Persistently high antibody responses after AS03-adjuvanted H1N1pdm09 vaccine: Dissecting the HA specific antibody response.

Current influenza vaccines have a suboptimal effectiveness. The introduction of a novel A/H1N1 influenza virus in 2009 (H1N1pdm09) provided a unique opportunity to study the humoral response to the AS03-adjuvanted H1N1pdm09 vaccine and repeated annual vaccination with the homologous virus in subsequent influenza seasons. Thirty-two HCWs immunized with the AS03-adjuvanted H1N1pdm09 vaccine in 2009 were divided into four groups based on the longevity of their antibody responses (persistently high or transient), and whether they were repeatedly annually vaccinated in the subsequent four influenza seasons or not. Serological assays were utilized to measure the quantity, quality and functionality of antibodies targeting the major surface glycoprotein hemagglutinin (HA). Persistent high responders (hemagglutination inhibition (HI) titre ≥ 80 at 12 months after H1N1pdm09 vaccination) had protective levels of HI antibodies throughout the study period. In addition, the quality and functionality of these antibodies were greater than the individuals who had a transient antibody response to the pandemic vaccine (HI titre < 40 at 12 months after H1N1pdm09 vaccination). All groups had similar levels of antibodies towards the conserved HA stalk domain. The level of HA head-specific antibodies gradually increased over time with annual vaccination in the transient responders. The AS03-adjuvanted H1N1pdm09 vaccine elicited a robust humoral response that persisted up to 5 years in some individuals. Seasonal annual vaccination boosted the HA-antibodies over time in individuals with a transient response to the pandemic H1N1pdm09 vaccine.

No evidence of antigenic seniority in hemagglutinin specific antibody responses after adjuvanted pandemic 2009 influenza vaccination.

Influenza is a contagious respiratory illness caused by the influenza virus. The pandemic outbreak of influenza A H1N1 in 2009 (H1N1pdm09) gave us a unique opportunity to study humoral immune responses to a novel influenza vaccine strain. Here, we investigate how an individual's previous encounter with different influenza subtypes influences the humoral response after pandemic vaccination in 2009. We retrospectively chose and grouped 80 vaccinated healthcare workers (HCWs) based on their year of birth into 4 groups, reflecting which influenza subtype they were likely first exposed to during childhood. Pre- and 21 days post- vaccination sera were analyzed. We investigated antibodies to the major surface protein hemagglutinin (HA), and specifically antibodies binding to the conserved stalk domain of the HA-protein. Serological assays were used to assess the quantity and functionality of the influenza-specific antibodies, including virus neutralization and activation of natural killer (NK) cells involved in antibody-dependent cell-mediated cytotoxicity (ADCC). The AS03-adjuvanted H1N1pdm09 vaccine elicited robust antibody responses in all groups of HCWs. We found that the more antigenically experienced individuals had higher pre-vaccination antibody-levels towards the stalk domain of the HA. We also demonstrated that despite their inferior pre-vaccination antibody levels, the younger individuals reached similar antibody levels as the older birth-cohorts after pandemic vaccination. Our findings are important for understanding the effect of AS03 adjuvant on the antibody response in individuals exposed to different influenza viruses during their early childhood years, which is crucial for developing vaccine strategies against influenza.

Antibody Responses to Influenza A/H1N1pdm09 Virus After Pandemic and Seasonal Influenza Vaccination in Healthcare Workers: A 5-Year Follow-up Study.

Background: The 2009 influenza pandemic was caused by the A/H1N1pdm09 virus, which was subsequently included in the seasonal vaccine, up to 2016/2017, as the A/H1N1 strain. This provided a unique opportunity to investigate the antibody response to H1N1pdm09 over time. Methods: Healthcare workers (HCWs) were immunized with the AS03-adjuvanted H1N1pdm09 vaccine in 2009 (N = 250), and subsequently vaccinated with seasonal vaccines containing H1N1pdm09 for 4 seasons (repeated group), <4 seasons (occasional group), or no seasons (single group). Blood samples were collected pre and at 21 days and 3, 6, and 12 months after each vaccination, or annually (pre-season) from 2010 in the single group. The H1N1pdm09-specific antibodies were measured by the hemagglutination inhibition (HI) assay. Results: Pandemic vaccination robustly induced HI antibodies that persisted above the 50% protective threshold (HI titers ≥ 40) over 12 months post-vaccination. Previous seasonal vaccination and the duration of adverse events after the pandemic vaccination influenced the decision to vaccinate in subsequent seasons. During 2010/2011-2013/2014, antibodies were boosted after each seasonal vaccination, although no significant difference was observed between the repeated and occasional groups. In the single group without seasonal vaccination, 32% of HCWs seroconverted (≥4-fold increase in HI titers) during the 4 subsequent years, most of whom had HI titers <40 prior to seroconversion. When excluding these seroconverted HCWs, HI titers gradually declined from 12 to 60 months post-pandemic vaccination. Conclusions: Pandemic vaccination elicited durable antibodies, supporting the incorporation of adjuvant. Our findings support the current recommendation of annual influenza vaccination in HCWs. Clinical Trials Registration: NCT01003288.

