Åsne Jul-Larsen

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.

Promyelocytic leukemia nuclear bodies are predetermined processing sites for damaged DNA.

The promyelocytic leukemia protein (PML) participates in several cellular functions, including transcriptional regulation, apoptosis and maintenance of genomic stability. A key feature of this protein is its ability to induce the assembly of nuclear compartments termed PML-nuclear bodies (PML-NBs). Here we show that these nuclear structures recruit single-stranded DNA (ssDNA) molecules in response to exogenous DNA damage. ssDNA was readily detected in PML-NBs within 1 hour following exposure of cells to UV light. Confocal real-time imaging of cells expressing YFP-tagged PML did not reveal de novo formation of new PML-NBs following UV-irradiation, which shows that ssDNA focus formation occurred within pre-existing PML-NBs. Moreover, siRNA-mediated depletion of PML prevented ssDNA focus formation and sensitized cells to UV-induced apoptosis. PML-dependent ssDNA focus formation was found to be particularly efficient during S-phase of the cell cycle, and PML-depleted cells became retarded in S-phase upon growth in the presence of etoposide. In addition, we found that caffeine and the poly(ADP-ribose) polymerase (PARP) inhibitor NU1027 enhanced UV-induced recruitment of ssDNA to PML-NBs. Together, our results show that PML-NBs have the capacity to accommodate DNA metabolic activities that are associated with processing of damaged DNA.

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.

Replication protein A prevents accumulation of single-stranded telomeric DNA in cells that use alternative lengthening of telomeres

The activation of a telomere maintenance mechanism is required for cancer development in humans. While most tumors achieve this by expressing the enzyme telomerase, a fraction (5–15%) employs a recombination-based mechanism termed alternative lengthening of telomeres (ALT). Here we show that loss of the single-stranded DNA-binding protein replication protein A (RPA) in human ALT cells, but not in telomerase-positive cells, causes increased exposure of single-stranded G-rich telomeric DNA, cell cycle arrest in G2/M phase, accumulation of single-stranded telomeric DNA within ALT-associated PML bodies (APBs), and formation of telomeric aggregates at the ends of metaphase chromosomes. This study demonstrates differences between ALT cells and telomerase-positive cells in the requirement for RPA in telomere processing and implicates the ALT mechanism in tumor cells as a possible therapeutic target.

Cell-cycle regulation and dynamics of cytoplasmic compartments containing the promyelocytic leukemia protein and nucleoporins.

Nucleoporins and the promyelocytic leukemia protein (PML) represent structural entities of nuclear pore complexes and PML nuclear bodies, respectively. In addition, these proteins might function in a common biological mechanism, because at least two different nucleoporins, Nup98 and Nup214, as well as PML, can become aberrantly expressed as oncogenic fusion proteins in acute myeloid leukemia (AML) cells. Here we show that PML and nucleoporins become directed to common cytoplasmic compartments during the mitosis-to-G1 transition of the cell cycle. These protein assemblies, which we have termed CyPNs (cytoplasmic assemblies of PML and nucleoporins), move on the microtubular network and become stably connected to the nuclear membrane once contact with the nucleus has been made. The ability of PML to target CyPNs depends on its nuclear localization signal, and loss of PML causes an increase in cytoplasmic-bound versus nuclear-membrane-bound nucleoporins. CyPNs are also targeted by the acute promyelocytic leukemia (APL) fusion protein PML-RARα and can be readily detected within the APL cell line NB4. These results provide insight into a dynamic pool of cytoplasmic nucleoporins that form a complex with the tumor suppressor protein PML during the G1 phase of the cell cycle.

Subcellular distribution of nuclear import-defective isoforms of the promyelocytic leukemia protein

