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1.  In Vitro Assessment of the Immunological Significance of a Human Monoclonal Antibody Directed to the Influenza A Virus Nucleoprotein 
Influenza A viruses cause annual epidemics and occasionally pandemics. Antibodies directed to the conserved viral nucleoprotein (NP) may play a role in immunity against various influenza A virus subtypes. Here, we assessed the immunological significance of a human monoclonal antibody directed to NP in vitro. This antibody bound to virus-infected cells but did not display virus-neutralizing activity, complement-dependent cell cytotoxicity, or opsonization of viral antigen for improved antigen presentation to CD8+ T cells by dendritic cells.
PMCID: PMC3754521  PMID: 23761662
2.  Induction of Influenza (H5N8) Antibodies by Modified Vaccinia Virus Ankara H5N1 Vaccine 
Emerging Infectious Diseases  2015;21(6):1086-1088.
PMCID: PMC4451911  PMID: 25988813
vaccine; avian influenza; MVA; clinical trial; antibodies; zoonoses; viruses
3.  Human Cytotoxic T Lymphocytes Directed to Seasonal Influenza A Viruses Cross-React with the Newly Emerging H7N9 Virus 
Journal of Virology  2014;88(3):1684-1693.
In February 2013, zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported, a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population, there is interest in identifying other correlates of protection, such as cross-reactive CD8+ T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8+ T cells are known to recognize conserved internal proteins of influenza A viruses predominantly, but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here, we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8+ T cells, obtained from HLA-typed study subjects, with the novel H7N9 virus. The cross-reactivity of CD8+ T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that, apart from recognition of individual H7N9 variant epitopes, CD8+ T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8+ T cells may afford some protection against infection with the new virus.
PMCID: PMC3911609  PMID: 24257602
4.  Infection of the Upper Respiratory Tract with Seasonal Influenza A(H3N2) Virus Induces Protective Immunity in Ferrets against Infection with A(H1N1)pdm09 Virus after Intranasal, but Not Intratracheal, Inoculation 
Journal of Virology  2013;87(8):4293-4301.
The clinical symptoms caused by infection with influenza A virus vary widely and depend on the strain causing the infection, the dose and route of inoculation, and the presence of preexisting immunity. In most cases, seasonal influenza A viruses cause relatively mild upper respiratory tract disease, while sometimes patients develop an acute severe pneumonia. Heterosubtypic immunity induced by previous infections with influenza A viruses may dampen the development of clinical symptoms caused by infection with influenza A viruses of another subtype, as is the case during influenza pandemics. Here we show that ferrets acquire protective immunity after infection of the upper respiratory tract with a seasonal influenza A(H3N2) virus against subsequent infection with influenza A(H1N1)pdm09 virus inoculated by the intranasal route. However, protective heterosubtypic immunity was afforded locally, since the prior infection with the A(H3N2) virus did not provide protection against the development of pneumonia induced after intratracheal inoculation with the A(H1N1)pdm09 virus. Interestingly, some of these animals developed more severe disease than that observed in naïve control animals. These findings are of interest in light of the development of so-called universal influenza vaccines that aim at the induction of cross-reactive T cell responses.
PMCID: PMC3624397  PMID: 23365444
5.  Reassortment between Avian H5N1 and Human Influenza Viruses Is Mainly Restricted to the Matrix and Neuraminidase Gene Segments 
PLoS ONE  2013;8(3):e59889.
Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus.
PMCID: PMC3604002  PMID: 23527283
6.  Induction of Virus-Specific Cytotoxic T Lymphocytes as a Basis for the Development of Broadly Protective Influenza Vaccines 
There is considerable interest in the development of broadly protective influenza vaccines because of the continuous emergence of antigenic drift variants of seasonal influenza viruses and the threat posed by the emergence of antigenically distinct pandemic influenza viruses. It has been recognized more than three decades ago that influenza A virus-specific cytotoxic T lymphocytes recognize epitopes located in the relatively conserved proteins like the nucleoprotein and that they cross-react with various subtypes of influenza A viruses. This implies that these CD8+ T lymphocytes may contribute to protective heterosubtypic immunity induced by antecedent influenza A virus infections. In the present paper, we review the evidence for the role of virus-specific CD8+ T lymphocytes in protective immunity against influenza virus infections and discuss vaccination strategies that aim at the induction of cross-reactive virus-specific T-cell responses.
