Ramona Trebbien

Influenza vaccine effectiveness in adults 65 years and older, Denmark, 2015/16 - a rapid epidemiological and virological assessment.

In Denmark, both influenza A(H1N1)pdm09 and influenza B co-circulated in the 2015/16 season. We estimated the vaccine effectiveness (VE) of the trivalent influenza vaccine in patientsu200965 years and older using the test-negative case-control design. The adjusted VE against influenza A(H1N1)pdm09 was 35.0% (95% confidence interval (CI): 11.1-52.4) and against influenza B 4.1% (95% CI: -22.0 to 24.7). The majority of influenza A(H1N1)pdm09 circulating in 2015/16 belonged to the new genetic subgroup subclade 6B.1.

Changes in genetically drifted H3N2 influenza A viruses and vaccine effectiveness in adults 65 years and older during the 2016/17 season in Denmark

BACKGROUNDnIn Denmark, influenza A virus of the subtype H3N2 has been dominating the 2016/17 season, as in most countries of the Northern Hemisphere.nnnOBJECTIVESnThis study was conducted as part of the Danish seasonal influenza surveillance programme to genetically characterize circulating H3N2 viruses and determine the seasonal vaccine effectiveness (VE) overall in the Danish population and further on the virus cluster level.nnnSTUDY DESIGNnInfluenza virus positive samples submitted for the national surveillance programme were genetically characterized by sequencing. VE estimates against influenza A and the circulating virus clusters were determined in patients above 65 years using the test-negative case-control design.nnnRESULTSnThe genetic characterization revealed several genetically drifted viruses, which could be divided into four main clusters by the defining amino acid substitutions: 3C.2a/N121K/S144K, 3C.2a/T131K/R142K, 3C.2a1, and 3C.2a1/N121K. Some of the drifted viruses appeared to be more prominent in vaccinated or non-vaccinated individuals, respectively. Overall the adjusted VE was 7.4% (95% confidence interval (CI): -6.0-19.2) among inpatients and 19.3% (95% CI: -5.7-38.4) among outpatients, respectively. VE for the four main virus clusters was; cluster 3C.2a1: 38.8% (95% CI: -29.8-71.1), cluster 3C.2a/N121K/S144K: 9.2% (95% CI: -63.0-49.4), cluster 3C.2a/T131K/R142K: 19.0% (95% CI: -85.3-64.6), and cluster 3C.2a1/N121K: -12.2% (95%CI: -129.7-45.2).nnnCONCLUSIONSnSeveral genetically drifted H3N2 viruses have been circulating in Denmark in the 2016-17 influenza season. An overall low VE was estimated and VE for the four main virus cluster indicate different VEs between the circulating drifted H3N2 viruses.

Interim 2017/18 influenza seasonal vaccine effectiveness: combined results from five European studies

Between September 2017 and February 2018, influenza A(H1N1)pdm09, A(H3N2) and B viruses (mainly B/Yamagata, not included in 2017/18 trivalent vaccines) co-circulated in Europe. Interim results from five European studies indicate that, in all age groups, 2017/18 influenza vaccine effectiveness was 25 to 52% against any influenza, 55 to 68% against influenza A(H1N1)pdm09, −42 to 7% against influenza A(H3N2) and 36 to 54% against influenza B. 2017/18 influenza vaccine should be promoted where influenza still circulates.

Development of oseltamivir and zanamivir resistance in influenza A(H1N1)pdm09 virus, Denmark, 2014

Antiviral treatment of immunocompromised patients with prolonged influenza virus infection can lead to multidrug resistance. This study reveals the selection of antiviral resistance mutations in influenza A(H1N1)pdm09 virus in an immunocompromised patient during a 6-month period. The patient was treated with two courses of oseltamivir (5 days and 2 months, respectively), with the first course starting at symptom onset, and subsequently zanamivir (2 months and 10 days, respectively). Respiratory samples were investigated by Sanger and next generation sequencing (NGS) and, for NGS data, low-frequency-variant-detection analysis was performed. Neuraminidase-inhibition tests were conducted for samples isolated in Madin-Darby canine kidney cells. In a sample collected 15 days after the end of the first treatment with oseltamivir (Day 20 post-symptom onset), oseltamivir resistance was detected (mutation H275Y with 60.3% frequency by NGS). Day 149 when the patient had almost completed the second zanamivir treatment, mixes of the following resistance mutations were detected; H275Y(65.1%), I223R(9.2%), and E119G(89.6%), accompanied by additional mutations, showing a more complex viral population in the long-term treated patient. Two samples obtained on Day 151 from bronchoalveolar lavage (BAL) and nasopharyngeal swab, respectively, showed different mutation profiles, with a higher frequency of antiviral resistance mutations in BAL. The results emphasise the importance of timely antiviral resistance testing both for treatment of individual patients as well as for preventive measures to control the development and transmission of antiviral resistant viruses.

