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Background: Influenza A viruses (IAVs) represent repeatedly emerging pathogens with near worldwide distribution and an unclear nonavian-host spectrum. While the natural hosts for IAV are among waterfowl species, certain mammals can be productively infected. Southern Africa is home to diverse avian and mammalian fauna for which almost no information exists on IAV dynamics., Methods: We evaluated 111 serum samples from 14 mammalian species from Namibia for the presence of IAV-specific antibodies and tested whether host phylogeny, sociality, or diet influence viral prevalence and diversity., Results: Free-ranging African mammals are exposed to diverse IAV subtypes. Herbivores developed antibodies against 3 different hemagglutinin (HA) subtypes, at low prevalence, while carnivores showed a higher prevalence and diversity of HA-specific antibody responses against 11 different subtypes. Host phylogeny and sociality were not significantly associated with HA antibody prevalence or subtype diversity. Both seroprevalence and HA diversity were significantly increased in carnivores regularly feeding on birds., Conclusions: The risk of infection and transmission may be driven by diet and ecological factors that increase contact with migratory and resident waterfowl. Consequently, wild mammals, particularly those that specialize on hunting and scavenging birds, could play an important but overlooked role in influenza epizootics., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Objective: To investigate the efficacy and safety of an influenza vaccination in patients with myasthenia gravis with acetylcholine receptor antibodies (AChR MG)., Methods: An influenza vaccination or placebo was administered to 47 AChR MG patients. Before and 4 weeks after administration blood samples and clinical outcome scores were obtained. Antibodies to the vaccine strains A/California/7/2009 (H1N1)pdm09, A/Hong Kong/4801/14 (H3N2) and B/Brisbane/060/08 were measured using the hemagglutination-inhibition (HI) assay and disease-specific AChR antibody titers were measured with a radio-immunoprecipitation assay. Forty-seven healthy controls (HC) were vaccinated with the same influenza vaccine to compare antibody titers., Results: A post-vaccination, seroprotective titer (HI ≥ 1:40) was achieved in 89.4% of MG patients vs. 93.6% in healthy controls for the H3N2 strain, 95.7% vs 97.9% for the H1N1 strain and 46.8 vs 51% for the B-strain. A seroprotective titer for all three strains of the seasonal influenza vaccine was reached in 40.4% (19/47) of the MG group and in 51% (24/47) of the HC group. Immunosuppressive medication did not significantly influence post geomean titers (GMT). The titers of disease-specific AChR antibodies were unchanged 4 weeks after vaccination. The clinical outcome scores showed no exacerbation of MG symptoms., Conclusion: The antibody response to an influenza vaccination in patients with AChR MG was not different from that in healthy subjects, even in AChR MG patients using immunosuppressive medication. Influenza vaccination does not induce an immunological or clinical exacerbation of AChR MG., Clinical Trial Registry: The influenza trial is listed on clinicaltrialsregister.eu under 2016-003138-26., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The hemagglutination inhibition (HI) assay has been used for the antigenic characterization of influenza viruses for decades. However, the majority of recent seasonal influenza A viruses of the H3N2 subtype has lost the capacity to agglutinate erythrocytes of various species. The hemagglutination (HA) activity of other A(H3N2) strains is generally sensitive to the action of the neuraminidase inhibitor oseltamivir, which indicates that the neuraminidase and not the hemagglutinin is responsible for the HA activity. These findings complicate the antigenic characterization and selection of A(H3N2) vaccine strains, calling for alternative antigenic characterization assays. Here we describe the development and use of the ViroSpot microneutralization (MN) assay as a reliable and robust alternative for the HI assay. Serum neutralization of influenza A(H3N2) reference virus strains and epidemic isolates was determined by automated readout of immunostained cell monolayers, in a format designed to minimize the influence of infectious virus doses on serum neutralization titers. Neutralization of infection was largely independent from rates of viral replication and cell-to-cell transmission, facilitating the comparison of different virus isolates. Other advantages of the ViroSpot MN assay include its relative insensitivity to variation in test dose of infectious virus, automated capture and analyses of residual infection patterns, and compatibility with standardized large scale analyses. Using this assay, a number of epidemic influenza A(H3N2) strains that failed to agglutinate erythrocytes, were readily characterized antigenically., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Antigenic characterization of influenza viruses is typically based on hemagglutination inhibition (HI) assay data for viral isolates tested against strain-specific postinfection ferret antisera. Here, similar virus characterizations were performed using serological data from humans with primary influenza A(H3N2) infection., Methods: We screened sera collected between 1995 and 2011 from children between 9 and 24 months of age for influenza virus antibodies, performed HI tests for the positive sera against 23 influenza viruses isolated between 1989 and 2011, and measured HI titers of antisera against influenza A(H3N2) from 24 ferrets against the same panel of viruses., Results: Of the 17 positive human sera, 6 had a high response, showing HI patterns that would be expected from primary infection antisera, while 11 sera had lower, more dispersed patterns of reactivity that are not easily explained. The antigenic map based on the high-response human HI data was similar to the map created using ferret data., Conclusions: Although the overall structure of the ferret and human antigenic maps is similar, local differences in virus positions indicate that the human and ferret immune system might see antigenic properties of viruses differently. Further studies are needed to establish the degree of similarity between serological patterns in ferret and human data., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America.)