BackgroundThe promyelocytic leukemia (PML) protein participates in a number of cellular processes, including transcription regulation, apoptosis, differentiation, virus defense and genome maintenance. This protein is structurally organized into a tripartite motif (TRIM) at its N-terminus, a nuclear localization signal (NLS) at its central region and a C-terminus that varies between alternatively spliced isoforms. Most PML splice variants target the nucleus where they define sub-nuclear compartments termed PML nuclear bodies (PML NBs). However, PML variants that lack the NLS are also expressed, suggesting the existence of PML isoforms with cytoplasmic functions. In the present study we expressed PML isoforms with a mutated NLS in U2OS cells to identify potential cytoplasmic compartments targeted by this protein.ResultsExpression of NLS mutated PML isoforms in U2OS cells revealed that PML I targets early endosomes, PML II targets the inner nuclear membrane (partially due to an extra NLS at its C-terminus), and PML III, IV and V target late endosomes/lysosomes. Clustering of PML at all of these subcellular locations depended on a functional TRIM domain.ConclusionsThis study demonstrates the capacity of PML to form macromolecular protein assemblies at several different subcellular sites. Further, it emphasizes a role of the variable C-terminus in subcellular target selection and a general role of the N-terminal TRIM domain in promoting protein clustering.

The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine

Please cite this paper as: Svindland et al. The mucosal and systemic immune responses elicited by a chitosan‐adjuvanted intranasal influenza H5N1 vaccine. Influenza and Other Respiratory Viruses DOI:10.1111/j.1750‐2659.2011.00271.x.

A study of Chitosan and c-di-GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine

Please cite this paper as: Svindland et al. (2012) A study of Chitosan and c‐di‐GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine. Influenza and Other Respiratory Viruses 10.1111/irv.12056000(000), 000–000.

Live Attenuated Influenza Vaccine in Children Induces B-Cell Responses in Tonsils

Background. Tonsils play a key role in eliciting immune responses against respiratory pathogens. Little is known about how tonsils contribute to the local immune response after intranasal vaccination. Here, we uniquely report the mucosal humoral responses in tonsils and saliva after intranasal live attenuated influenza vaccine (LAIV) vaccination in children. Methods. Blood, saliva, and tonsils samples were collected from 39 children before and after LAIV vaccination and from 16 age-matched, nonvaccinated controls. Serum antibody responses were determined by a hemagglutination inhibition (HI) assay. The salivary immunoglobulin A (IgA) level was measured by an enzyme-linked immunosorbent assay. Antibody-secreting cell (ASC) and memory B-cell (MBC) responses were enumerated in tonsils and blood. Results. Significant increases were observed in levels of serum antibodies and salivary IgA to influenza A(H3N2) and influenza B virus strains as early as 14 days after vaccination but not to influenza A(H1N1). Influenza virus–specific salivary IgA levels correlated with serum HI responses, making this a new possible indicator of vaccine immunogenicity in children. LAIV augmented influenza virus–specific B-cell responses in tonsils and blood. Tonsillar MBC responses correlated with systemic MBC and serological responses. Naive children showed significant increases in MBC counts after LAIV vaccination. Conclusions. This is the first study to demonstrate that LAIV elicits humoral B-cell responses in tonsils of young children. Furthermore, salivary IgA analysis represents an easy method for measuring immunogenicity after vaccination.

The human potential of a recombinant pandemic influenza vaccine produced in tobacco plants

Rapid production of influenza vaccine antigen is an important challenge when a new pandemic occurs. Production of recombinant antigens in plants is a quick, cost effective and up scalable new strategy for influenza vaccine production. In this study, we have characterized a recombinant influenza haemagglutinin antigen (HAC1) that was derived from the 2009 pandemic H1N1 (pdmH1N1) virus and expressed in tobacco plants. Volunteers vaccinated with the 2009 pdmH1N1 oil-in-water adjuvanted vaccine provided serum and lymphocyte samples that were used to study the immunogenic properties of the HAC1 antigen in vitro. By 7 d post vaccination, the vaccine fulfilled the licensing criteria for antibody responses to the HA detected by haemagglutination inhibition and single radial hemolysis. By ELISA and ELISPOT analysis we showed that HAC1 was recognized by specific serum antibodies and antibody secreting cells, respectively. We conducted a kinetic analysis and found a peak of serum HAC1 specific antibody response between day 14 and 21 post vaccination by ELISA. We also detected elevated production of IL-2 and IFNγ and low frequencies of CD4+ T cells producing single or multiple Th1 cytokines after stimulating PBMCs (peripheral blood mononuclear cells) with the HAC1 antigen in vitro. This indicates that the antigen can interact with T cells, although confirming that an effective adjuvant would be required to improve the T-cell stimulation of plant based vaccines. We conclude that the tobacco derived recombinant HAC1 antigen is a promising vaccine candidate recognized by both B and T cells.