Impact of pre-existing immunity on the induction of functional cross-reactive anti-hemagglutinin stalk antibodies following vaccination with an AS03 adjuvanted pandemic H1N1 vaccine.

The 2009 pandemic H1N1 (A(H1N1)pdm09) virus had a highly divergent hemagglutinin (HA) compared to pre-2009 seasonal H1N1 strains. Most peoples were immunologically naïve to the A(H1N1)pdm09, although hospital workers were exposed early in the pandemic before pandemic vaccines became available. Here, we evaluated how pre-existing antibodies influence the induction of cross-functional HA stalk antibodies following A(H1N1)pdm09 vaccination. Fifty-six healthcare workers vaccinated with AS03 adjuvanted A(H1N1)pdm09 vaccine were chosen by their pre-vaccination priming status (primed HI titers ≥ 40 or unprimed < 40). We analyzed the HA head- and stalk-specific serum IgG subclasses at pre- and 21 days post-vaccination. We also assessed the functionality of the HA stalk-specific antibodies to neutralize virus and mediate antibody dependent cellular cytotoxicity (ADCC). Primed individuals had higher pre-existing HA head- and stalk-specific IgG1, as well as higher ADCC functionality of stalk antibodies. However, following vaccination with the adjuvanted pandemic vaccine, only the quantity of HA head specific IgG1 antibodies were significantly higher than in unprimed individuals. The priming status did not impact upon the cross-reactive HA stalk specific IgG antibodies or their ability to neutralize virus or induce ADCC post-vaccination. In conclusion, a single dose of AS03 adjuvanted pandemic vaccine elicited similar levels of functional antibodies in naïve and primed individuals. These findings are important for understanding the immunological factors that impact or modulate pandemic vaccine responses in humans.

Influenza A haemagglutinin specific IgG responses in children and adults after seasonal trivalent live attenuated influenza vaccination.

Influenza is a major respiratory pathogen and vaccination is the main method of prophylaxis. In 2012, the trivalent live attenuated influenza vaccine (LAIV3) was licensed in Europe for use in children. Vaccine-induced antibodies directed against the main viral surface glycoprotein, haemagglutinin (HA), play an important role in virus neutralization through different mechanism. The objective of this study was to dissect the HA specific antibody responses induced after LAIV3 immunization to the influenza A viruses in children and adults. Plasma was collected from 20 children and 20 adults pre- and post-LAIV3 vaccination (up to ayear) and analysed by the haemagglutination inhibition (HI) and ELISA assays. We found that LAIV3 boosted the HA specific IgG response against the head and the full-length of H3N2 in children, but not adults. Adults had higher levels of pre-existing stalk antibodies (towards H3N2 and H1N1), but these were not boosted by LAIV3. Importantly, we observed a trend in boosting of H1N1 HA stalk specific antibodies in children after LAIV3. Whereas, heterosubtypic H5 and H7 full-length HA specific antibodies were not boosted in either children or adults. In conclusion, LAIV3 elicited H3-head and low levels of H1 stalk specific antibody responses in children, supporting the prophylactic use of LAIV in children.

Comparative analysis of influenza A(H3N2) virus hemagglutinin specific IgG subclass and IgA responses in children and adults after influenza vaccination.

