The virosomal influenza vaccine Invivac: immunogenicity and tolerability compared to an adjuvanted influenza vaccine (Fluad in elderly subjects.

Several approaches are currently being pursued in order to improve the efficacy of influenza vaccines in elderly individuals and others who have impaired immune responses to conventional influenza vaccines. There are two influenza vaccines available for elderly subjects: Fluad (Chiron) and Invivac (Solvay Pharmaceuticals). The present clinical study was a randomized, endpoint-blind, parallel group study in elderly subjects aged 61 years and older to investigate the safety and immunogenicity of these vaccines as compared to a standard influenza vaccine Invivac (Solvay Pharmaceuticals). The three vaccines had similar immunogenicity results, whereas the tolerability profile of Invivac was better as compared to Fluad.

Clinical experience with inactivated, virosomal influenza vaccine.

Current available influenza vaccines are safe and effective in preventing influenza. Nevertheless, there is a need for influenza vaccines with improved efficacy in the elderly. This need is underscored by both the observation that influenza has a major clinical and economic impact in the elderly and the fact that currently available vaccines are generally less effective in elderly than in younger subjects. Several approaches are currently being pursued in order to improve the efficacy of influenza vaccines in elderly individuals and others who have impaired immune responses to conventional influenza vaccines. A novel antigen-presenting strategy to overcome impaired immune responses is the use of virosomes. Previously, data on safety and reactogenicity have been published regarding the use of virosomal influenza vaccines. Data from three recent clinical trials are presented here. The first of these was a comparative study of a virosomal vaccine and a conventional subunit vaccine in "at-risk" adults with underlying chronic illness. The virosomal vaccine demonstrated comparable tolerability to the subunit vaccine, with about 98% of patients reporting tolerability to be good or very good. The vast majority of adverse events reported were mild to moderate in severity. With both vaccine types, mean HI titres decreased with age for both the A-H1N1 and B influenza virus strains, but for the A-H3N2 strain (the most virulent of the three strains), mean HI titres did not decrease with age, suggesting a better response with the virosomal vaccine when compared to the subunit vaccine. All three studies explored the long-term persistence of antibodies after vaccination with virosomal influenza vaccines. Immunogenicity declined over time but remained high at 4, 6 and 12 months post-vaccination compared to baseline, indicating that adequate seroprotection is achievable for the duration of the influenza season. Virosomal vaccines may induce better immunity in elderly subjects and may be more effective in reducing morbidity and mortality in this age group.

Virosomal influenza vaccine: a safe and effective influenza vaccine with high efficacy in elderly and subjects with low pre-vaccination antibody titers.

In 14 clinical studies, various efficacy and safety aspects of a new virosomal influenza vaccine (Invivac) were assessed in 2865 subjects. The virosomal influenza vaccine fully complies with the Committee for Proprietary Medicinal Products (CPMP) requirement for immunogenicity of influenza vaccines. In particular, in a subset of subjects with low pre-vaccination titers (thus those persons who actually need protection by a vaccine), between 76 and 99% of subjects (dependent on age, health status and vaccine components) achieved protective hemagglutination inhibiting (HI) antibody titers after vaccination with the virosomal influenza vaccine. Acceptable frequencies of well-known local and systemic reactions were observed in healthy adults and risk subjects in clinical studies and in a post-marketing study population. These reactions were transient and generally not severe, and did not cause major inconvenience. In conclusion, Invivac is an efficacious and safe vaccine for the protection against influenza in healthy and chronically ill adult subjects. The vaccine is especially efficacious in subjects with low pre-vaccination immunity.

+1 Proteins and aging.

