Pandemic H1N1 Strain Research Articles

Highly Pathogenic H5N1 and Novel H7N9 Influenza A Viruses Induce More Profound Proteomic Host Responses than Seasonal and Pandemic H1N1 Strains.

Influenza A viruses (IAV) are important human and animal pathogens with potential for causing pandemics. IAVs exhibit a wide spectrum of clinical illness in humans, from relatively mild infections by seasonal strains to acute respiratory distress syndrome during infections with some highly pathogenic avian influenza (HPAI) viruses. In the present study, we infected A549 human cells with seasonal H1N1 (sH1N1), 2009 pandemic H1N1 (pdmH1N1), or novel H7N9 and HPAI H5N1 strains. We used multiplexed isobaric tags for relative and absolute quantification to measure proteomic host responses to these different strains at 1, 3, and 6 h post-infection. Our analyses revealed that both H7N9 and H5N1 strains induced more profound changes to the A549 global proteome compared to those with low-pathogenicity H1N1 virus infection, which correlates with the higher pathogenicity these strains exhibit at the organismal level. Bioinformatics analysis revealed important modulation of the nuclear factor erythroid 2-related factor 2 (NRF2) oxidative stress response in infection. Cellular fractionation and Western blotting suggested that the phosphorylated form of NRF2 is not imported to the nucleus in H5N1 and H7N9 virus infections. Fibronectin was also strongly inhibited in infection with H5N1 and H7N9 strains. This is the first known comparative proteomic study of the host response to H7N9, H5N1, and H1N1 viruses and the first time NRF2 is shown to be implicated in infection with highly pathogenic strains of influenza.

ID: 157: Mapping of amino acids in the nucleoprotein of influenza A viruses responsible for the interaction with MxA

The MxA protein belongs to the family of dynamin-like, large GTPases and exerts antiviral activity against a variety of RNA and DNA viruses, including influenza A (IAV). MxA restricts infection with zoonotic avian IAV, while human IAV have adapted and exhibit a largely resistant phenotype. Recent evidence reveals that the resistance phenotype of human IAV segregates with a discrete cluster of surface-exposed amino acids in the nucleoprotein (NP) of human IAV, indicating that MxA targets NP. Thus, we addressed the question whether MxA has to form a stable complex with NP to interfere with its function. Co-immunoprecipitation experiments with cytoplasmic extracts of cells either infected with IAV or ectopically transfected with plasmids encoding NP revealed that interaction of MxA and NP was only detected when MxA was expressed in its dimeric form. As expected, MxA bound to NP of all Mx-sensitive avian strains tested, while the NP of a human MxA-resistant IAV strain derived from the 1918 IAV (H1N1) strain did not bind. Intriguingly, the NP from the MxA-resistant human pandemic H1N1 (2009) strain still exhibited pronounced binding to MxA. However, the resistance-associated amino acids in NP differ between these two human two strains, suggesting that MxA binding to NP is required but not sufficient for activity. Hence, we currently map the amino acids of NP from sensitive and resistant strains responsible for binding and/or susceptibilities to MxA exploiting their differences in binding-capacities and sensitivity to MxA.

Difluorosialic acids, potent novel influenza virus neuraminidase inhibitors, induce fewer drug resistance-associated neuraminidase mutations than does oseltamivir

Neuraminidase inhibitors (NAIs), including the most frequently prescribed oral therapeutic oseltamivir, play a critical role in the control of severe influenza virus (IFV) infections. However, recent reports of spread of an oseltamivir-resistant H1N1 pandemic strain in individuals who have never been exposed to oseltamivir highlight an urgent need for new antivirals against NAI-resistant IFVs. Difluorosialic acids (DFSAs) are a novel class of anti-IFV NAIs designed based on the mechanism of action of IFV NA, and distinguished by their covalent inhibition mode and their high structural similarity to the natural substrate, sialic acid. These characteristics should render the development of resistance a less rapid process. In this report, we evaluated the relative propensity of influenza A virus (IFV-A) NA to develop resistance against the DFSA class of inhibitor by passaging IFV-A strains in vitro in the presence of either oseltamivir or a representative DFSA (FeqGuDFSA). All the passage-selected lines gained mutations in hemagglutinin. Among the 12 oseltamivir-resistant passaged lines, five gained NA mutations and four of these were the well-defined H275Y mutation that causes oseltamivir resistance. In contrast, out of 15 DFSA-passaged lines, only 2 lines gained NA mutations. Further, NA inhibition assays indicated that these mutations did not change the sensitivity of NA to DFSA and thus the resistance to DFSA was not conferred by these NA mutations. These results strongly suggest that, compared to oseltamivir, IFV is less prone to development of resistance against DFSAs through NA mutations.