Two different influenza vaccines are generally used in many countries; trivalent live attenuated influenza vaccine (LAIV3) and trivalent inactivated influenza vaccine (IIV3). Studies comparing the antibody response to IIV3 and LAIV3 commonly investigate the seroprotective response by hemagglutination-inhibition (HI) assay. However, there is limited data regarding comparative analysis of IgG subclass and IgA responses induced by LAIV3 and IIV3. Fifteen children <5years received 2 doses of LAIV3 while 14 children aged 10-17years received one dose. In addition, 15 adults were vaccinated with either intranasal LAIV3 or intramuscular IIV3. We analyzed the H3N2 humoral responses by HI assay and the hemagglutinin (HA) specific IgG1, IgG2, IgG3, IgG4 and IgA1 responses by ELISA. Furthermore, we investigated the avidity of induced IgG antibodies. Pre-existing seroprotective HI antibodies were present in adults (73%) previously vaccinated with IIV3. Vaccination resulted in a significant increase in HI titers in all groups, except LAIV3 vaccinated adults. Furthermore, a negative correlation between age and HI titers in LAIV3 vaccinated subjects was observed post-vaccination. LAIV3 in children and IIV3 in adults induced HA-specific IgG1, low IgG3 but no IgG2 or IgG4. Moreover, significant IgA1 responses were only induced in children. Interestingly, IIV3 and LAIV3 induced IgG antibodies with comparable and significantly augmented avidity post-vaccination in children and adults. Our results suggest that age and/or exposure history play a significant role in determining the antibody response. Clinical trial registry: ClinicalTrials.gov NCT01003288 and NCT01866540.

Long-term Maintenance of the Influenza-Specific Cross-Reactive Memory CD4+ T-Cell Responses Following Repeated Annual Influenza Vaccination.

Background: Annual vaccination for healthcare workers and other high-risk groups is the mainstay of the public health strategy to combat influenza. Inactivated influenza vaccines confer protection by inducing neutralizing antibodies efficiently against homologous and closely matched virus strains. In the absence of neutralizing antibodies, cross-reactive T cells have been shown to limit disease severity. However, animal studies and a study in immunocompromised children suggested that repeated vaccination hampers CD8+ T cells. Yet the impact of repeated annual influenza vaccination on both cross-reactive CD4+ and CD8+ T cells has not been explored, particularly in healthy adults. Methods: We assembled a unique cohort of healthcare workers who received a single AS03-adjuvanted H1N1pdm09 vaccine in 2009 and subsequently either repeated annual vaccination or no further vaccination during 2010-2013. Blood samples were collected before the influenza season or vaccination to assess antibody and T-cell responses. Results: Antibody titers to H1N1pdm09 persisted above the protective level in both the repeated- and single-vaccination groups. The interferon γ+ (IFN-γ+) and multifunctional CD4+ T-cell responses were maintained in the repeated group but declined significantly in the single-vaccination group. The IFN-γ+CD8+ T cells remained stable in both groups. Conclusions: This study provides the immunological evidence base for continuing annual influenza vaccination in adults.

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.

Immune Responses in Acute and Convalescent Patients with Mild, Moderate and Severe Disease during the 2009 Influenza Pandemic in Norway.

Increased understanding of immune responses influencing clinical severity during pandemic influenza infection is important for improved treatment and vaccine development. In this study we recruited 46 adult patients during the 2009 influenza pandemic and characterized humoral and cellular immune responses. Those included were either acute hospitalized or convalescent patients with different disease severities (mild, moderate or severe). In general, protective antibody responses increased with enhanced disease severity. In the acute patients, we found higher levels of TNF-α single-producing CD4+T-cells in the severely ill as compared to patients with moderate disease. Stimulation of peripheral blood mononuclear cells (PBMC) from a subset of acute patients with peptide T-cell epitopes showed significantly lower frequencies of influenza specific CD8+ compared with CD4+ IFN-γ T-cells in acute patients. Both T-cell subsets were predominantly directed against the envelope antigens (HA and NA). However, in the convalescent patients we found high levels of both CD4+ and CD8+ T-cells directed against conserved core antigens (NP, PA, PB, and M). The results indicate that the antigen targets recognized by the T-cell subsets may vary according to the phase of infection. The apparent low levels of cross-reactive CD8+ T-cells recognizing internal antigens in acute hospitalized patients suggest an important role for this T-cell subset in protective immunity against influenza.

Persistence and avidity maturation of antibodies to A(H1N1)pdm09 in healthcare workers following repeated annual vaccinations.

