Haakon Sjursen

A phase I clinical trial of a PER.C6 cell grown influenza H7 virus vaccine.

Avian influenza H7 viruses have transmitted from poultry to man causing human illness and fatality, highlighting the need for pandemic preparedness against this subtype. We have developed and tested the first cell-based human vaccine against H7 avian influenza virus in a phase I clinical trial. Sixty healthy volunteers were intramuscularly vaccinated with two doses of split H7N1 virus vaccine containing 12 microg or 24 microg haemagglutinin alone or with aluminium hydroxide adjuvant (300 microg or 600 microg, respectively). The vaccine was well tolerated in all subjects and no serious adverse events occurred. The vaccine elicited low haemagglutination inhibition and microneutralisation titres, although the addition of aluminium adjuvant augmented the antibody response. We found a higher number of antibody secreting cells and an association with IL-2 production in subjects with antibody response. In conclusion, our study shows that producing effective H7 pandemic vaccines is as challenging as has been observed for H5 vaccines.

Induction of broadly-reactive anti-hemagglutinin stalk antibodies by an H5N1 vaccine in humans

ABSTRACT Influenza virus infections are a major public health concern and cause significant morbidity and mortality worldwide. Current vaccines are effective but strain specific due to their focus on the immunodominant globular head domain of the hemagglutinin (HA). It has been hypothesized that sequential exposure of humans to hemagglutinins with divergent globular head domains but conserved stalk domains could refocus the immune response to broadly neutralizing epitopes in the stalk. Humans have preexisting immunity against H1 (group 1 hemagglutinin), and vaccination with H5 HA (also group 1)—which has a divergent globular head domain but a similar stalk domain—represents one such sequential-exposure scenario. To test this hypothesis, we used novel reagents based on chimeric hemagglutinins to screen sera from an H5N1 clinical trial for induction of stalk-specific antibodies by quantitative enzyme-linked immunosorbent assay (ELISA) and neutralization assays. Importantly, we also investigated the biological activity of these antibodies in a passive transfer in a mouse challenge model. We found that the H5N1 vaccine induced high titers of stalk-reactive antibodies which were biologically active and protective in the passive-transfer experiment. The induced response showed exceptional breadth toward divergent group 1 hemagglutinins but did not extend to group 2 hemagglutinins. These data provide evidence for the hypothesis that sequential exposure to hemagglutinins with divergent globular head domains but conserved stalk domains can refocus the immune response toward the conserved stalk domain. Furthermore, the results support the concept of a chimeric hemagglutinin universal influenza virus vaccine strategy that is based on the same principle. IMPORTANCE Influenza virus vaccines have to be reformulated and readministered on an annual basis. The development of a universal influenza virus vaccine could abolish the need for this cumbersome and costly process and would also enhance our pandemic preparedness. This study addressed the following questions, which are essential for the development of a hemagglutinin stalk-based universal influenza virus vaccine. (i) Can stalk-reactive antibodies be boosted by vaccination with divergent HAs that share conserved epitopes? (ii) How long-lived are these vaccine-induced stalk-reactive antibody responses? (iii) What is the breadth of this reactivity? (iv) Are these antibodies functional and protective? Our results further strengthen the concept of induction of stalk-reactive antibodies by sequential exposure to hemagglutinin immunogens with conserved stalk and divergent head domains. A universal influenza virus vaccine based on the same principles seems possible and might have a significant impact on global human health.