Molecular misreading is an expression used to describe errors in RNA that lead to the translation of mutated proteins. We have shown that dinucleotide deletions (delta GA, delta GU) are introduced in simple sequence repeats (e.g. GAGAG) of mRNA. If the resulting mutant transcripts escape RNA quality control systems, they are translated into +1 proteins. If functional domains are located downstream of the frameshift site, the result will be a protein with either a partial or complete loss of function. A clear example is ubiquitin(+1) (UBB(+1)), which has lost its capacity to ubiquitinate, i.e. tagging proteins destined for proteasomal degradation. This is an important step in regulating the degradation of misfolded proteins and transcription factors. In fact, UBB(+1) seems to block the proteasome. UBB(+1) and other proteins accumulate in the neuropathological hallmarks of Alzheimer's disease (AD), which suggests a causal relationship. We have hypothesized that quality control mechanisms for both transcripts and proteins work less efficiently during aging. In this manner +1 proteins may become manifest and contribute to age-related diseases.

Accumulation of rab4GTP in the cytoplasm and association with the peptidyl-prolyl isomerase pin1 during mitosis.

Transport through the endocytic pathway is inhibited during mitosis. The mechanism responsible for this inhibition is not understood. Rab4 might be one of the proteins involved as it regulates transport through early endosomes, is phosphorylated by p34(cdc2) kinase, and is translocated from early endosomes to the cytoplasm during mitosis. We investigated the perturbation of the rab4 GTPase cycle during mitosis. Newly synthesized rab4 was less efficiently targeted to membranes during mitosis. By subcellular fractionation of mitotic cells, we found a large increase of cytosolic rab4 in the active GTP-form, an increase not associated with the cytosolic rabGDP chaperone GDI. Instead, phosphorylated rab4 is in a complex with the peptidyl-prolyl isomerase Pin1 during mitosis, but not during interphase. Our results show that less efficient recruitment of rab4 to membranes and a bypass of the normal GDI-mediated retrieval of rab4GDP from early endosomes reduce the amount of rab4GTP on membranes during mitosis. We propose that phosphorylation of rab4 inhibits both the recruitment of rab4 effector proteins to early endosomes and the docking of rab4-containing transport vesicles. This mechanism might contribute to the inhibition of endocytic membrane transport during mitosis.

Rabaptin4, a novel effector of the small GTPase rab4a, is recruited to perinuclear recycling vesicles.

The small GTPase rab4a is associated with early endocytic compartments and regulates receptor recycling from early endosomes. To understand how rab4a mediates its function, we searched for proteins which associate with this GTPase and regulate its activity in endocytic transport. Here we identified rabaptin4, a novel effector molecule of rab4a. Rabaptin4 is homologous with rabaptin5 and contains a C-terminal deletion with respect to rabaptin5. Rabaptin4 preferentially interacts with rab4a-GTP and to a lesser extent with rab5aGTP. We identified a rab4a-binding domain in the N-terminal region of rabaptin4, and two binding sites for rab5, including a novel N-terminal rab5a-binding site. Rabaptin4 is a cytosolic protein that inhibits the intrinsic GTP hydrolysis rate of rab4a and is recruited by rab4a-GTP to recycling endosomes enriched in cellubrevin and internalized indocarbocyanine-3 (Cy3)-labelled transferrin. We propose that rabaptin4 assists in the docking of transport vesicles en route from early endosomes to recycling endosomes.

Hyperinducible expression of the interferon-gamma (IFN-gamma) gene and its suppression in systemic lupus erythematosus (SLE).

