Protein and modified vaccinia virus Ankara-based influenza virus nucleoprotein vaccines are differentially immunogenic in BALB/c mice.

Because of the high variability of seasonal influenza viruses and the eminent threat of influenza viruses with pandemic potential, there is great interest in the development of vaccines that induce broadly protective immunity. Most probably, broadly protective influenza vaccines are based on conserved proteins, such as nucleoprotein (NP). NP is a vaccine target of interest as it has been shown to induce cross-reactive antibody and T cell responses. Here we tested and compared various NP-based vaccine preparations for their capacity to induce humoral and cellular immune responses to influenza virus NP. The immunogenicity of protein-based vaccine preparations with Matrix-M™ adjuvant as well as recombinant viral vaccine vector modified Vaccinia virus Ankara (MVA) expressing the influenza virus NP gene, with or without modifications that aim at optimization of CD8+ T cell responses, was addressed in BALB/c mice. Addition of Matrix-M™ adjuvant to NP wild-type protein-based vaccines significantly improved T cell responses. Furthermore, recombinant MVA expressing the influenza virus NP induced strong antibody and CD8+ T cell responses, which could not be improved further by modifications of NP to increase antigen processing and presentation.

A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication.

RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5' packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared with the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.

Detection of nonhemagglutinating influenza a(h3) viruses by enzyme-linked immunosorbent assay in quantitative influenza virus culture.

To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate.

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 occurred. Occasionally, influenza A viruses that circulate in other species cross the species barrier and infect humans. Virus reassortment (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. The identification and characterisation 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.

Use of influenza A viruses expressing reporter genes to assess the frequency of double infections in vitro.

Exchange of gene segments between mammalian and avian influenza A viruses may lead to the emergence of potential pandemic influenza viruses. Since co-infection of single cells with two viruses is a prerequisite for reassortment to take place, we assessed frequencies of double-infection in vitro using influenza A/H5N1 and A/H1N1 viruses expressing the reporter genes eGFP or mCherry. Double-infected A549 and Madin-Darby canine kidney cells were detected by confocal microscopy and flow cytometry.

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets.

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.

Characterization of the human CD8⁺ T cell response following infection with 2009 pandemic influenza H1N1 virus.

The 2009 H1N1 influenza pandemic provided an opportunity to study human virus-specific T cell responses after infection with a novel influenza virus against which limited humoral immunity existed in the population. Here we describe the magnitude, kinetics, and nature of the virus-specific T cell response using intracellular gamma interferon (IFN-γ) staining and fluorochrome-labeled major histocompatibility complex (MHC) class I-peptide complexes. We demonstrate that influenza virus-infected patients develop recall T cell responses that peak within 1 week postinfection and that contract rapidly. In particular, effector cell frequencies declined rapidly postinfection in favor of relatively larger proportions of central memory cells.

Response to influenza virus vaccination during chemotherapy in patients with breast cancer.

BACKGROUND: Patients receiving chemotherapy are at increased risk for influenza virus infection. Little is known about the preferred moment of vaccination during chemotherapy. PATIENTS AND METHODS: Breast cancer patients received influenza vaccination during FEC (5-fluorouracil, epirubicin and cyclophosphamide)-containing chemotherapy regimens. Patients were randomised for early (day 4) or late (day 16) vaccination during the chemotherapy cycle. Influenza virus-specific antibody titres were determined before and 3 weeks after vaccination by haemagglutination inhibition. RESULTS: We included 38 breast cancer patients (20 in the early and 18 in the late group) and 21 healthy controls. The overall patient group had significant lower responses to the vaccine compared with healthy controls. Patients vaccinated at day 4 tended to have higher antibody titres as compared with patients vaccinated at day 16, although the difference in post-vaccination titres is not statistically significant. Geometric mean titres post-vaccination for day 4 versus day 16 were 63.7 versus 29.5 (H3N2), 28.2 versus 19.6 (H1N1) and 29.8 versus 16.0 (B/Brisbane), respectively. CONCLUSIONS: Patients on chemotherapy have significantly lower responses to influenza virus vaccination compared with healthy controls. Vaccination early during the chemotherapy cycle induces better responses than does vaccination at day 16 of the cycle. Follow-up studies are needed to confirm this effect.

A single immunization with CoVaccine HT-adjuvanted H5N1 influenza virus vaccine induces protective cellular and humoral immune responses in ferrets.

Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 microg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.

Pandemic 2009 H1N1 influenza virus causes diffuse alveolar damage in cynomolgus macaques.

The pathogenesis of lower respiratory tract disease from the pandemic 2009 H1N1 (H1N1v) influenza A virus is poorly understood. Therefore, either H1N1v virus or a seasonal human H1N1 influenza A virus was inoculated into cynomolgus macaques as a nonhuman primate model of influenza pneumonia, and virological, pathological, and microarray analyses were performed. Macaques in the H1N1v group had virus-associated diffuse alveolar damage involving both type I and type II alveolar epithelial cells and affecting an average of 16% of the lung area. In comparison, macaques in the seasonal H1N1 group had milder pulmonary lesions. H1N1v virus tended to be reisolated from more locations in the respiratory tract and at higher titers than seasonal H1N1 virus. In contrast, differential expression of messenger RNA transcripts between H1N1v and seasonal H1N1 groups did not show significant differences. The most upregulated genes in H1N1v lung samples with lesions belonged to the innate immune response and proinflammatory pathways and correlated with histopathological results. Our results demonstrate that the H1N1v virus infects alveolar epithelial cells and causes diffuse alveolar damage in a nonhuman primate model. Its higher pathogenicity compared with a seasonal H1N1 virus may be explained in part by higher replication in the lower respiratory tract.

Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus.

Since the number of human cases of infection with avian H5N1 influenza viruses is ever increasing, a pandemic outbreak caused by these viruses is feared. Therefore, in addition to virus-specific antibodies, there is considerable interest in immune correlates of protection against these viruses, which could be a target for the development of more universal vaccines. After infection with seasonal influenza A viruses of the H3N2 and H1N1 subtypes, individuals develop virus-specific cytotoxic T-lymphocyte responses, which are mainly directed against the relatively conserved internal proteins of the virus, like the nucleoprotein (NP). Virus-specific cytotoxic T lymphocytes (CTL) are known to contribute to protective immunity against infection, but knowledge about the extent of cross-reactivity with avian H5N1 influenza viruses is sparse. In the present study, we evaluated the cross-reactivity with H5N1 influenza viruses of polyclonal CTL obtained from a group of well-defined HLA-typed study subjects. To this end, the recognition of synthetic peptides representing H5N1 analogues of known CTL epitopes was studied. In addition, the ability of CTL specific for seasonal H3N2 influenza virus to recognize the NP of H5N1 influenza virus or H5N1 virus-infected cells was tested. It was concluded that, apart from some individual epitopes that displayed amino acid variation between H3N2 and H5N1 influenza viruses, considerable cross-reactivity exists with H5N1 viruses. This preexisting cross-reactive T-cell immunity in the human population may dampen the impact of a next pandemic.

Influenza B virus in seals.

Influenza B virus is a human pathogen whose origin and possible reservoir in nature are not known. An influenza B virus was isolated from a naturally infected harbor seal (Phoca vitulina) and was found to be infectious to seal kidney cells in vitro. Sequence analyses and serology indicated that influenza virus B/Seal/Netherlands/1/99 is closely related to strains that circulated in humans 4 to 5 years earlier. Retrospective analyses of sera collected from 971 seals showed a prevalence of antibodies to influenza B virus in 2% of the animals after 1995 and in none before 1995. This animal reservoir, harboring influenza B viruses that have circulated in the past, may pose a direct threat to humans.

Avian influenza viruses in mammals.

Highly pathogenic avian influenza viruses of subtype H5N1 are remarkable because of their expanding non-avian host range and wide tissue tropism. They have caused severe or fatal respiratory and extra-respiratory disease in seven naturally infected species of carnivore. However, they are not unique in their ability to cross the species barrier, to cause clinical disease and mortality, or to replicate in extra-respiratory organs. Low pathogenic avian influenza viruses have crossed from birds to swine, horses, harbour seals, whales and mink; have resulted in severe respiratory disease and mortality; and may have spread beyond the respiratory tract in some of these species. They are also transmitted from mammal to mammal in most species, and have become endemic in swine and horse populations, demonstrating their ability to adapt to and become sustained in mammals. Until now, highly pathogenic avian influenza viruses H5N1 have not acquired this ability, but there are concerns that they may adapt to mammalian species and, thus, could spark an influenza pandemic in humans.

The effect of anti-tumour necrosis factor alpha treatment on the antibody response to influenza vaccination.

OBJECTIVES: The effect of anti-tumour necrosis factor (TNF) therapy on the antibody responses to vaccines is the subject of ongoing debate. Therefore, we investigated the effect of the three currently available anti-TNF agents on influenza vaccination outcomes in a patient population with long-standing disease. METHODS: In a prospective cohort study, we assessed the antibody response upon influenza vaccination in 112 patients with long-standing autoimmune disease treated with immunosuppressive medication either with anti-TNF (etanercept, adalimumab or infliximab; n = 64) or without anti-TNF (n = 48) and a control group of 18 healthy individuals. Antibody responses were determined by haemagglutination inhibition assay, before and 4 weeks after vaccination. RESULTS: The proportion of individuals with a protective titre (>or=40) after vaccination was large (80-94%) and did not significantly differ between the three groups. Post-vaccination geometric mean antibody titres against influenza (A/H3N2 and B) were significantly lower in the 64 patients treated with anti-TNF compared with the 48 patients not receiving anti-TNF, and the healthy controls. CONCLUSIONS: The antibody response to influenza vaccination in patients treated with anti-TNF is only modestly impaired. The proportion of patients that achieves a protective titre is not significantly diminished by the use of TNF blocking therapies.

[Influenza season 2007/'08 in the Netherlands: antigenic variation, oseltamivir resistance and vaccine composition for the 2008/'09 season]. / Het influenzaseizoen 2007/'08 in Nederland: antigene variatie, resistentie tegen oseltamivir en de vaccinsamenstelling voor het seizoen 2008/'09.