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.

Unlabelled: 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.

Recently, it was shown that interferon-γ mediated immune responses, which play a major role in the control of infection with Mycobacterium tuberculosis (Mtb), can be inhibited by type I interferons. Since type I interferons are abundantly induced during viral infections, we hypothesized that infections with influenza viruses might play a role in the development of active TB disease either directly after exposure to Mtb or through reactivation of latent Mtb infection. To explore this hypothesis we investigated in a retrospective study whether newly diagnosed adult tuberculosis patients from Indonesia had had recent influenza infection. Plasma samples from TB patients and controls were assayed for antibodies against two subtypes of at that time relevant, seasonal influenza A viruses. Overall, no correlation was observed with the presence of antibodies and manifest tuberculosis. Still, antibody titers against circulating A/H3N2 influenza virus were slightly enhanced in tuberculosis patients as compared to controls, and highest in cases of advanced tuberculosis. This suggests that tuberculosis patients were recently infected with influenza, before clinical manifestation of the disease. Alternatively, the production of antibodies and susceptibility to tuberculosis may be influenced by a common confounding factor, for example the ability of patients to induce interferon-α. We conclude that in an endemic country like Indonesia, an influenza virus infection is not a major determinant for developing clinically manifest tuberculosis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

The emergence of a new influenza A virus (H1N1) variant in 2009 led to a worldwide vaccination program, which was prepared in a relatively short period of time. This study investigated the humoral immunity against this virus before and after vaccination with a 2009 influenza A virus (H1N1) monovalent MF59-adjuvanted vaccine, as well as the persistence of vaccine-induced antibodies. Our prospective longitudinal study included 498 health care workers (mean age, 43 years; median age, 44 years). Most (89%) had never or only occasionally received a seasonal influenza virus vaccine, and 11% were vaccinated annually (on average, for >10 years). Antibody titers were determined by a hemagglutination inhibition (HI) assay at baseline, 3 weeks after the first vaccination, and 5 weeks and 7 months after the second vaccination. Four hundred thirty-five persons received two doses of the 2009 vaccine. After the first dose, 79.5% developed a HI titer of ≥40. This percentage increased to 83.3% after the second dose. Persistent antibodies were found in 71.9% of the group that had not received annual vaccinations and in 43.8% of the group that had received annual vaccinations. The latter group tended to have lower HI titers (P=0.09). With increasing age, HI titers decreased significantly, by 2.4% per year. A single dose of the 2009 vaccine was immunogenic in almost 80% of the study population, whereas an additional dose resulted in significantly increased titers only in persons over 50. Finally, a reduced HI antibody response against the 2009 vaccine was found in adults who had previously received seasonal influenza virus vaccination. More studies on the effect of yearly seasonal influenza virus vaccination on the immune response are warranted.

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.

