Geir Bredholt

Evaluation of the Sublingual Route for Administration of Influenza H5N1 Virosomes in Combination with the Bacterial Second Messenger c-di-GMP

Avian influenza A H5N1 is a virus with pandemic potential. Mucosal vaccines are attractive as they have the potential to block viruses at the site of entry, thereby preventing both disease and further transmission. The intranasal route is safe for the administration of seasonal live-attenuated influenza vaccines, but may be less suitable for administration of pandemic vaccines. Research into novel mucosal routes is therefore needed. In this study, a murine model was used to compare sublingual administration with intranasal and intramuscular administration of influenza H5N1 virosomes (2 µg haemagglutinin; HA) in combination with the mucosal adjuvant (3′,5′)-cyclic dimeric guanylic acid (c-di-GMP). We found that sublingual immunisation effectively induced local and systemic H5N1-specific humoral and cellular immune responses but that the magnitude of response was lower than after intranasal administration. However, both the mucosal routes were superior to intramuscular immunisation for induction of local humoral and systemic cellular immune responses including high frequencies of splenic H5N1-specific multifunctional (IL-2+TNF-α+) CD4+ T cells. The c-di-GMP adjuvanted vaccine elicited systemic haemagglutination inhibition (HI) antibody responses (geometric mean titres ≥40) both when administered sublingually, intranasally and inramuscularly. In addition, salivary HI antibodies were elicited by mucosal, but not intramuscular vaccination. We conclude that the sublingual route is an attractive alternative for administration of pandemic influenza vaccines.

Longevity of B-Cell and T-Cell Responses After Live Attenuated Influenza Vaccination in Children

Background. The live attenuated influenza vaccine (LAIV) is the preferred vaccine for children, but the mechanisms behind protective immune responses are unclear, and the duration of immunity remains to be elucidated. This study reports on the longevity of B-cell and T-cell responses elicited by the LAIV. Methods. Thirty-eight children (3–17 years old) were administered seasonal LAIV. Blood samples were collected before vaccination with sequential sampling up to 1 year after vaccination. Humoral responses were evaluated by a hemagglutination inhibition assay, and memory B-cell responses were evaluated by an enzyme-linked immunosorbent spot assay (ELISpot). T-cell responses were evaluated by interferon γ (IFN-γ) ELISpot analysis, and intracellular cytokine staining of CD4+ T cells for detection of IFN-γ, interleukin 2, and tumor necrosis factor α was performed using flow cytometry. Results. LAIV induced significant increases in B-cell and T-cell responses, which were sustained at least 1 year after vaccination. Strain variations were observed, in which the B strain elicited stronger responses. IFN-γ–expressing T cell counts increased significantly, and remained higher than prevaccination levels 1 year later. Expression of T-helper type 1 intracellular cytokines (interleukin 2, IFN-γ, and tumor necrosis factor α) increased after 1 dose and were boosted after the second dose. Hemagglutination inhibition titers were sustained for 1 year. Vaccine-induced memory B cell counts were significantly increased, and the response persisted for one year. Conclusions. LAIV elicited B-cell and T-cell responses that persisted for at least 1 year in children. This is a novel finding that will aid future vaccine policy.

Gene expression patterns related to vascular invasion and aggressive features in endometrial cancer.

The presence of tumor cells entering vascular channels is a prognostic marker for many cancers, including endometrial carcinoma. Vascular invasion is considered to be an early step in the metastatic process and important for the progress of malignant tumors. Here, we investigated the gene expression patterns related to vascular involvement in 57 primary endometrial cancers, using DNA microarray and quantitative PCR techniques. A vascular invasion signature of 18 genes was significantly associated with patient survival and clinicopathological phenotype. Vascular involvement was also related to gene sets for epithelial-mesenchymal transition, wound response, endothelial cells, and vascular endothelial growth factor (VEGF) activity. With immunohistochemical validation, both collagen 8 and matrix metalloproteinase 3 (MMP3) were associated with vascular invasion, whereas ANGPTL4 and IL-8 were associated with patient survival. Our findings indicate that vascular involvement within primary tumors is associated with gene expression profiles related to angiogenesis and epithelial-mesenchymal transition. These data could contribute to an improved understanding of potential targets for metastatic spread and may provide clinically important information for better management of endometrial cancer.

Serum IgG titres, but not avidity, correlates with neutralizing antibody response after H5N1 vaccination

BACKGROUND Influenza H5N1 virus constitutes a pandemic threat and development of effective H5N1 vaccines is a global priority. Anti-influenza antibodies directed towards the haemagglutinin (HA) define a correlate of protection. Both antibody concentration and avidity may be important for virus neutralization and resolving influenza disease. METHODS We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with the immunostimulating complex Matrix M™. Sixty adults were intramuscularly immunized with two vaccine doses (21 days apart) of 30 μg HA alone or 1.5, 7.5 or 30 μg HA adjuvanted with Matrix M™. Serum H5 HA1-specific antibodies and virus neutralization were determined at days 0, 21, 42, 180 and 360 and long-term memory B cells at day 360 post-vaccination. The binding of the HA specific antibodies was measured by avidity NaSCN-elution ELISA and surface plasmon resonance (SPR). RESULTS The H5 HA1-specific IgG response peaked after the second dose (day 42), was dominated by IgG1 and IgG3 and was highest in the adjuvanted vaccine groups. IgG titres correlated significantly with virus neutralization at all time points (Spearman r≥0.66, p<0.0001). By elution ELISA, serum antibody avidity was highest at days 180 and 360 post vaccination and did not correlate with virus neutralization. Long-lasting H5 HA1-specific memory B cells produced high IgG antibody avidity similar to serum IgG. CONCLUSIONS Maturation of serum antibody avidity continued up to day 360 after influenza H5N1 vaccination. Virus neutralization correlated with serum H5 HA1-specific IgG antibody concentrations and not antibody avidity.