Healthcare workers are at increased risk of influenza infection through direct patient care, particularly during the early stages of a pandemic. Although influenza vaccination is widely recommended in Healthcare workers, data on long-term immunogenicity of vaccination in healthcare workers are lacking. The present study was designed to assess the persistence of the humoral response after pandemic vaccination as well as the impact of repeated annual vaccination in healthcare workers (n=24). Pandemic influenza vaccination resulted in a significant increase in haemagglutination inhibition (HI) antibody titers with 93-100% of subjects achieving protective titers 21-days post each of the three annual vaccinations. Seroprotective antibodies measured by HI, microneutralization and single radial hemolysis assays were present in 77-94% of healthcare workers 6 months post-vaccination. Repeated vaccination resulted in an increased duration of seroprotective antibodies with seroprotective titers increasing from 35-62% 12 months after 2009 pandemic vaccination to 50-75% 12 months after 2010 vaccination. Furthermore, repeated annual vaccination augmented the avidity of influenza-specific IgG antibodies. In conclusion, we have shown that A(H1N1)pdm09 vaccination induces high seroprotective titers that persist for at least 6 months. We demonstrate that repeated vaccination is beneficial to healthcare workers and results in further avidity maturation of vaccine-induced antibodies.

Single dose vaccination of the ASO3-adjuvanted A(H1N1)pdm09 monovalent vaccine in health care workers elicits homologous and cross-reactive cellular and humoral responses to H1N1 strains.

Healthcare workers (HCW) were prioritized for vaccination during the 2009 influenza A(H1N1)pdm09 pandemic. We conducted a clinical trial in October 2009 where 237 HCWs were immunized with a AS03-adjuvanted A(H1N1)pdm09 monovalent vaccine. In the current study, we analyzed the homologous and cross-reactive H1N1 humoral responses using prototype vaccine strains dating back to 1977 by the haemagglutinin inhibition (HI), single radial hemolysis SRH), antibody secreting cell (ASC) and memory B cell (MBC) assays. The cellular responses were assessed by interferon-γ (IFN-γ) ELISPOT and by intracellular staining (ICS) for the Th1 cytokines IFN-γ, interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). All assays were performed using blood samples obtained prior to (day 0) and 7, 14 and 21 d post-pandemic vaccination, except for ASC (day 7) and ICS (days 0 and 21). Vaccination elicited rapid HI, SRH and ASC responses against A(H1N1)pdm09 which cross reacted with seasonal H1N1 strains. MBC responses were detected against the homologous and seasonal H1N1 strains before vaccination and were boosted 2 weeks post-vaccination. An increase in cellular responses as determined by IFN-γ ELISPOT and ICS were observed 1-3 weeks after vaccination. Collectively, our data show that the AS03-adjuvanted A(H1N1)pdm09 vaccine induced rapid cellular and humoral responses against the vaccine strain and the response cross-reacted against prototype H1N1 strains dating back to 1977.

Matrix M(TM) adjuvanted virosomal H5N1 vaccine induces balanced Th1/Th2 CD4(+) T cell responses in man.

T cellular responses play a significant role in mediating protective immune responses against influenza in humans. In the current study, we evaluated the ability of a candidate virosomal H5N1 vaccine adjuvanted with Matrix M(TM) to induce CD4(+) and CD8(+) T cell responses in a phase 1 clinical trial. We vaccinated 60 healthy adult volunteers (at days 0 and 21) with 30 µg haemagglutinin (HA) alone or 1.5, 7.5, or 30 µg HA formulated with Matrix M(TM). To evaluate the T cellular responses, lymphocytes were stimulated in vitro with homologous (A/Vietnam/1194/2004 [H5N1]) and heterologous H5N1 (A/Anhui/1/05 or A/Bar-headed Goose/Qinghai/1A/05) antigens. The antigen-specific cytokine responses were measured by intracellular cytokine staining and by multiplex (Luminex) assays. An increase in CD4(+) Th1 and Th2 cytokines was detected 21 days after the first vaccine dose. No increase in Th cytokine responses was observed after the second dose, although it is possible that the cytokine levels peaked earlier than sampling point at day 42. Formulation with the Matrix M(TM) adjuvant augmented both the homologous and cross-reactive cytokine response. Antigen-specific CD8(+) T cell responses were detected only in a few vaccinated individuals. The concentrations of Th1 and to a lesser extent, Th2 cytokines at 21 days post-vaccination correlated moderately with subsequent days 35 and 180 serological responses as measured by the microneutralisation, haemagglutination inhibition, and single radial hemolysis assays. Results presented here show that the virosomal H5N1 vaccine induced balanced Th1/Th2 cytokine responses and that Matrix M(TM) is a promising adjuvant for future development of candidate pandemic influenza vaccines.

