this antibody response, influenza A viruses undergo continuous genetic variation as they replicate, enabling viruses with advantageous antigenic mutations to spread and cause disease in naïve or previously immune or vaccinated individuals. To date, the 2009 pandemic virus (A(H1N1)pdm09) has not undergone significant antigenic drift, with the result that the vaccine remains well-matched and should provide good protection to A(H1N1)pdm09 circulating viruses. In this study, we induced antigenic drift in an A(H1N1)pdm09 virus in the ferret model. A single amino acid mutation emerged in the dominant surface glycoprotein, hemagglutinin, which had a multifaceted effect, altering both antigenicity and virus receptor specificity. The mutant virus could not be isolated using routine cell culture methods without the virus acquiring additional amino acid changes, yet was fit in vivo. The implications for surveillance of circulating influenza virus are significant as current assays commonly used to assess vaccine mismatch, as well as to produce isolates for vaccine manufacture, are biased against identification of viruses containing only this mutation.
doi:10.1371/journal.ppat.1003354
PMCID: PMC3649996  PMID: 23671418
17.  Comparative Study of Influenza Virus Replication in MDCK Cells and in Primary Cells Derived from Adenoids and Airway Epithelium 
Journal of Virology  2012;86(21):11725-11734.
Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is desirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bronchial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive immune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live attenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of viruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates.
doi:10.1128/JVI.01477-12
PMCID: PMC3486302  PMID: 22915797
18.  Cooperativity Between CD8+ T Cells, Non-Neutralizing Antibodies, and Alveolar Macrophages Is Important for Heterosubtypic Influenza Virus Immunity 
PLoS Pathogens  2013;9(3):e1003207.
Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential “universal” vaccine.
Author Summary
Influenza virus continues to pose a significant risk to global health and is responsible for thousands of deaths each year in the United States. This threat is largely due to the ability of the influenza virus to undergo rapid changes, allowing it to escape from immune responses elicited by previous infections or vaccinations. Certain internal determinants of the influenza virus are largely conserved across different viral strains and represent attractive targets for potential “universal” influenza vaccines. Here, we demonstrated that cross-subtype protection against the influenza virus could be obtained through simultaneous priming of multiple arms of the immune response against conserved elements of the influenza virus. These results suggest a novel strategy that could potentially form a primary component of a universal influenza vaccine capable of providing long-lasting protection.
doi:10.1371/journal.ppat.1003207
PMCID: PMC3597515  PMID: 23516357
19.  Effect of Priming with H1N1 Influenza Viruses of Variable Antigenic Distances on Challenge with 2009 Pandemic H1N1 Virus 
Journal of Virology  2012;86(16):8625-8633.
Compared to seasonal influenza viruses, the 2009 pandemic H1N1 (pH1N1) virus caused greater morbidity and mortality in children and young adults. People over 60 years of age showed a higher prevalence of cross-reactive pH1N1 antibodies, suggesting that they were previously exposed to an influenza virus or vaccine that was antigenically related to the pH1N1 virus. To define the basis for this cross-reactivity, ferrets were infected with H1N1 viruses of variable antigenic distance that circulated during different decades from the 1930s (Alaska/35), 1940s (Fort Monmouth/47), 1950s (Fort Warren/50), and 1990s (New Caledonia/99) and challenged with 2009 pH1N1 virus 6 weeks later. Ferrets primed with the homologous CA/09 or New Jersey/76 (NJ/76) virus served as a positive control, while the negative control was an influenza B virus that should not cross-protect against influenza A virus infection. Significant protection against challenge virus replication in the respiratory tract was observed in ferrets primed with AK/35, FM/47, and NJ/76; FW/50-primed ferrets showed reduced protection, and NC/99-primed ferrets were not protected. The hemagglutinins (HAs) of AK/35, FM/47, and FW/50 differ in the presence of glycosylation sites. We found that the loss of protective efficacy observed with FW/50 was associated with the presence of a specific glycosylation site. Our results suggest that changes in the HA occurred between 1947 and 1950, such that prior infection could no longer protect against 2009 pH1N1 infection. This provides a mechanistic understanding of the nature of serological cross-protection observed in people over 60 years of age during the 2009 H1N1 pandemic.
doi:10.1128/JVI.00147-12
PMCID: PMC3421718  PMID: 22674976
20.  Memory CD4+ T cells: beyond “helper” functions  
The Journal of Clinical Investigation  2012;122(8):2768-2770.
In influenza virus infection, antibodies, memory CD8+ T cells, and CD4+ T cells have all been shown to mediate immune protection, but how they operate and interact with one another to mediate efficient immune responses against virus infection is not well understood. In this issue of the JCI, McKinstry et al. have identified unique functions of memory CD4+ T cells beyond providing “help” for B cell and CD8+ T cell responses during influenza virus infection.
doi:10.1172/JCI65208
PMCID: PMC3408759  PMID: 22820285
21.  The Contribution of Systemic and Pulmonary Immune Effectors to Vaccine-Induced Protection from H5N1 Influenza Virus Infection 
Journal of Virology  2012;86(9):5089-5098.
