[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.

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.

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.

Universal influenza vaccines: a realistic option?

The extensive antigenic drift displayed by seasonal influenza viruses and the risk of pandemics caused by newly emerging antigenically distinct influenza A viruses of novel subtypes has raised considerable interest in the development of so-called universal influenza vaccines. We review options for the development of universal flu vaccines and discuss progress that has been made recently.

Antibody landscapes after influenza virus infection or vaccination.

We introduce the antibody landscape, a method for the quantitative analysis of antibody-mediated immunity to antigenically variable pathogens, achieved by accounting for antigenic variation among pathogen strains. We generated antibody landscapes to study immune profiles covering 43 years of influenza A/H3N2 virus evolution for 69 individuals monitored for infection over 6 years and for 225 individuals pre- and postvaccination. Upon infection and vaccination, titers increased broadly, including previously encountered viruses far beyond the extent of cross-reactivity observed after a primary infection. We explored implications for vaccination and found that the use of an antigenically advanced virus had the dual benefit of inducing antibodies against both advanced and previous antigenic clusters. These results indicate that preemptive vaccine updates may improve influenza vaccine efficacy in previously exposed individuals.

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.

Serum antibody response to influenza virus vaccination during chemotherapy treatment in adult patients with solid tumours.

BACKGROUND: Higher rates of hospitalization and mortality are described in oncology patients with influenza virus infection compared to the general population. Yearly influenza vaccination is recommended for patients treated with chemotherapy. The optimal moment to administer the vaccine during a treatment cycle has not been studied extensively. PATIENTS AND METHODS: During the influenza season 2011-2012 we conducted a multicenter randomized controlled trial (OFLUVAC, NTR2858, no sponsoring) in the Netherlands. Patients receiving adjuvant chemotherapy for breast or colorectal cancer were randomized between early (day 5 after chemotherapy) and late (day 16 after chemotherapy) vaccination with the influenza virus vaccine (Influvac(®) 2011/2012-Vaxigrip(®) 2011/2012). Influenza virus-specific antibody titres were determined before, 3 and 12 weeks after vaccination by haemagglutination inhibition. RESULTS: Thirty-eight breast cancer patients (early=21; late=17) and 18 colorectal cancer patients (early=8; late=10) were analyzed. In breast cancer patients overall serologic responses were adequate. A statistically significant higher response in patients who received early compared to late vaccination in the chemotherapy cycle was observed. Geometric mean titres post vaccination on day 5 versus day 16 were 69.3 versus 27.4 (H3N2), 76.4 versus 17.5 (H1N1) and 34.4 versus 26.0 (B/Brisbane), respectively. In colorectal cancer patients overall serologic responses were adequate, no significant difference was found between early and late vaccination. Geometric mean titres post vaccination on day 5 versus day 16 were 170.1 versus 192.4 (H3N2), 233.0 versus 280.8 (H1N1) and 62.6 versus 75.9 (B/Brisbane), respectively. CONCLUSION: Overall antibody response to the influenza virus vaccine in patients treated with chemotherapy for breast or colorectal cancer patients is adequate. Breast cancer patients seem to mount the best antibody response when vaccinated early after a chemotherapy cycle (≤day 5). No difference was found between early and late vaccination in colorectal cancer patients.

Low pathogenic avian influenza A(H7N9) virus causes high mortality in ferrets upon intratracheal challenge: a model to study intervention strategies.

Infections with low pathogenic avian influenza (LPAI) A(H7N9) viruses have caused more than 100 hospitalized human cases of severe influenza in China since February 2013 with a case fatality rate exceeding 25%. Most of these human infections presented with severe viral pneumonia, while limited information is available currently on the occurrence of mild and subclinical cases. In the present study, a ferret model for this virus infection in humans is presented to evaluate the pathogenesis of the infection in a mammalian host, as ferrets have been shown to mimic the pathogenesis of human infection with influenza viruses most closely. Ferrets were inoculated intratracheally with increasing doses (>10 e5 TCID50) of H7N9 influenza virus A/Anhui/1/2013 and were monitored for clinical and virological parameters up to four days post infection. Virus replication was detected in the upper and lower respiratory tracts while animals developed fatal viral pneumonia. This study illustrates the high pathogenicity of LPAI-H7N9 virus for mammals. Furthermore, the intratracheal inoculation route in ferrets proofs to offer a solid model for LPAI-H7N9 virus induced pneumonia in humans. This model will facilitate the development and assessment of clinical intervention strategies for LPAI-H7N9 virus infection in humans, such as preventive vaccination and the use of antivirals.