Evaluation of a virosomal H5N1 vaccine formulated with Matrix M™ adjuvant in a phase I clinical trial

The avian influenza H5 virus epizootic continues to cause zoonosis with human fatalities, highlighting the continued need for pandemic preparedness against this subtype. This study evaluated the tolerability and immunogenicity of a Matrix M™ adjuvanted virosomal H5N1 vaccine in a phase I clinical trial. Sixty healthy adults were vaccinated intramuscularly with two doses of influenza H5N1 (NIBRG-14) virosomal vaccine alone (30 μg haemagglutinin (HA)) or 1.5, 7.5 or 30 μg HA formulated with 50 μg Matrix M™ adjuvant. The antibody response was analysed by haemagglutination inhibition (HI), microneutralisation (MN) and single radial haemolysis (SRH) assays. The vaccine was well tolerated in all groups but injection site pain was more frequently observed in the Matrix M™ adjuvanted groups. The vaccine elicited homologous and heterologous H5N1-specific antibody responses and the Matrix M™ adjuvanted formulations met all the EU regulatory criteria. In conclusion, Matrix M™ adjuvant was well tolerated and augmented the antibody response allowing considerable dose sparing down to 1.5 μg HA.

An adjuvanted pandemic influenza H1N1 vaccine provides early and long term protection in health care workers

Mass vaccination was the most effective prophylaxis for protecting the population during the influenza H1N1 pandemic. We have evaluated the tolerability, immunogenicity and kinetics of the antibody response to a monovalent oil-in-water (AS03) adjuvanted human pandemic split influenza A/California/7/2009 H1N1 (3.75 μg haemagglutinin) vaccine in health care workers. Vaccination elicited a rapid and early protective level of haemagglutination inhibition antibody from 6 to 7 days post vaccination, and by 14 to 21 days post vaccination, up to 98% of vaccinees had protective antibody titres which persisted for at least 3 months in 84-92% of subjects. A rapid induction of protective antibody is important in reducing community spread of pandemic influenza and in helping maintain the integrity of the health care system during the pandemic.

T-Helper 1 Cells Elicited by H5N1 Vaccination Predict Seroprotection

BACKGROUND Vaccination is the best measure to protect the population against a potential influenza H5N1 pandemic, but 2 doses of vaccine are needed to elicit protective immune responses. An immunological marker for H5N1 vaccine effectiveness is needed for early identification of the best vaccine candidate. METHODS We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with Matrix M. Sixty adult volunteers were vaccinated intramuscularly with 2 doses of either 30 μg hemagglutinin (HA) alone or with 1.5, 7.5, or 30 μg HA and Matrix M adjuvant (50 μg). The humoral response was measured by the hemagglutination inhibition (HI), microneutralization (MN), and single radial hemolysis (SRH) assays, and the CD4(+) T-helper 1 (Th1)-cell response was measured by intracellular staining for the cytokines interleukin 2, interferon γ, and tumor necrosis factor α. RESULTS The adjuvanted vaccine effectively induced CD4(+) Th1-cell responses, and the frequency of influenza-specific Th1 cells after the first vaccine dose predicted subsequent HI, MN, and SRH seroprotective responses after the second vaccination. CONCLUSIONS These results support early identification of Th1-cell responses as a predictive biomarker for an efficient vaccine response, which could have great implications for early identification of persons with low or no response to vaccine when evaluating future pandemic influenza vaccines.

Tickborne encephalitis virus, Norway and Denmark.

Serum from 2 Norwegians with tickborne encephalitis (TBE) (1 of whom was infected in Denmark) and 810 Norwegian ticks were tested for TBE virus (TBEV) RNA by reverse transcription–polymerase chain reaction. Sequencing and phylogenetic analysis were performed. This is the first genome detection of TBEV in serum from Norwegian patients.

An H7N1 Influenza Virus Vaccine Induces Broadly Reactive Antibody Responses against H7N9 in Humans

ABSTRACT Emerging H7N9 influenza virus infections in Asia have once more spurred the development of effective prepandemic H7 vaccines. However, many vaccines based on avian influenza viruses—including H7—are poorly immunogenic, as measured by traditional correlates of protection. Here we reevaluated sera from an H7N1 human vaccine trial performed in 2006. We examined cross-reactive antibody responses to divergent H7 strains, including H7N9, dissected the antibody response into head- and stalk-reactive antibodies, and tested the in vivo potency of these human sera in a passive-transfer H7N9 challenge experiment with mice. Although only a low percentage of vaccinees induced neutralizing antibody responses against the homologous vaccine strain and also H7N9, we detected strong cross-reactivity to divergent H7 hemagglutinins (HAs) in a large proportion of the cohort with a quantitative enzyme-linked immunosorbent assay. Furthermore, H7N1 vaccination induced antibodies to both the head and stalk domains of the HA, which is in sharp contrast to seasonal inactivated vaccines. Finally, we were able to show that both neutralizing and nonneutralizing antibodies improved in vivo virus clearance in a passive-transfer H7N9 challenge mouse model.