Introduction of influenza viruses with gene segments of avian origin into the human population may result in the emergence of new pathogenic human influenza viruses. The recent infection of a 3-year-old boy with an influenza A (H5N1) virus of avian origin can be considered as an example of such an event. However, this virus, influenza A/Hong Kong/156/97 (H5N1) and the 17 additional H5N1 viruses isolated from humans by the end of 1997 lack the ability to spread efficiently amongst humans and therefore have limited pandemic potential. However, the possibility of reassortment of these viruses with currently circulating human viruses illustrates the need for pandemic preparedness.

Background: In May, 1997, a 3-year-old boy in Hong Kong was admitted to the hospital and subsequently died from influenza pneumonia, acute respiratory distress syndrome, Reye's syndrome, multiorgan failure, and disseminated intravascular coagulation. An influenza A H5N1 virus was isolated from a tracheal aspirate of the boy. Preceding this incident, avian influenza outbreaks of high mortality were reported from three chicken farms in Hong Kong, and the virus involved was also found to be of the H5 subtype., Methods: We carried out an antigenic and molecular comparison of the influenza A H5N1 virus isolated from the boy with one of the viruses isolated from outbreaks of avian influenza by haemagglutination-inhibition and neuraminidase-inhibition assays and nucleotide sequence analysis., Findings: Differences were observed in the antigenic reactivities of the viruses by the haemagglutination-inhibition assay. However, nucleotide sequence analysis of all gene segments revealed that the human virus A/Hong Kong/156/97 was genetically closely related to the avian A/chicken/Hong Kong/258/97., Interpretation: Although direct contact between the sick child and affected chickens has not been established, our results suggest transmission of the virus from infected chickens to the child without another intermediate mammalian host acting as a "mixing vessel". This event illustrates the importance of intensive global influenza surveillance.

Cynomolgus macaque monkeys (Macaca fascicularis) were immunized twice intramuscularly, either with a conventional non-adjuvanted subunit vaccine or with a candidate immune-stimulating complex (iscom) vaccine, each containing 10 micrograms envelope glycoprotein of a recent human influenza A(H3N2) virus (A/Netherlands/18/94). In contrast to the macaques vaccinated with the classical subunit vaccine, those immunized with the iscom vaccine developed high titres of specific IgM, IgA and IgG serum antibodies, as well as high titres of haemagglutination-inhibiting and virus-neutralizing serum antibodies. Also, specific proliferative T cell responses were only found in the iscom-vaccinated monkeys and their levels were similar to those found in monkeys experimentally infected with the homologous virus. Upon intratracheal challenge with the homologous virus, the iscom-vaccinated monkeys were completely protected from detectable virus replication in lungs, pharynx and nose, whereas those vaccinated with the classical subunit vaccines were not, or were only partially protected. The kinetics of specific serum antibody development in the iscom-vaccinated monkeys after challenge were quite similar to those of monkeys after secondary infection with the same virus. In contrast, the post-challenge kinetics of serum antibody development in the monkeys vaccinated with the classical subunit vaccines resembled those of naive monkeys, confirming that these vaccines only provided limited protection in such animals.

In the autumn of 1992 two-thirds of the population of a nursing home in Amsterdam was vaccinated against influenza. However, in March 1993 an outbreak of an influenza like illness occurred with a morbidity rate of 49% and a mortality rate of 10%. There was sufficient serological evidence to show that the vaccine as such had induced adequate immunity. As the causative agent an influenza A/H3N2 virus was identified. The failing activity of the vaccine in this instance was apparently caused by the absence of sufficient antigen similarity between the A/H3N2 vaccine component and the epidemic virus ('vaccine mismatch').

The cytotoxic T-cell and humoral immune response to a commercially available influenza A-H1N1 subunit vaccine in 14 patients with type 1 diabetes mellitus was compared with the response in 13 healthy volunteers. Cytotoxic T-cell response to vaccination was poor in both patients and controls. At a calculated 50: 1 effector-target cell ratio, however, significantly more controls than patients showed an increase of over 5% cytotoxic T-cell mediated lysis after vaccination (P less than 0.05). In patients the cytotoxic T-cell response decreased with higher percentages of glycosylated haemoglobin (regression coefficient not equal to 0 with P less than 0.05). No significant difference was found between diabetic patients and control subjects with respect to antibody response after vaccination. Implications for vaccination strategy are discussed.