Clearance of influenza virus infections by T cells: risk of collateral damage?

Influenza A viruses are a major cause of respiratory infections in humans. To protect against influenza, vaccines mainly aim at the induction of antibodies against the two surface proteins and do not protect against influenza A viruses from other subtypes. There is an increasing interest in heterosubtypic immunity that does protect against different subtypes. CD8 and CD4 T cells have a beneficial effect on the course of influenza A virus infection and can recognize conserved IAV epitopes. The T cell responses are tightly regulated to avoid collateral damage due to overreaction. Different studies have shown that an aberrant T cell response to an influenza virus infection could be harmful and could contribute to immunopathology. Here we discuss the recent findings on the balance between the beneficial and detrimental effects of T cell responses in influenza virus infections.

Pathogenesis of influenza virus infections: the good, the bad and the ugly.

The clinical outcome of different influenza virus infections ranges from subclinical upper respiratory tract disease to fatal lower respiratory tract disease. An important determinant in the pathogenesis of these diseases is the tissue tropism of the influenza virus. Furthermore, virulence is often correlated with virus replication and is regulated by multiple virus genes. Host defense against virus infection consists of both innate and adaptive immune responses. However, excessive or dysbalanced immune response may result in lung tissue damage, reduced respiratory capacity, and severe disease or even death. By interdisciplinary efforts to better understand the intricate interaction between virus, tissue, and immune response, we may be able to find new ways to improve the outcome of influenza virus infections.

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.

Profiling of humoral immune responses to influenza viruses by using protein microarray.

The emergence of pandemic A(H1N1) 2009 influenza showed the importance of rapid assessment of the degree of immunity in the population, the rate of asymptomatic infection, the spread of infection in households, effects of control measures, and ability of candidate vaccines to produce a response in different age groups. A limitation lies in the available assay repertoire: reference standard methods for measuring antibodies to influenza virus are haemagglutination inhibition (HI) assays and virus neutralization tests. Both assays are difficult to standardize and may be too specific to assess possible partial humoral immunity from previous exposures. Here, we describe the use of antigen-microarrays to measure antibodies to HA1 antigens from seven recent and historical seasonal H1, H2 and H3 influenza viruses, the A(H1N1) 2009 pandemic influenza virus, and three avian influenza viruses. We assessed antibody profiles in 18 adult patients infected with A(H1N1) 2009 influenza virus during the recent pandemic, and 21 children sampled before and after the pandemic, against background reactivity observed in 122 persons sampled in 2008, a season dominated by seasonal A(H1N1) influenza virus. We show that subtype-specific and variant-specific antibody responses can be measured, confirming serological responses measured by HI. Comparison of profiles from persons with similar HI response showed that the magnitude and broadness of response to individual influenza subtype antigens differs greatly between individuals. Clinical and vaccination studies, but also exposure studies, should take these findings into consideration, as they may indicate some level of humoral immunity not measured by HI assays.

Immune responses to influenza virus infection.

Influenza viruses cause annual outbreaks of respiratory tract infection with attack rates of 5-10%. This means that humans are infected repeatedly with intervals of, on average, 10-20 years. Upon each infection subjects develop innate and adaptive immune responses which aim at clearing the infection. Strain-specific antibody responses are induced, which exert selective pressure on circulating influenza viruses and which drive antigenic drift of seasonal influenza viruses, especially in the hemagglutinin molecule. This antigenic drift necessitates updating of seasonal influenza vaccines regularly in order to match the circulating strains. Upon infection also virus-specific T cell responses are induced, including CD4+ T helper cells and CD8+ cytotoxic T cells. These cells are mainly directed to conserved proteins and therefore display cross-reactivity with a variety of influenza A viruses of different subtypes. T cell mediated immunity therefore may contribute to so-called heterosubtypic immunity and may afford protection against antigenically distinct, potentially pandemic influenza viruses. At present, novel viral targets are identified that may help to develop broad-protective vaccines. Here we review the various arms of the immune response to influenza virus infections and their viral targets and discuss the possibility of developing universal vaccines. The development of such novel vaccines would imply that also new immune correlates of protection need to be established in order to facilitate assessment of vaccine efficacy.

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.