Possible Impact of Yearly Childhood Vaccination With Trivalent Inactivated Influenza Vaccine (TIV) on the Immune Response to the Pandemic Strain H1N1.

Annual vaccination of children against seasonal influenza with trivalent inactivated influenza vaccine (TIV) has shown to be beneficial. However, this yearly practice may have unintended effect. Studies have shown that infection with wild type influenza A viruses can stimulate protective heterotypic immunity against unrelated or new influenza subtypes. We hypothesized that a consequence of yearly TIV vaccination is lack of induction of heterotypic immunity against the recent H1N1 pandemic. This was a retrospective case-control study. We reviewed the medical records of polymerase chain reaction-confirmed cases of 2009 H1N1 influenza infection in children 6 months to 18 years and a matched control group seen during the pandemic. We identified 353 polymerase chain reaction-confirmed H1N1 cases and 396 matching control subjects. Among the H1N1 group, 202/353 (57%) cases received a total of 477 doses of seasonal TIV compared with 218/396 (55%) in the control group who received a total of 435 doses. Seasonal TIV uptake was significantly higher in the H1N1 group 477/548 (87%) than in the control group, 435/532 (81%) (P = .017). Seasonal TIV uptake was significantly higher in H1N1-infected group. The finding suggests that the practice of yearly vaccination with TIV might have negatively affected the immune response against the novel pandemic H1N1 strain. Given the rarity of pandemic novel influenza viruses, and the high predictability of seasonal influenza occurrence, the practice of yearly influenza vaccination should be continued. However, the use of live attenuated intranasal vaccine, as opposed to TIV, may allow for the desirable development of a vigorous heterotypic immune response against future pandemics.

CD8+ cytotoxic T lymphocytes in human influenza virus infection.

CD8+ cytotoxic T lymphocytes in human influenza virus infection.

Pandemic Swine-Origin H1N1 Influenza Virus Replicates to Higher Levels and Induces More Fever and Acute Inflammatory Cytokines in Cynomolgus versus Rhesus Monkeys and Can Replicate in Common Marmosets.

The close immunological and physiological resemblance with humans makes non-human primates a valuable model for studying influenza virus pathogenesis and immunity and vaccine efficacy against infection. Although both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of susceptibility to infection and disease has not yet been performed. In the current study a head-to-head comparison was made between these species, by using a recently described swine-origin pandemic H1N1 strain, A/Mexico/InDRE4487/2009. In comparison to rhesus macaques, cynomolgus macaques developed significantly higher levels of virus replication in the upper airways and in the lungs, involving both peak level and duration of virus production, as well as higher increases in body temperature. In contrast, clinical symptoms, including respiratory distress, were more easily observed in rhesus macaques. Expression of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, was 50 to 73 times more abundant in trachea and bronchus of cynomolgus macaques relative to rhesus macaques. The study also shows that common marmosets, a New World non-human primate species, are susceptible to infection with pandemic H1N1. The study results favor the cynomolgus macaque as model for pandemic H1N1 influenza virus research because of the more uniform and high levels of virus replication, as well as temperature increases, which may be due to a more abundant expression of the main human influenza virus receptor in the trachea and bronchi.

Adaptive Mutations That Occurred during Circulation in Humans of H1N1 Influenza Virus in the 2009 Pandemic Enhance Virulence in Mice.