PMCID: PMC3189652  PMID: 22007149
7.  Low Virulence and Lack of Airborne Transmission of the Dutch Highly Pathogenic Avian Influenza Virus H5N8 in Ferrets 
PLoS ONE  2015;10(6):e0129827.
Highly pathogenic avian influenza (HPAI) H5N8 viruses that emerged in poultry in East Asia spread to Europe and North America by late 2014. Here we show that the European HPAI H5N8 viruses differ from the Korean and Japanese HPAI H5N8 viruses by several amino acids and that a Dutch HPAI H5N8 virus had low virulence and was not transmitted via the airborne route in ferrets. The virus did not cross-react with sera raised against pre-pandemic H5 vaccine strains. This data is useful for public health risk assessments.
PMCID: PMC4474857  PMID: 26090682
8.  A central role for Notch in effector CD8+ T cell differentiation 
Nature immunology  2014;15(12):1143-1151.
Activated CD8+ T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We show that Notch controls this choice. Notch promoted differentiation of immediately protective TECs and was correspondingly required for clearance of an acute influenza virus infection. Notch activated a major portion of the TEC-specific gene expression program and suppressed the MPC-specific program. Expression of Notch receptors was induced on naïve CD8+ T cells by inflammatory mediators and interleukin 2 (IL-2) via mTOR and T-bet dependent pathways. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of the infection.
PMCID: PMC4232996  PMID: 25344724
CD8 T cell; effector; memory; inflammation; MPC; TEC
9.  Identification, Characterization, and Natural Selection of Mutations Driving Airborne Transmission of A/H5N1 virus 
Cell  2014;157(2):329-339.
Recently, A/H5N1 influenza viruses were shown to acquire airborne transmissibility between ferrets upon targeted mutagenesis and virus passage. The critical genetic changes in airborne A/Indonesia/5/05 were not yet identified. Here, five substitutions proved to be sufficient to determine this airborne transmission phenotype. Substitutions in PB1 and PB2 collectively caused enhanced transcription and virus replication. One substitution increased HA thermostability and lowered the pH of membrane fusion. Two substitutions independently changed HA binding preference from α2,3 linked to α2,6 linked sialic acid receptors. The loss of a glycosylation site in HA enhanced overall binding to receptors. The acquired substitutions emerged early during ferret passage as minor variants and became dominant rapidly. Identification of substitutions that are essential for airborne transmission of avian influenza viruses between ferrets and their associated phenotypes advances our fundamental understanding of virus transmission and will increase the value of future surveillance programs and public health risk assessments.
PMCID: PMC4003409  PMID: 24725402
10.  An autotransporter display platform for the development of multivalent recombinant bacterial vector vaccines 
The Autotransporter pathway, ubiquitous in Gram-negative bacteria, allows the efficient secretion of large passenger proteins via a relatively simple mechanism. Capitalizing on its crystal structure, we have engineered the Escherichia coli autotransporter Hemoglobin protease (Hbp) into a versatile platform for secretion and surface display of multiple heterologous proteins in one carrier molecule.
As proof-of-concept, we demonstrate efficient secretion and high-density display of the sizeable Mycobacterium tuberculosis antigens ESAT6, Ag85B and Rv2660c in E. coli simultaneously. Furthermore, we show stable multivalent display of these antigens in an attenuated Salmonella Typhimurium strain upon chromosomal integration. To emphasize the versatility of the Hbp platform, we also demonstrate efficient expression of multiple sizeable antigenic fragments from Chlamydia trachomatis and the influenza A virus at the Salmonella cell surface.
The successful efficient cell surface display of multiple antigens from various pathogenic organisms highlights the potential of Hbp as a universal platform for the development of multivalent recombinant bacterial vector vaccines.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-014-0162-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4252983  PMID: 25421093
Antigen delivery; Recombinant live vaccine; Surface display; Autotransporter; Multivalent
11.  Genomewide Analysis of Reassortment and Evolution of Human Influenza A(H3N2) Viruses Circulating between 1968 and 2011 
Journal of Virology  2014;88(5):2844-2857.