Transient expression of IFN-gamma and IL-2 mRNA and its control by post-transcriptional and suppressive mechanisms were analysed in phytohaemagglutinin-induced peripheral blood mononuclear cells (PBMC) from 47 patients with SLE and 31 age-matched normal donors, using quantitative hybridization with antisense RNA probes. In SLE, basal levels of gene expression did not deviate from those of normal donors, but strongly aberrant patterns were obtained upon induction. The ratio of subjects exhibiting highly inducible IFN-gamma gene expression in their PBMC to those showing moderate or low inducibility was increased five-fold in SLE (P = 0.003). High inducibility was observed for 43% of SLE patients and was equally pronounced in partial remission, mild or active disease. Inducibility of IL-2 mRNA, by contrast, remained similar to that for normal donors. However, regulation of IFN-gamma gene expression differed for mild SLE. Patients with mild disease showing high inducibility of IFN-gamma mRNA in their PBMC not only had the highest frequency of responders, but also the highest extent of an individual response, defined by superinduction of mRNA, to agents that relieve suppression (gamma-irradiation) or post-transcriptional down-regulation (cycloheximide). By contrast, patients with active SLE showing high IFN-gamma mRNA inducibility had normal suppressive capacity as well as post-transcriptional control. Hence, both high inducibility of the IFN-gamma gene and its suppression are relevant to disease. Hyperactivation of the IFN-gamma gene may be alleviated in mild SLE by a vigorous, concomitant activation of post-transcriptional control and of cell-mediated suppression.

A novel epitope tag for the detection of rabGTPases.

Rab GTPases are localized on the cytoplasmic surface of most intracellular organelles where they play a role in the regulation of vesicular transport. As it has been difficult to detect endogenous rab proteins by morphological methods, their localizations were often inferred from transfection experiments using epitope-tagged constructs. Because most of the available epitope tags are only recognitzed by mouse monoclonal antibodies they are often not suitable for double or triple label immunocytochemistry. To overcome this problem, we generated antibodies against a novel 10 amino acid X31 influenza hemagglutin epitope (NH). We here characterized these antibodies and document their utility for detecting early endosomal rab proteins N-terminally tagged with the NH decapeptide in morphological and biochemical assays.

Adjuvancy and reactogenicity of N-acetylglucosaminyl-N-acetylmuramyl-dipeptide (GMDP) orally administered just prior to trivalent influenza subunit vaccine. A double-blind placebo-controlled study in nursing home residents.

One hundred and fifty-three nursing home residents received 0, 5, 25 or 50 mg N-acetylglucosaminyl-N-acetylmuramyl-dipeptide (GMDP) orally, and trivalent influenza subunit vaccine intramuscularly. One day after intervention, there was a strong increase of total leucocytes, monocytes and neutrophils in the groups receiving 25 or 50 mg GMDP. A GMDP dose dependent increase in systemic, but not in local, vaccine side-effects was observed. No significant differences in post-vaccination haemagglutination inhibiting serum antibody titres were observed between the four groups, indicating that oral administration of GMDP together with influenza vaccination, does not lead to a higher vaccine efficacy.

Prediction of response to treatment in superficial bladder carcinoma through pattern of interleukin-2 gene expression.

PURPOSE: Superficial bladder carcinoma, treated by resection and intravesical administration of bacillus Calmette-Guérin (BCG), yields a remission rate that approaches 70%. We examined whether expression of interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) genes can serve to predict response. PATIENTS AND METHODS: During BCG treatment, we analyzed induction of IL-2 and IFN-gamma mRNA in peripheral-blood mononuclear cells (PBMC) from 73 patients: 51 with papillary tumors and 22 with carcinoma-in-situ (CIS). Results were correlated with remission, relapse, or tumor persistence over a 4-year follow-up period. RESULTS: Independent of tumor type, induction of IL-2 mRNA was observed for patients who responded with remission, but not for those who relapsed (P = .0001). Multivariate logistic analysis showed that inducibility of IL-2 mRNA is the discriminating parameter, which yields a predictive value of 97% for remission. Of 23 patients with relapse/persistence, 22 lacked inducibility of IL-2 mRNA (sensitivity, 95.6%), while 35 of 50 patients in remission exhibited inducibility (specificity, 70%). For patients with carcinoma-in-situ, in which remission or failure depends solely on response to BCG, sensitivity and specificity were 88% and 86%, respectively; for patients with papillary tumors, they were 100% and 64%. IFN-gamma mRNA, by contrast, was clearly inducible in PBMC from all patients (P = .51). The disease-free interval increased progressively with inducibility of IL-2 mRNA; this trend was highly significant (P = .0001). CONCLUSION: IL-2 gene expression is essential for mounting an antitumor response in superficial bladder carcinoma. Inducibility of IL-2 mRNA is an independent prognostic parameter and useful predictive indicator of remission versus relapse.