During the 2009 H1N1 influenza pandemic, infection attack rates were particularly high among young individuals who suffered from pneumonia with occasional death. Moreover, previously reported determinants of mammalian adaptation and pathogenicity were not present in 2009 pandemic H1N1 influenza A viruses. Thus, it was proposed that unknown viral factors might have contributed to disease severity in humans. In this study, we performed a comparative analysis of two clinical 2009 pandemic H1N1 strains that belong to the very early and later phases of the pandemic. We identified mutations in the viral hemagglutinin (HA) and the nucleoprotein (NP) that occurred during pandemic progression and mediate increased virulence in mice. Lethal disease outcome correlated with elevated viral replication in the alveolar epithelium, increased proinflammatory cytokine and chemokine responses, pneumonia, and lymphopenia in mice. These findings show that viral mutations that have occurred during pandemic circulation among humans are associated with severe disease in mice. In this study, novel determinants of 2009 pandemic H1N1 influenza pathogenicity were identified in the viral hemagglutinin (HA) and the nucleoprotein (NP) genes. In contrast to highly pathogenic avian influenza viruses, increased virulence in mice did not correlate with enhanced polymerase activity but with reduced activity. Lethal 2009 pandemic H1N1 infection in mice correlated with lymphopenia and severe pneumonia. These studies suggest that molecular mechanisms that mediate 2009 pandemic H1N1 influenza pathogenicity are distinct from those that mediate avian influenza virus pathogenicity in mice.

Distribution of antibodies against influenza virus in pigs from farrow‐to‐finish farms in M inas G erais state, B razil

BackgroundSwine influenza virus (SIV) is the cause of an acute respiratory disease that affects swine worldwide. In Brazil, SIV has been identified in pigs since 1978. After the emergence of pandemic H1N1 in 2009 (H1N1pdm09), few studies reported the presence of influenza virus in Brazilian herds.ObjectivesThe objective of this study was to evaluate the serological profile for influenza virus in farrow-to-finish pig farms in Minas Gerais state, Brazil.MethodsThirty farms with no SIV vaccination history were selected from the four larger pig production areas in Minas Gerais state (Zona da Mata, Triângulo Mineiro/Alto Paranaíba, South/Southwest and the Belo Horizonte metropolitan area). At each farm, blood samples were randomly collected from 20 animals in each production cycle category: breeding animals (sows and gilts), farrowing crate (2–3 weeks), nursery (4–7 weeks), grower pigs (8–14 weeks), and finishing pigs (15–16 weeks), with 100 samples per farm and a total of 3000 animals in this study. The samples were tested for hemagglutination inhibition activity against H1N1 pandemic strain (A/swine/Brazil/11/2009) and H3N2 SIV (A/swine/Iowa/8548-2/98) reference strain.ResultsThe percentages of seropositive animals for H1N1pdm09 and H3N2 were 26·23% and 1·57%, respectively, and the percentages of seropositive herds for both viruses were 96·6% and 13·2%, respectively.ConclusionsThe serological profiles differed for both viruses and among the studied areas, suggesting a high variety of virus circulation around the state, as well as the presence of seronegative animals susceptible to influenza infection and, consequently, new respiratory disease outbreaks.

Similarity of currently circulating H1N1 virus with the 2009 pandemic clone: Viability of an imminent pandemic

The first influenza pandemic in the 21st century commenced in March, 2009 causing nearly 300,000 deaths globally within the first year of the pandemic. In late 2013 and in early 2014, there was gradual increase in the reported case of H1N1 infection and according to World Health Organization (WHO) report, influenza activity increased in several areas of the Southern Hemisphere and was dominated by the H1N1 pandemic strain of 2009. In the present study, a comprehensive comparison of the global amino acid composition and the structural features of all HA gene sequences of H1N1, available in the Flu Database (NCBI), from 1918 to December, 2014 has been performed to trace out the possibility of a further H1N1 pandemic in near future. The results suggest that the increased potential to enhance pathogenicity for the H1N1 samples of 2013 (latter part) and 2014 could lead to a more severe outbreak in the near future.