Influenza A(H3N2) viruses became widespread in humans during the 1968 H3N2 virus pandemic and have been a major cause of influenza epidemics ever since. These viruses evolve continuously by reassortment and genomic evolution. Antigenic drift is the cause for the need to update influenza vaccines frequently. Using two data sets that span the entire period of circulation of human influenza A(H3N2) viruses, it was shown that influenza A(H3N2) virus evolution can be mapped to 13 antigenic clusters. Here we analyzed the full genomes of 286 influenza A(H3N2) viruses from these two data sets to investigate the genomic evolution and reassortment patterns. Numerous reassortment events were found, scattered over the entire period of virus circulation, but most prominently in viruses circulating between 1991 and 1998. Some of these reassortment events persisted over time, and one of these coincided with an antigenic cluster transition. Furthermore, selection pressures and nucleotide and amino acid substitution rates of all proteins were studied, including those of the recently discovered PB1-N40, PA-X, PA-N155, and PA-N182 proteins. Rates of nucleotide and amino acid substitutions were most pronounced for the hemagglutinin, neuraminidase, and PB1-F2 proteins. Selection pressures were highest in hemagglutinin, neuraminidase, matrix 1, and nonstructural protein 1. This study of genotype in relation to antigenic phenotype throughout the period of circulation of human influenza A(H3N2) viruses leads to a better understanding of the evolution of these viruses.
IMPORTANCE Each winter, influenza virus infects approximately 5 to 15% of the world's population, resulting in significant morbidity and mortality. Influenza A(H3N2) viruses evolve continuously by reassortment and genomic evolution. This leads to changes in antigenic recognition (antigenic drift) which make it necessary to update vaccines against influenza A(H3N2) viruses frequently. In this study, the relationship of genetic evolution to antigenic change spanning the entire period of A(H3N2) virus circulation was studied for the first time. The results presented in this study contribute to a better understanding of genetic evolution in correlation with antigenic evolution of influenza A(H3N2) viruses.
PMCID: PMC3958060  PMID: 24371052
13.  Modified Vaccinia Virus Ankara (MVA) as Production Platform for Vaccines against Influenza and Other Viral Respiratory Diseases 
Viruses  2014;6(7):2735-2761.
Respiratory viruses infections caused by influenza viruses, human parainfluenza virus (hPIV), respiratory syncytial virus (RSV) and coronaviruses are an eminent threat for public health. Currently, there are no licensed vaccines available for hPIV, RSV and coronaviruses, and the available seasonal influenza vaccines have considerable limitations. With regard to pandemic preparedness, it is important that procedures are in place to respond rapidly and produce tailor made vaccines against these respiratory viruses on short notice. Moreover, especially for influenza there is great need for the development of a universal vaccine that induces broad protective immunity against influenza viruses of various subtypes. Modified Vaccinia Virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform. MVA can encode one or more foreign antigens and thus functions as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant capacities and induces humoral and cellular immune responses. However, there are some practical and regulatory issues that need to be addressed in order to develop MVA-based vaccines on short notice at the verge of a pandemic. In this review, we discuss promising novel influenza virus vaccine targets and the use of MVA for vaccine development against various respiratory viruses.
PMCID: PMC4113791  PMID: 25036462
modified vaccinia virus ankara; vaccine development; influenza virus; respiratory syncytial virus; parainfluenza virus; coronavirus
14.  Antigenic Variation of Clade 2.1 H5N1 Virus Is Determined by a Few Amino Acid Substitutions Immediately Adjacent to the Receptor Binding Site 
mBio  2014;5(3):e01070-14.
Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are genetically highly variable and have diversified into multiple phylogenetic clades over the past decade. Antigenic drift is a well-studied phenomenon for seasonal human influenza viruses, but much less is known about the antigenic evolution of HPAI H5N1 viruses that circulate in poultry. In this study, we focused on HPAI H5N1 viruses that are enzootic to Indonesia. We selected representative viruses from genetically distinct lineages that are currently circulating and determined their antigenic properties by hemagglutination inhibition assays. At least six antigenic variants have circulated between 2003, when H5N1 clade 2.1 viruses were first detected in Indonesia, and 2011. During this period, multiple antigenic variants cocirculated in the same geographic regions. Mutant viruses were constructed by site-directed mutagenesis to represent each of the circulating antigenic variants, revealing that antigenic differences between clade 2.1 viruses were due to only one or very few amino acid substitutions immediately adjacent to the receptor binding site. Antigenic variants of H5N1 virus evaded recognition by both ferret and chicken antibodies. The molecular basis for antigenic change in clade 2.1 viruses closely resembled that of seasonal human influenza viruses, indicating that the hemagglutinin of influenza viruses from different hosts and subtypes may be similarly restricted to evade antibody recognition.
Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for severe outbreaks in both commercial and backyard poultry, causing considerable economic losses and regular zoonotic transmissions to humans. Vaccination is used increasingly to reduce the burden of HPAI H5N1 virus in poultry. Influenza viruses can escape from recognition by antibodies induced upon vaccination or infection through genetic changes in the hemagglutinin protein. The evolutionary patterns and molecular basis of antigenic change in HPAI H5N1 viruses are poorly understood, hampering formulation of optimal vaccination strategies. We have shown here that HPAI H5N1 viruses in Indonesia diversified into multiple antigenic variants, that antigenic differences were due to one or a very few substitutions near the receptor binding site, and that the molecular basis for antigenic change was remarkably similar to that for seasonal human influenza viruses. These findings have consequences for future vaccination and surveillance considerations and contribute to the understanding of the antigenic evolution of influenza viruses.
PMCID: PMC4056550  PMID: 24917596
15.  Advances in influenza vaccination 
F1000Prime Reports  2014;6:47.
Influenza virus infections yearly cause high morbidity and mortality burdens in humans, and the development of a new influenza pandemic continues to threaten mankind as a Damoclean sword. Influenza vaccines have been produced by using egg-based virus growth and passaging techniques that were developed more than 60 years ago, following the identification of influenza A virus as an etiological agent of seasonal influenza. These vaccines aimed mainly at eliciting neutralizing antibodies targeting antigenically variable regions of the hemagglutinin (HA) protein, which requires regular updates to match circulating seasonal influenza A and B virus strains. Given the relatively limited protection induced by current seasonal influenza vaccines, a more universal influenza vaccine that would protect against more—if not all—influenza viruses is among the largest unmet medical needs of the 21st century. New insights into correlates of protection from influenza and into broad B- and T-cell protective anti-influenza immune responses offer promising avenues for innovative vaccine development as well as manufacturing strategies or platforms, leading to the development of a new generation of vaccines. These aim at the rapid and massive production of influenza vaccines that provide broad protective and long-lasting immunity. Recent advances in influenza vaccine research demonstrate the feasibility of a wide range of approaches and call for the initiation of preclinical proof-of-principle studies followed by clinical trials in humans.
PMCID: PMC4047948  PMID: 24991424
16.  Determinants of virulence of influenza A virus 
Influenza A viruses cause yearly seasonal epidemics and occasional global pandemics in humans. In the last century, four human influenza A virus pandemics have occured. Ocasionally, influenza A viruses that circulate in other species, cross the species barrier and infect humans. Virus re-assortment (i.e. mixing of gene segments of multiple viruses) and the accumulation of mutations contribute to the emergence of new influenza A virus variants. Fortunately, most of these variants do not have the ability to spread among humans and subsequently cause a pandemic. In this review we focus on the threat of animal influenza A viruses which have shown the ability to infect humans. In addition, genetic factors which could alter the virulence of influenza A viruses are discussed. Identification and characterization of these factors may provide insights into genetic traits which change virulence and help us to understand which genetic determinants are of importance for the pandemic potential of animal influenza A viruses.
PMCID: PMC3969785  PMID: 24078062
Influenza A viruses; virulence factors; pandemic threat; transmission
18.  Genetic evolution of the neuraminidase of influenza A (H3N2) viruses from 1968 to 2009 and its correspondence to haemagglutinin evolution 
The Journal of General Virology  2012;93(Pt 9):1996-2007.