Live attenuated influenza vaccines (LAIVs) are effective in providing protection against influenza challenge in animal models and in preventing disease in humans. We previously showed that LAIVs elicit a range of immune effectors and that successful induction of pulmonary cellular and humoral immunity in mice requires pulmonary replication of the vaccine virus. An upper respiratory tract immunization (URTI) model was developed in mice to mimic the human situation, in which the vaccine virus does not replicate in the lower respiratory tract, allowing us to assess the protective efficacy of an H5N1 LAIV against highly pathogenic H5N1 virus challenge in the absence of significant pulmonary immunity. Our results show that, after one dose of an H5N1 LAIV, pulmonary influenza-specific lymphocytes are the main contributors to clearance of challenge virus from the lungs and that contributions of influenza-specific enzyme-linked immunosorbent assay (ELISA) antibodies in serum and splenic CD8+ T cells were negligible. Complete protection from H5N1 challenge was achieved after two doses of H5N1 LAIV and was associated with maturation of the antibody response. Although passive transfer of sera from mice that received two doses of vaccine prevented lethality in naive recipients following challenge, the mice showed significant weight loss, with high pulmonary titers of the H5N1 virus. These data highlight the importance of mucosal immunity in mediating optimal protection against H5N1 infection. Understanding the requirements for effective induction and establishment of these protective immune effectors in the respiratory tract paves the way for a more rational and effective vaccine approach in the future.
doi:10.1128/JVI.07205-11
PMCID: PMC3347357  PMID: 22379093
22.  Influenza Virus Vaccines: Lessons from the 2009 H1N1 pandemic 
Current opinion in virology  2011;1(4):254-262.
Reflecting on the 2009 H1N1 pandemic, we summarize lessons regarding influenza vaccines that can be applied in the future. The two major challenges to vaccination during the 2009 H1N1 pandemic were timing and availability of vaccine. Vaccines were, however, well-tolerated and immunogenic, with inactivated vaccines containing 15μg of HA generally inducing antibody titers ≥1:40 in adults within 2 weeks of the administration of a single dose. Moreover, the use of oil-in-water adjuvants in Europe permitted dose- reduction, with vaccines containing as little as 3.75 or 7.5μg HA being immunogenic. Case-control studies demonstrated that monovalent 2009 H1N1 vaccines were effective in preventing infection with the 2009 H1N1 virus, but preliminary data suggests that it is important for individuals to be re-immunized annually.
doi:10.1016/j.coviro.2011.08.002
PMCID: PMC3224079  PMID: 22125588
2009 H1N1; pandemic; influenza; vaccines
23.  The Multibasic Cleavage Site of the Hemagglutinin of Highly Pathogenic A/Vietnam/1203/2004 (H5N1) Avian Influenza Virus Acts as a Virulence Factor in a Host-Specific Manner in Mammals 
Journal of Virology  2012;86(5):2706-2714.
Highly pathogenic avian influenza (HPAI) viruses of the H5 and H7 subtypes typically possess multiple basic amino acids around the cleavage site (MBS) of their hemagglutinin (HA) protein, a recognized virulence motif in poultry. To determine the importance of the H5 HA MBS as a virulence factor in mammals, recombinant wild-type HPAI A/Vietnam/1203/2004 (H5N1) viruses that possessed (H5N1) or lacked (ΔH5N1) the H5 HA MBS were generated and evaluated for their virulence in BALB/c mice, ferrets, and African green monkeys (AGMs) (Chlorocebus aethiops). The presence of the H5 HA MBS was associated with lethality, significantly higher virus titers in the respiratory tract, virus dissemination to extrapulmonary organs, lymphopenia, significantly elevated levels of proinflammatory cytokines and chemokines, and inflammation in the lungs of mice and ferrets. In AGMs, neither H5N1 nor ΔH5N1 virus was lethal and neither caused clinical symptoms. The H5 HA MBS was associated with mild enhancement of replication and delayed virus clearance. Thus, the contribution of H5 HA MBS to the virulence of the HPAI H5N1 virus varies among mammalian hosts and is most significant in mice and ferrets and less remarkable in nonhuman primates.
doi:10.1128/JVI.05546-11
PMCID: PMC3302284  PMID: 22205751
24.  Attacking the flu 
Nature medicine  2009;15(11):1251-1252.
doi:10.1038/nm1109-1251
PMCID: PMC3415221  PMID: 19893556
25.  Cellular targets for influenza drugs 
Nature Biotechnology  2010;28(3):239-240.
doi:10.1038/nbt0310-239
PMCID: PMC3397661  PMID: 20212486

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