Influenza A(H1N1)pdm09 Infection With Simultaneous Comorbidity of Severe Pneumonia and Acute Disseminated Encephalomyelitis: A Case Report.

The 2009 H1N1 pandemic strain of influenza A(H1N1)pdm09 was first detected in Mexico and the United States in the spring of 2009 and, thereafter, spread to across the world. In Japan, the number of patients affected by the virus increased dramatically since September 2009.1 The clinical profile of (H1N1)pdm09 infection varies from asymptomatic or mild to severe cases, resulting in severe respiratory failure, encephalitis or encephalopathy, and death in the worst scenario.2 Acute disseminated encephalomyelitis (ADEM) is an inflammatory demyelinating disorder of the central nervous system following a viral infection or vaccination. The pathogenesis of ADEM is proposed to be autoimmune mechanisms triggered by infectious agents or vaccines. Childhood ADEM occurs more often after infection, and its prognosis is considered to be generally favorable.3 There are several reports of severe pneumonia caused by (H1N1)pdm09 infection, while there are only 7 pediatric case reports of ADEM following (H1N1)pdm09 until recently: none of these cases were complicated by pneumonia.4-6 Here we describe the case of 5-year-old boy who developed ADEM secondary to (H1N1)pdm09 severe pneumonia. To the best of our knowledge, this is the first case report of a pediatric patient with both comorbidities.

Evaluation of a fully human monoclonal antibody against multiple influenza A viral strains in mice and a pandemic H1N1 strain in nonhuman primates

Influenza virus is a global health concern due to its unpredictable pandemic potential. Frequent mutations of surface molecules, hemagglutinin (HA) and neuraminidase (NA), contribute to low efficacy of the annual flu vaccine and therapeutic resistance to standard antiviral agents. The populations at high risk of influenza virus infection, such as the elderly and infants, generally mount low immune responses to vaccines, and develop severe disease after infection. Novel therapeutics with high effectiveness and mutation resistance are needed. Previously, we described the generation of a fully human influenza virus matrix protein 2 (M2) specific monoclonal antibody (mAb), Z3G1, which recognized the majority of M2 variants from natural viral isolates, including highly pathogenic avian strains. Passive immunotherapy with Z3G1 significantly protected mice from the infection when administered either prophylactically or 1–2days post infection. In the present study, we showed that Z3G1 significantly protected mice from lethal infection when treatment was initiated 3days post infection. In addition, therapeutic administration of Z3G1 reduced lung viral titers in mice infected with different viral strains, including amantadine and oseltamivir-resistant strains. Furthermore, prophylactic and therapeutic administration of Z3G1 sustained O2 saturation and reduced lung pathology in monkeys infected with a pandemic H1N1 strain. Finally, de-fucosylated Z3G1 with an IgG1/IgG3 chimeric Fc region was generated (AccretaMab® Z3G1), and showed increased ADCC and CDC in vitro. Our data suggest that the anti-M2 mAb Z3G1 has great potential as a novel anti-flu therapeutic agent.

Demographic distribution and transmission potential of influenza A and 2009 pandemic influenza A H1N1 in pilgrims.

The World Health Organization's persistent reporting of global outbreaks of influenza A viruses, including the 2009 pandemic swine A H1N1 strain (H1N1pdm09), justified the targeted surveillance of pilgrims during their annual congregation that pools more than two million people from around 165 nations in a confined area of Makkah city in the Kingdom of Saudi Arabia (KSA). A total of 1,600 pilgrims were included in the targeted surveillance of influenza A and the 2009 pandemic swine H1N1 strain in the Hajj (pilgrimage) season of 2010. Each pilgrim responded to a demographic and health questionnaire. Collected oropharyngeal swabs were analyzed by real-time PCR for influenza A viruses, and positive samples were further analyzed for the presence of H1N1pdm09. Fisher's exact test was applied in the analysis of the significance of the distribution of influenza-positive pilgrims according to demographic characters. A total of 120 pilgrims (7.5%) tested positive for influenza A viruses by real-time PCR. Nine out of the 120 influenza-A-positive pilgrims (7.5%) were positive for H1N1pdm09. Demographics played a significant role in those pilgrims who tested positive for influenza A. The detection of H1N1pdm09 in pilgrims at their port of entry to the KSA was alarming, due to the high potential of trans-boundary transmission. This situation necessitates the implementation of specific prevention and control programs to limit infection by influenza A viruses.