Antibodies to CRMP3-4 associated with limbic encephalitis and thymoma.

We present a case with subacute limbic encephalitis (LE) and thymoma. Neither classical onconeural antibodies nor antibodies to voltage gated potassium channels (VGKC) were detected, but the serum was positive for anti‐glutamic acid decarboxylase (GAD). The patient serum also stained synaptic boutons of pyramidal cells and nuclei of granule cells of rat hippocampus. The objective of the study was to identify new antibodies associated with LE. Screening a cDNA expression library identified collapsin response mediator protein 3 (CRMP3), a protein involved in neurite outgrowth. The serum also reacted with both CRMP3 and CRMP4 by Western blot. Similar binding pattern of hippocampal granule cells was obtained with the patient serum and rabbit anti‐serum against CRMP1–4. The CRMP1–4 antibodies stained neuronal nuclei of a biopsy from the patients temporal lobe, but CRMP1–4 expression in thymoma could only be detected by immunoblotting. Absorption studies with recombinant GAD failed to abolish the staining of the hippocampal granule cells. Our findings illustrate that CRMP3–4 antibodies can be associated with LE and thymoma. This has previously been associated with CRMP5.

Microarray analysis reveals down-regulation of the tumour suppressor gene WWOX and up-regulation of the oncogene TYMS in intracranial sporadic meningiomas

Background In order to investigate pathways that may influence on tumour development in meningiomas, we performed high throughput microarray analysis of the RNA expression and DNA copy number of 22 WHO grade I and five WHO grade II meningiomas. Since meningiomas derive from arachnoid cap cells, we used samples from four patients operated for arachnoid cysts as control tissue. Results The expression of the tumour suppressor gene WW containing oxidoreductase (WWOX) was down-regulated, and the thymidylate synthase (TYMS) oncogene was up-regulated in all meningiomas as compared to arachnoid cysts. Unsupervised RNA cluster analysis showed that fibrous meningiomas gathered in two clusters, and thus were more homogeneous than the other meningiomas. The other histological subgroups could not be linked to any uniform gene expression signatures. Rearrangements were most abundant on chromosomes 1 and 22, but were identified on all except chromosome 16. The fibrous and mixed meningiomas generally had chromosomal deletions. Duplications were more frequent in the meningothelial meningiomas. WHO grade II meningiomas had increased chromosomal instability. Conclusion Decreased expression of the WWOX tumour suppressor gene and increased expression of the TYMS oncogene may be of importance for the development of human intracranial meningiomas. We have identified several genes (BMPR1B, DMD, RAMP1) with expression signatures specific for fibrous meningiomas. CGH analysis revealed distinct chromosomal patterns in relation to the histological subtypes of the meningiomas.

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.

Tumor necrosis is an important hallmark of aggressive endometrial cancer and associates with hypoxia, angiogenesis and inflammation responses.

Aims Tumor necrosis is associated with aggressive features of endometrial cancer and poor prognosis. Here, we investigated gene expression patterns and potential treatment targets related to presence of tumor necrosis in primary endometrial cancer lesions. Methods and Results By DNA microarray analysis, expression of genes related to tumor necrosis reflected multiple tumor-microenvironment interactions like tissue hypoxia, angiogenesis and inflammation pathways. A tumor necrosis signature of 38 genes and a related patient cluster (Cluster I, 67% of the cases) were associated with features of aggressive tumors such as type II cancers, estrogen receptor negative tumors and vascular invasion. Further, the tumor necrosis signature was increased in tumor cells grown in hypoxic conditions in vitro. Multiple genes with increased expression are known to be activated by HIF1A and NF-kB. Conclusions Our findings indicate that the presence of tumor necrosis within primary tumors is associated with hypoxia, angiogenesis and inflammation responses. HIF1A, NF-kB and PI3K/mTOR might be potential treatment targets in aggressive endometrial cancers with presence of tumor necrosis.

A(H1N1)pdm09 Vaccination of Health Care Workers: Improved Immune Responses in Low Responders Following Revaccination

BACKGROUND We conducted a clinical trial in October 2009 to evaluate the immunogenicity of the AS03-adjuvanted influenza vaccine (pH1N1 vaccine) in health care workers (HCWs). By 2 weeks after vaccination, 97% had protective hemagglutinin inhibition (HI) titers (≥ 40) however, 16% were low responders (LR) and failed to maintain a protective response 90 days after vaccination. METHODS We analyzed the humoral responses (HI, antibody-secreting cell [ASC], and serum immunoglobulin G [IgG]) in 15 LRs and 25 control HCWs. Twelve LRs were revaccinated with the pH1N1 vaccine, and 7 were subsequently vaccinated with the 2010 seasonal trivalent influenza vaccine. We conducted a long-term analysis of the humoral and CD4(+) T-helper (Th) 1 responses. RESULTS The LRs had a slower HI antibody response than the control HCWs, with protective antibody titers not reached until 2 weeks after vaccination in the majority of the participants. The LRs also had significantly lower IgG ASCs at day 7 and HA1-specific serum IgG responses at day 21, compared with the control HCWs. Revaccination with the pH1N1 vaccine elicited rapid HI antibody, ASC, memory B cell, and multifunctional CD4(+) Th1 cell responses. CONCLUSION This study shows that revaccination of low-responding HCWs with the pH1N1 vaccine is required for maintaining long-term protection. CLINICAL TRIALS REGISTRATION NCT01003288.