A polyvalent influenza DNA vaccine applied by needle-free intradermal delivery induces cross-reactive humoral and cellular immune responses in pigs

Background Pigs are natural hosts for influenza A viruses, and the infection is widely prevalent in swine herds throughout the world. Current commercial influenza vaccines for pigs induce a narrow immune response and are not very effective against antigenically diverse viruses. To control influenza in pigs, the development of more effective swine influenza vaccines inducing broader cross-protective immune responses is needed. Previously, we have shown that a polyvalent influenza DNA vaccine using vectors containing antibiotic resistance genes induced a broadly protective immune response in pigs and ferrets using intradermal injection followed by electroporation. However, this vaccination approach is not practical in large swine herds, and DNA vaccine vectors containing antibiotic resistance genes are undesirable. Objectives To investigate the immunogenicity of an optimized version of our preceding polyvalent DNA vaccine, characterized by a next-generation expression vector without antibiotic resistance markers and delivered by a convenient needle-free intradermal application approach. Methods The humoral and cellular immune responses induced by three different doses of the optimized DNA vaccine were evaluated in groups of five to six pigs. The DNA vaccine consisted of six selected influenza genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase. Results Needle-free vaccination of growing pigs with the optimized DNA vaccine resulted in specific, dose-dependent immunity down to the lowest dose (200 μg DNA/vaccination). Both the antibody-mediated and the recall lymphocyte immune responses demonstrated high reactivity against vaccine-specific strains and cross-reactivity to vaccine-heterologous strains. Conclusion The results suggest that polyvalent DNA influenza vaccination may provide a strong tool for broad protection against swine influenza strains threatening animal as well as public health. In addition, the needle-free administration technique used for this DNA vaccine will provide an easy and practical approach for the large-scale vaccination of swine.

Co-circulation of multiple subtypes of enterovirus A71 (EV- A71) genotype C, including novel recombinants characterised by use of whole genome sequencing (WGS), Denmark 2016

In Europe, enterovirus A71 (EV-A71) has primarily been associated with sporadic cases of neurological disease. The recent emergence of new genotypes and larger outbreaks with severely ill patients demonstrates a potential for the spread of new, highly pathogenic EV-A71 strains. Detection and characterisation of these new emerging EV variants is challenging as standard EV assays may not be adequate, necessitating the use of whole genome analysis.

Antiviral resistance due to deletion in the neuraminidase gene and defective interfering-like viral polymerase basic 2 RNA of influenza A virus subtype H3N2

BACKGROUND AND OBJECTIVEnAntiviral treatment of influenza virus infections can lead to drug resistance of virus. This study investigates a selection of mutations in the full genome of H3N2 influenza A virus isolated from a patient in treatment with oseltamivir.nnnSTUDY DESIGNnRespiratory samples from a patient were collected before, during, and after antiviral treatment. Whole genome sequencing of the influenza virus by next generation sequencing, and low-frequency-variant analysis was performed. Neuraminidase-inhibition tests were performed with oseltamivir and zanamivir, and viruses were propagated in sial-transferase gene transfected Madin-Darby Canine Kidney cells.nnnRESULTSnA deletion at amino acid position 245-248 in the neuraminidase gene occurred after initiation of treatment with oseltamivir. The deleted virus had highly reduced inhibition against oseltamivir but was sensitive to zanamivir. Nine days after discontinuation of oseltamivir treatment the deleted H3N2 virus was still present in the patient. After three passages of the deleted virus in cell culture, the deletion was retained. Several variant mutations appeared in the other genes of the H3N2 virus, where most striking were two major out-of-frame deletions in the polymerase basic 2 (PB2) gene, indicating defective interfering-like viral RNA.nnnCONCLUSIONSnThe viruses harboring the 245-248 deletion in the neuraminidase gene were still present after discontinuation of oseltamivir treatment and passages in cell cultures, indicating a potential risk for transmission of the deleted virus. Full genome deep sequencing was useful to reveal variant mutations that might be selected due to antiviral treatment, and defective interfering-like viral PB2 RNA in the respiratory samples was detected.