Superior in vitro stimulation of human CD8+ T-cells by whole virus versus split virus influenza vaccines.

Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8+ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8+ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4+ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8+ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8+ T cells should include whole virus rather than split virus formulations.

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.

Molecular characterization of circulating pandemic strains of influenza A virus during 2012 to 2013 in Lucknow (India).

The pandemic H1N1 strain of Influenza A virus [A(H1N1)pdm09] is now well adapted in human populations. However, it is still causing sporadic outbreaks worldwide with different severity. The present study was planned to understand the genetic diversity (based on the HA1 gene) of influenza A(H1N1)pdm09 strains circulating during the post pandemic period. The HA1 gene was selected because the HA1 protein is immunogenic, functions as a receptor binding site and indirectly affects transmission and pathogenicity of virus. A total of 2,818 cases were enrolled. Nasal/throat swabs from all cases were tested by one-step real time PCR for detection of influenza virus types and subtypes according to the CDC protocol. Of these, 134 cases were A(H1N1)pdm09 positive, 34 of which were screened for HA1 gene (position 434-905) sequencing (Big-Dye terminator using 3130 ABI, Genetic analyzer). Molecular and phylogenetic analysis was performed using PhyML approach (v. 3.0). All A(H1N1)pdm09 positive and negative cases were clinically characterized. Phylogentically, all Lucknow strains (n = 33) except one fall with the clade seven reference strain. One strain showed 99.9% similarities with clade one reference strain A/California/07/2009. In mutational analysis, 33 strains had the S220T mutation, which is at an antigenic site and characteristic of clade seven along with few minor mutations; K180I/T/Q, V190I, S200P, S202T, A203T, A214T, S220T, V251I, and A273T. These results suggest that clade seven was the most widely circulating clade in Lucknow and A(H1N1)pdm09 cases showed mild clinical symptoms as compared to A(H3N2) or influenza B cases.

Mutations flanking the carbohydrate binding site of surfactant protein D confer antiviral activity for pandemic influenza A viruses

We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan-rich viral envelope proteins including hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or increased neutralizing activity compared with D325A+R343V. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild-type SP-D NCRD did not. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan side on the head region of the HA and are fully resistant to inhibition by native SP-D. Importantly, we now show that D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains, and reduced uptake of Cal09 by epithelial cells. Inhibition of Cal09 was mediated by the lectin activity of the NCRDs. All known human pandemic strains have at least one glycan attachment on the top or side of the HA head, and our results indicate that they may be susceptible to inhibition by modified host defense lectins.

Influenza A H1N1 pandemic strain evolution--divergence and the potential for antigenic drift variants.

The emergence of a novel A(H1N1) strain in 2009 was the first influenza pandemic of the genomic age, and unprecedented surveillance of the virus provides the opportunity to better understand the evolution of influenza. We examined changes in the nucleotide coding regions and the amino acid sequences of the hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) segments of the A(H1N1)pdm09 strain using publicly available data. We calculated the nucleotide and amino acid hamming distance from the vaccine strain A/California/07/2009 for each sequence. We also estimated Pepitope–a measure of antigenic diversity based on changes in the epitope regions–for each isolate. Finally, we compared our results to A(H3N2) strains collected over the same period. Our analysis found that the mean hamming distance for the HA protein of the A(H1N1)pdm09 strain increased from 3.6 (standard deviation [SD]: 1.3) in 2009 to 11.7 (SD: 1.0) in 2013, while the mean hamming distance in the coding region increased from 7.4 (SD: 2.2) in 2009 to 28.3 (SD: 2.1) in 2013. These trends are broadly similar to the rate of mutation in H3N2 over the same time period. However, in contrast to H3N2 strains, the rate of mutation accumulation has slowed in recent years. Our results are notable because, over the course of the study, mutation rates in H3N2 similar to that seen with A(H1N1)pdm09 led to the emergence of two antigenic drift variants. However, while there has been an H1N1 epidemic in North America this season, evidence to date indicates the vaccine is still effective, suggesting the epidemic is not due to the emergence of an antigenic drift variant. Our results suggest that more research is needed to understand how viral mutations are related to vaccine effectiveness so that future vaccine choices and development can be more predictive.