The first signs of influenza activity during the 2007/'08 influenza season in the Netherlands were sporadic isolations of influenza viruses between week 40 and week 52 of 2007. The frequency of virus isolations and clinical influenza activity increased after week 1 of 2008 and peaked around week 9. In this week, 7.2 patients with influenza-like illness were recorded per 10,000 inhabitants. The influenza epidemic was caused primarily by influenza A/H1N1 viruses and influenza B viruses. Two antigenically distinct variants of influenza A/H1N1 viruses were isolated, which resembled the 2007/'08 vaccine reference strain A/Solomon Islands/3/06 and the new vaccine reference strain A/Brisbane/59/07, respectively. The most remarkable finding was that 27% of the A/H1N1 viruses isolated in the Netherlands during the 2007/'08 epidemic were resistant to the neuraminidase inhibitor oseltamivir. The isolated influenza B viruses originated from the B/Yamagata/16/88 lineage and did not match the vaccine strain, which originated from a different and antigenically distinct lineage of influenza B viruses (B/Victoria/2/87). Only a small number of influenza A/H3N2 viruses was isolated, which were related to the vaccine strain for this subtype (A/Wisconsin/67/05). Thus in contrast to previous influenza seasons, A/H3N2 viruses did not play a major role in the 2007/'08 influenza season in the Netherlands. For the 2008/'09 influenza season, the World Health Organization has recommended the following vaccine composition: A/Brisbane/59/07 (H1N1), A/Brisbane/10/07 (H3N2) and B/Florida/4/06.

[The 2006/'07 influenza season in the Netherlands and the vaccine composition for the 2007/'08 season]. / Het influenzaseizoen 2006/'07 in Nederland en de vaccinsamenstelling voor het seizoen 2007/'08.

The influenza epidemic of 2006/'07 began late in the season, like the two previous influenza epidemics. In week 8 a peak of modest height was reached. As usual, the causal strains were mainly A/H3N2 viruses and to a lesser extent A/H1N1 and B viruses. A new A/H1N1 virus variant has emerged, an event that on average takes place only every 10 years. However, almost all A/H1N1 virus isolates belonged to the old variant and were similar to the vaccine virus. The A/H3N2 virus isolates appeared to deviate from the vaccine strain, but after antigenic cartographic analysis and correction for low avidity they proved also closely related to the vaccine strain. The few type B virus isolates belonged to the B/Yamagata/16/88 lineage, whereas the used B vaccine virus had been chosen from the B/Victoria/2/87 lineage. The vaccine therefore will have provided almost optimal protection against the circulating influenza A/H1N1 and A/H3N2 viruses but not against the influenza B viruses. For the 2007/'08 influenza season the World Health Organization has recommended the following vaccine composition: A/Solomon Islands/3/06 (H1N1) (new), A/Wisconsin/67/05 (H3N2), and B/Malaysia/2506/04.

[Influenza vaccination of patients with solid tumours during chemotherapy: efficacy and safety]. / Griepvaccinatie tijdens chemotherapie bij patiënten met solide tumoren.

- Adult patients with solid tumours receiving chemotherapy have reduced immunity against infections and are at increased risk of influenza infection and its complications. However, many of said patients are not vaccinated for influenza.- Limited observational research in this patient group has given some indication of the protective effects of the influenza vaccine on clinical outcome measures.- Serological studies have shown that the antibody response following influenza vaccination is often less pronounced in patients with solid tumours compared to healthy individuals. Nonetheless, in most cases a timely protective antibody response can be achieved.- The inactivated influenza vaccine is safe in immunosuppressed patients, irrespective of the moment at which it is administered. Side-effects are similar, both in nature and number, to those seen in healthy individuals.- Influenza vaccination can be offered to all adult patients with solid tumours, preferably before chemotherapy is commenced. Vaccination during chemotherapy, however, usually also generates sufficient vaccination response and can reduce the risk of influenza-related complications. Therefore, chemotherapy should not preclude patients from being administered the influenza vaccine.

Microarray profile of the humoral immune response to influenza vaccination in breast cancer patients treated with chemotherapy.

BACKGROUND: Patients treated with chemotherapy have an impaired response to influenza virus vaccination compared to healthy controls. Little is known about the broadness of the antibody response in these patients. METHODS: Breast cancer patients on FEC (5-fluorouracil, epirubicin and cyclophosphamide) chemotherapy regimens were vaccinated with influenza virus vaccine. Sera were obtained before and three weeks after vaccination. In addition to the determination of virus-specific antibody titres by hemagglutination inhibition assay, the broadness of the response was assessed by the use of a protein microarray and baseline titres were compared with an age-matched reference group. RESULTS: We included 38 breast cancer patients and found a wide variety in serum antibody response after vaccination. Patients with a history of influenza vaccination had higher pre-vaccination titres, which were comparable to the reference group. Increasing number of cycles of chemotherapy did not have a negative effect on influenza array antibody levels, nor on the HI antibody response. CONCLUSIONS: Overall there was a broad serum antibody response to the influenza virus vaccine in patients treated with chemotherapy for breast cancer.