Protective effect of a polyvalent influenza DNA vaccine in pigs

Background Influenza A virus in swine herds represents a major problem for the swine industry and poses a constant threat for the emergence of novel pandemic viruses and the development of more effective influenza vaccines for pigs is desired. By optimizing the vector backbone and using a needle-free delivery method, we have recently demonstrated a polyvalent influenza DNA vaccine that induces a broad immune response, including both humoral and cellular immunity. Objectives To investigate the protection of our polyvalent influenza DNA vaccine approach in a pig challenge study. Methods By intradermal needle-free delivery to the skin, we immunized pigs with two different doses (500 μg and 800 μg) of an influenza DNA vaccine based on six genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase as previously demonstrated. Two weeks following immunization, the pigs were challenged with the 2009 pandemic H1N1 virus. Results When challenged with 2009 pandemic H1N1, 0/5 vaccinated pigs (800 μg DNA) became infected whereas 5/5 unvaccinated control pigs were infected. The pigs vaccinated with the low dose (500 μg DNA) were only partially protected. The DNA vaccine elicited binding-, hemagglutination inhibitory (HI) − as well as cross-reactive neutralizing antibody activity and neuraminidase inhibiting antibodies in the immunized pigs, in a dose-dependent manner. Conclusion The present data, together with the previously demonstrated immunogenicity of our influenza DNA vaccine, indicate that naked DNA vaccine technology provides a strong approach for the development of improved pig vaccines, applying realistic low doses of DNA and a convenient delivery method for mass vaccination.

ESwab challenges influenza virus propagation in cell cultures

Although the ESwab kit (Copan, Brescia, Italy) is intended for sampling bacteria for culture, this kit is increasingly also used for virus sampling. The effect of ESwab medium on influenza virus detection by real-time reverse transcription-polymerase chain reaction (RT-PCR) or virus propagation in Madin-Darby canine kidney (MDCK) cell culture was investigated. The ESwab medium was suitable for viral RNA detection but not for viral propagation due to cytotoxicity. Sampling influenza viruses with ESwab challenges influenza surveillance by strongly limiting the possibility of antigenic characterisation.

Comparative evaluation of the CerTest VIASURE flu A, B & RSV real time RT-PCR detection kit on the BD MAX system versus a routine in-house assay for detection of influenza A and B virus during the 2016/17 influenza season

BACKGROUNDnDiagnosing of influenza rapidly and accurately helps clinicians to initiate appropriate treatment options and isolation protocols. Unnecessary antimicrobial treatment and laboratory testing can also be reduced. Assess commercial alternatives to in-house assays that may not only reduce laboratory technician hands on time but also the laboratory turnaround time is of interest.nnnOBJECTIVESnWe evaluated the performance of the VIASURE Flu A, B & RSV Real Time RT-PCR Detection Kit (CerTest Biotec) for detecting Influenza A and B viruses.nnnSTUDY DESIGNnDuring the 2016/17 influenza season 532 clinical samples were tested with the VIASURE assay on the BD MAX™ system versus an in-house real time RT-PCR assay with discrepant results resolved by a real time RT-PCR assay at a national reference laboratory.nnnRESULTSnThe VIASURE assay on the BD MAX showed a sensitivity of 99.5% (95% CI: 97.3-100) and a specificity of 99.1% (95% CI: 97.4-99.8) for detection of Influenza A virus. The positive predictive and negative predictive values were 98.5% (95% CI: 95.8-99.7) and 99.7% (95% CI: 98.3-100) respectively. Influenza B virus detection could not be evaluated due to a low positivity rate. The BD MAX platform offered the flexibility of several daily runs, shorter hands-on-time and shorter turnaround time than the in-house assay.nnnCONCLUSIONSnThe VIASURE assay on the BD MAX performed well and is now implemented in our clinical laboratory.