Each year, influenza viruses cause epidemics by evading pre-existing humoral immunity through mutations in the major glycoproteins: the haemagglutinin (HA) and the neuraminidase (NA). In 2004, the antigenic evolution of HA of human influenza A (H3N2) viruses was mapped (Smith et al., Science 305, 371–376, 2004) from its introduction in humans in 1968 until 2003. The current study focused on the genetic evolution of NA and compared it with HA using the dataset of Smith and colleagues, updated to the epidemic of the 2009/2010 season. Phylogenetic trees and genetic maps were constructed to visualize the genetic evolution of NA and HA. The results revealed multiple reassortment events over the years. Overall rates of evolutionary change were lower for NA than for HA1 at the nucleotide level. Selection pressures were estimated, revealing an abundance of negatively selected sites and sparse positively selected sites. The differences found between the evolution of NA and HA1 warrant further analysis of the evolution of NA at the phenotypic level, as has been done previously for HA.
PMCID: PMC3542130  PMID: 22718569
19.  Effect of Travel on Influenza Epidemiology 
Emerging Infectious Diseases  2013;19(6):925-931.
To assess the attack and incidence rates for influenza virus infections, during October 2006–October 2007 we prospectively studied 1,190 adult short-term travelers from the Netherlands to tropical and subtropical countries. Participants donated blood samples before and after travel and kept a travel diary. The samples were serologically tested for the epidemic strains during the study period. The attack rate for all infections was 7% (86 travelers) and for influenza-like illness (ILI), 0.8%. The incidence rate for all infections was 8.9 per 100 person-months and for ILI, 0.9%. Risk factors for infection were birth in a non-Western country, age 55–64 years, and ILI. In 15 travelers with fever or ILI, influenza virus infection was serologically confirmed; 7 of these travelers were considered contagious or incubating the infection while traveling home. Given the large number of travelers to (sub)tropical countries, travel-related infection most likely contributes to importation and further influenza spread worldwide.
PMCID: PMC3713810  PMID: 23735636
Influenza; travel; prospective study; attack rate; incidence; viruses; epidemiology
20.  Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis 
PLoS Pathogens  2013;9(3):e1003229.
Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza.
Author Summary
Influenza viruses, including H5N1 bird influenza viruses continue to form a major threat for public health. Available antiviral drugs for the treatment of influenza are effective to a limited extent and the emergence of resistant viruses may further undermine their use. The symptoms associated with influenza are caused by replication of the virus in the respiratory tract and the host immune response. Here, we report that a molecule of the fibrinolytic system, plasminogen, contributes to inflammation caused by influenza. Inhibiting the action of plasminogen protected mice from severe influenza infections, including those caused by H5N1 and H1N1 pandemic 2009 viruses and may be a promising novel strategy to treat influenza.
PMCID: PMC3605290  PMID: 23555246
21.  Recurring Influenza B Virus Infections in Seals  
Emerging Infectious Diseases  2013;19(3):511-512.
PMCID: PMC3647654  PMID: 23750359
influenza B virus; seals; serology; viruses; influenza; Suggested citation for this article: Bodewes R; Morick D; de Mutsert G; Osinga N; Bestebroer T; van der Vliet S; et al. Recurring influenza B virus infections in seals [letter]. Emerg Infect Dis [Internet]. 2013 Mar [date cited].
22.  Binding of DC-SIGN to the Hemagglutinin of Influenza A Viruses Supports Virus Replication in DC-SIGN Expressing Cells 
PLoS ONE  2013;8(2):e56164.
Dendritic cells express lectins receptors, like DC-SIGN, which allow these cells to sense glycans that are present on various bacterial and viral pathogens. Interaction of DC-SIGN with carbohydrate moieties induces maturation of dendritic cells and promotes endocytosis of pathogens which is an important property of these professional antigen presenting cells. Uptake of pathogens by dendritic cells may lead to cross-presentation of antigens or infection of these cells, which ultimately results in activation of virus-specific T cells in draining lymph nodes. Little is known about the interaction of DC-SIGN with influenza A viruses. Here we show that a virus with a non-functional receptor binding site in its hemagglutinin, can replicate in cells expressing DC-SIGN. Also in the absence of sialic acids, which is the receptor for influenza A viruses, these viruses replicate in DC-SIGN expressing cells including human dendritic cells. Furthermore, the efficiency of DC-SIGN mediated infection is dependent on the extent of glycosylation of the viral hemagglutinin.