Pandemic A/H1N1 2009 influenza vaccination, preceding infections and clinical findings in UK children with Guillain–Barré syndrome

ObjectiveTo record clinical findings in all new cases of Guillain–Barré syndrome (GBS) or Fisher syndrome (FS) in UK children in the 2 years following September 2009 and determine the proportion...

Immunization with cross-conserved H1N1 influenza CD4+ T-cell epitopes lowers viral burden in HLA DR3 transgenic mice

The emergence of the pandemic H1N1 strain of influenza in 2009 was associated with a unique w-shaped age-related susceptibility curve, with higher incidence of morbidity and mortality among young persons and lower incidence among older persons, also observed during the 1918 influenza pandemic. Pre-existing H1N1 antibodies were not cross-reactive with the prior seasonal vaccine, forcing influenza experts to scramble to develop a new vaccine specific for the pandemic virus. We hypothesized that response to T-cell epitopes that are cross-conserved between pandemic H1N1 and the 2008 seasonal influenza vaccine strains might have contributed to partial protection from clinical illness among older adults, despite the lack of cross-reactive humoral immunity. Using immunoinformatics tools, we previously identified hemagglutinin and neuraminidase epitopes that were highly conserved between seasonal and pandemic H1N1. Here, we validated predicted CD4+ T-cell epitopes for their ability to bind HLA and to stimulate interferon-γ production in peripheral blood mononuclear cells from a cohort of donors presenting with influenza-like illness during the 2009 pandemic and a separate cohort immunized with trivalent influenza vaccine in 2011. A limited-epitope heterologous DNA-prime/peptide-boost vaccine composed of these sequences stimulated immune responses and lowered lung viral loads in HLA DR3 transgenic mice challenged with pandemic 2009 H1N1 influenza. Cross-priming with conserved influenza T-cell epitopes such as these may be critically important to T cell-mediated protection against pandemic H1N1 in the absence of cross-protective antibodies.

Prevalence of antibodies to European porcine influenza viruses in humans living in high pig density areas of Germany

The risk of zoonotic human infection caused by European porcine influenza virus strains was estimated in German regions with a high pig density. Sera from 622 healthy volunteers were collected between April 2009 and November 2011, mainly in Westphalia and western Lower Saxony. These included 362 subjects with occupational contact to pigs and 260 blood donors without any direct exposition to pigs. Samples were analysed by the haemagglutination inhibition (HI) assay against a panel of six swine viruses of subtypes avian-like H1N1 and human-like H3N2 as well as against human H1N1 and H3N2 viruses including the pandemic H1N1 strain of 2009. Reciprocal HI titres ≥20 were quoted as seroreactive. Compared to the control group, a significantly higher proportion of subjects with direct contact to pigs exhibited seroreactivity against porcine antigens of the avian-like H1N1 (37.0 %/7.7 %), the human-like H3N2 (59.7 %/43.1 %), the pandemic H1N1 strain of 2009 (51.7 %/26.5 %) and against a historic seasonal H3N2 strain that is closely related antigenetically to currently circulating human-like H3N2 viruses of European pigs (57.5 %/36.5 %). This trend was also observed when a reciprocal HI titre ≥40 was chosen as cut-off. Particularly, in younger subjects, the differences in seroreactivity against porcine strains between the exposed and non-exposed group were significant. The data indicate a higher risk of infection in the exposed individuals.