An H7N1 influenza virus vaccine induces broadly reactive antibody responses against H7N9 in humans.

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.

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

BACKGROUND: Highly pathogenic avian influenza A/H5N1 virus remains a potential pandemic threat, and it is essential to continue vaccine development against this subtype. A local mucosal immune response in the upper respiratory tract may stop influenza transmission. It is therefore important to develop effective intranasal pandemic influenza vaccines that induce mucosal immunity at the site of viral entry. OBJECTIVES: We evaluated the humoral and cellular immune responses of two promising mucosal adjuvants (Chitosan and c-di-GMP) for intranasal influenza H5N1 vaccine in a murine model. Furthermore, we evaluated the concept of co-adjuvanting an experimental adjuvant (c-di-GMP) with chitosan. METHODS: BALB/c mice were intranasally immunised with two doses of subunit NIBRG-14 (H5N1) vaccine (7·5, 1·5 or 0·3 µg haemagglutinin (HA) adjuvanted with chitosan (CSN), c-di-GMP or both adjuvants. RESULTS: All adjuvant formulations improved the serum and local antibody responses, with the highest responses observed in the 7·5 µg HA CSN and c-di-GMP-adjuvanted groups. The c-di-GMP provided dose sparing with protective single radial haemolysis (SRH), and haemagglutination inhibition (HI) antibody responses found in the 0·3 µg HA group. CSN elicited a Th2 response, whereas c-di-GMP induced higher frequencies of virus-specific CD4+T cells producing one or more Th1 cytokines (IFN-γ+, IL-2+, TNF-α+). A combination of the two adjuvants demonstrated effectiveness at 7·5 µg HA and triggered a more balanced Th cytokine profile. CONCLUSION: These data show that combining adjuvants can modulate the Th response and in combination with ongoing studies of adjuvanted intranasal vaccines will dictate the way forward for optimal mucosal influenza vaccines.

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 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.

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

BACKGROUND: Development of influenza vaccines that induce mucosal immunity has been highlighted by the World Health Organisation as a priority (Vaccine 2005;23:1529). Dose-sparing strategies and an efficient mass-vaccination regime will be paramount to reduce the morbidity and mortality of a future H5N1 pandemic. OBJECTIVES: This study has investigated the immune response and the dose-sparing potential of a chitosan-adjuvanted intranasal H5N1 (RG-14) subunit (SU) vaccine in a mouse model. METHODS: Groups of mice were intranasally immunised once or twice with a chitosan (5 mg/ml)-adjuvanted SU vaccine [7·5, 15 or 30 µg haemagglutinin (HA)] or with a non-adjuvanted SU vaccine (30 µg HA). For comparison, another group of mice were intranasally immunised with a whole H5N1 (RG-14) virus (WV) vaccine (15 µg HA), and the control group consisted of unimmunised mice. RESULTS: The chitosan-adjuvanted SU vaccine induced an immune response superior to that of the non-adjuvanted SU vaccine. Compared with the non-adjuvanted SU group, the chitosan-adjuvanted SU vaccine elicited higher numbers of influenza-specific antibody-secreting cells (ASCs), higher concentrations of local and systemic antibodies and correspondingly an improved haemagglutination inhibition (HI) and single radial haemolysis (SRH) response against both the homologous vaccine strain and drifted H5 strains. We measured a mixed T-helper 1/T-helper 2 cytokine response in the chitosan-adjuvanted SU groups, and these groups had an increased percentage of virus-specific CD4(+) T cells producing two Thelper 1 (Th1) cytokines simultaneously compared with the non-adjuvanted SU group. Overall, the WV vaccine induced higher antibody concentrations in sera and an HI and SRH response similar to that of the chitosan-adjuvanted SU vaccine. Furthermore, the WV vaccine formulation showed a stronger bias towards a T-helper 1 profile than the SU vaccine and elicited the highest frequencies of CD4(+) Th1 cells simultaneously secreting three different cytokines (INFγ(+) , IL2(+) and INFα(+) ). As expected, two immunisations gave a better immune response than one in all groups. The control group had very low or not detectable results in the performed immunoassays. CONCLUSION: The cross-clade serum reactivity, improved B- and T-cell responses and dose-sparing potential of chitosan show that a chitosan-adjuvanted intranasal influenza vaccine is a promising candidate vaccine for further preclinical development.

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

BACKGROUND: The 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. RESULTS: Expression 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. CONCLUSIONS: This 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.

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.

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-RARalpha 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.

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.