Negative selection of human monocytes using magnetic particles covered by anti-lymphocyte antibodies

We have developed a standardized procedure for the isolation of monocytes from peripheral blood by negative selection using magnetic polymer particles coated with monoclonal antibodies against T and B lymphocytes. The average purity of the monocyte suspension was 85%, and monocyte recovery was 72% from Ficoll-Hypaque gradient separated mononuclear cells and 32% from whole blood. In a lucigenin enhanced chemiluminescence assay there was no significant difference between cells separated immunomagnetically and those separated on a gradient. Nor did electron microscopy show any significant difference in morphology between such monocytes. Negative selection using magnetic polymer particles is an efficient method for the separation of monocytes with intact morphology and function as measured by chemiluminescence.

BACTERIAL CONTAMINATION OF DROPS AND DROPPER TIPS OF IN‐USE MULTIDOSE EYE DROP BOTTLES

The type and frequency of bacterial contamination of 638 in‐use multidose eye drop bottles are reported. By dripping, bacteria were recovered from 82 bottles (12.9%). Bacterial growth considered to be clinically and microbiologically significant were found in 12 cases (1.9%). The swabbings from 38 of 180 dropper tips (21.1%) yielded bacteria, while none of the pipette aspirates from 196 bottles were positive. The flora obtained by dripping and swabbing was very similar to the normal conjunctival and skin flora. It is suggested that microorganisms isolated by dripping often originate from contaminated dropper tips. The frequency of contaminated drops did not increase with increasing duration of use of the bottles. Repeated examinations and inoculation studies indicated that the solutions were self‐sterilizing. This indicates that multidose eye drop bottles preserved and dispensed as in the present study may be used for more than the usual 4 weeks without increasing the risk of ocular infection.

IgG subclass antibodies to serogroup B meningococcal outer membrane antigens following infection and vaccination

IgG and IgG subclass antibodies to the outer membrane antigens from Neisseria meningitidis (serogroup B., serotype 15:P1.16) were quantitated by an enzyme‐linked immunosorbent assay (ELISA) in sera from 40 patients with group B:15:P1.16 meningococcal disease and 24 volunteers immunized with a serotype 15:P1.16 outer membrane vesicle vaccine. A second injection was given 6 weeks after the first immunization. Patient sera obtained two and six weeks after onset of the disease had significantly higher levels of total IgG, IgG1, IgG2, and IgG3 antibodies to the outer membrane antigens than acute sera, convalescent sera from patients with systemic non‐meningococcal bacterial infections and sera from healthy controls. The levels of total IgG and IgG 1 remained high one and three years later. Sera from the vaccinees showed high levels of total IgG and IgG 1 6, 12 and 26 weeks after the first immunization and high levels of IgG3 6 weeks after the second immunization. No increase of IgG2 or IgG4 levels was observed in the postimmunization sera. Immunoblotting of three convalescent sera demonstrated individual patterns of IgG subclass binding to various outer membrane antigens with most distinct binding of IgG 1 and IgG3 antibodies to the class 1 protein, the H.8 lipoprotein and the lipopolysaccharide. Since IgGl and IgG3 are the most effective antibodies for complement activation and phagocytosis, group B meningococcal disease and immunization with the serotype 15:P1.16 outer membrane vesicle vaccine stimulate production of those IgG subclasses which have the strongest opsonic and bactericidal activity.