PMCID: PMC3570528  PMID: 23424649
23.  Profiling of Humoral Response to Influenza A(H1N1)pdm09 Infection and Vaccination Measured by a Protein Microarray in Persons with and without History of Seasonal Vaccination 
PLoS ONE  2013;8(1):e54890.
The influence of prior seasonal influenza vaccination on the antibody response produced by natural infection or vaccination is not well understood.
We compared the profiles of antibody responses of 32 naturally infected subjects and 98 subjects vaccinated with a 2009 influenza A(H1N1) monovalent MF59-adjuvanted vaccine (Focetria®, Novartis), with and without a history of seasonal influenza vaccination. Antibodies were measured by hemagglutination inhibition (HI) assay for influenza A(H1N1)pdm09 and by protein microarray (PA) using the HA1 subunit for seven recent and historic H1, H2 and H3 influenza viruses, and three avian influenza viruses. Serum samples for the infection group were taken at the moment of collection of the diagnostic sample, 10 days and 30 days after onset of influenza symptoms. For the vaccination group, samples were drawn at baseline, 3 weeks after the first vaccination and 5 weeks after the second vaccination.
We showed that subjects with a history of seasonal vaccination generally exhibited higher baseline titers for the various HA1 antigens than subjects without a seasonal vaccination history. Infection and pandemic influenza vaccination responses in persons with a history of seasonal vaccination were skewed towards historic antigens.
Seasonal vaccination is of significant influence on the antibody response to subsequent infection and vaccination, and further research is needed to understand the effect of annual vaccination on protective immunity.
PMCID: PMC3554683  PMID: 23365683
24.  PAR1 contributes to influenza A virus pathogenicity in mice 
Influenza causes substantial morbidity and mortality, and highly pathogenic and drug-resistant strains are likely to emerge in the future. Protease-activated receptor 1 (PAR1) is a thrombin-activated receptor that contributes to inflammatory responses at mucosal surfaces. The role of PAR1 in pathogenesis of virus infections is unknown. Here, we demonstrate that PAR1 contributed to the deleterious inflammatory response after influenza virus infection in mice. Activating PAR1 by administering the agonist TFLLR-NH2 decreased survival and increased lung inflammation after influenza infection. Importantly, both administration of a PAR1 antagonist and PAR1 deficiency protected mice from infection with influenza A viruses (IAVs). Treatment with the PAR1 agonist did not alter survival of mice deficient in plasminogen (PLG), which suggests that PLG permits and/or interacts with a PAR1 function in this model. PAR1 antagonists are in human trials for other indications. Our findings suggest that PAR1 antagonism might be explored as a treatment for influenza, including that caused by highly pathogenic H5N1 and oseltamivir-resistant H1N1 viruses.
PMCID: PMC3533265  PMID: 23202729
25.  The Multibasic Cleavage Site in H5N1 Virus Is Critical for Systemic Spread along the Olfactory and Hematogenous Routes in Ferrets 
Journal of Virology  2012;86(7):3975-3984.
The route by which highly pathogenic avian influenza (HPAI) H5N1 virus spreads systemically, including the central nervous system (CNS), is largely unknown in mammals. Especially, the olfactory route, which could be a route of entry into the CNS, has not been studied in detail. Although the multibasic cleavage site (MBCS) in the hemagglutinin (HA) of HPAI H5N1 viruses is a major determinant of systemic spread in poultry, the association between the MBCS and systemic spread in mammals is less clear. Here we determined the virus distribution of HPAI H5N1 virus in ferrets in time and space—including along the olfactory route—and the role of the MBCS in systemic replication. Intranasal inoculation with wild-type H5N1 virus revealed extensive replication in the olfactory mucosa, from which it spread to the olfactory bulb and the rest of the CNS, including the cerebrospinal fluid (CSF). Virus spread to the heart, liver, pancreas, and colon was also detected, indicating hematogenous spread. Ferrets inoculated intranasally with H5N1 virus lacking an MBCS demonstrated respiratory tract infection only. In conclusion, HPAI H5N1 virus can spread systemically via two different routes, olfactory and hematogenous, in ferrets. This systemic spread was dependent on the presence of the MBCS in HA.
PMCID: PMC3302532  PMID: 22278228

Results 1-25 (58)