Post-transcriptional regulation of human interleukin-2 gene expression at processing of precursor transcripts.

Interleukin-2 (IL-2) regulates the clonal expansion of activated T cells and is produced in limited amounts during an immune response. Mitogenic induction of human IL-2 gene expression elicits a transient wave of unstable mRNA. We show here that transcription continues unabated during and well beyond the time when the wave is subsiding, yet few, if any, new mRNA molecules are generated once the wave has reached its maximum. Instead, IL-2 precursor transcripts accumulate, becoming the majority of expressed IL-2 RNA molecules. The flow of precursor transcripts into mature mRNA becomes inhibited in the course of induction. When translation is blocked (e.g. by cycloheximide), expression of IL-2 mRNA can be superinduced by 2 orders of magnitude. This superinduction is completely dependent upon transcription, yet is not accompanied by any significant increase in the rate of primary transcription or in mRNA stability. Instead, the processing of nuclear IL-2 precursor transcripts is greatly facilitated, resulting in pronounced superinduction of cytoplasmic mRNA. Once its transcription has been induced, therefore, expression of the IL-2 gene is down-regulated extensively at the level of precursor RNA processing.

Transient expression of human interleukin-2 and interferon-gamma genes is regulated by interaction between distinct cell subsets.

The level of transient expression of human IL-2 and IFN-gamma genes, we show, is regulated by dynamic interaction between two functionally distinct cell populations. One is able to express these genes, while the other, bearing one of several specific surface markers, actively inhibits their expression. Defined cell subsets were isolated from PBMC and tonsil cells using immunomagnetic beads coated with monoclonal antibodies directed against surface markers. Depletion of CD8, CD11a (Leu15), or Leu8 subsets led to a pronounced superinduction of IL-2 and IFN-gamma gene expression when the remaining cell population was stimulated with mitogen (PHA) or antigen (SEB). Thus, a 10-fold increase in production of IFN-gamma was observed after removal of CD11a (Leu15) cells constituting only a small percentage of the total cell population. By contrast, depletion of cells expressing CD19, a B cell marker, did not yield any superinduction. Conversely, CD8, CD11a (Leu15), or Leu8 cell subsets, but not CD19 cells, each inhibited the induction of IL-2 and IFN-gamma gene expression almost completely in depleted or total cell populations from which they were derived. Gene expression occurring within one cell subset could be effectively inhibited by cells from a second subset. Introduction of inhibitory cells (Leu8) into a population that actively expressed IL-2 and IFN-gamma mRNA resulted in an immediate cessation of gene expression. This suppression involves a soluble mediator, since the culture medium in which such cells were activated exerted a similarly effective inhibition.

Regulation of interleukin-2 and interferon-gamma gene expression in renal failure.

Regulated expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) genes, induced in cultured peripheral blood mononuclear cells from patients with end-stage renal disease on hemodialysis (HD; N = 13) or peritoneal dialysis (PD; N = 13), was compared to that of 32 normal donors. Culture conditions were chosen that measure the transient, phytohemagglutinin-induced expression of IL-2 and IFN-gamma messenger RNA (mRNA), as well the intactness of post-transcriptional and suppressor T cell-dependent mechanisms that control this expression. The latter was achieved by analyzing the superinduction of IL-2 and IFN-gamma mRNA occurring upon culture with cycloheximide or after low-dose gamma-irradiation, respectively. HD subjects showed a complete loss of inducibility of the IL-2 gene, concomitant with decreased inducibility of IFN-gamma mRNA. In PD subjects, by contrast, expression of IL-2 mRNA was as vigorous as in normal donors, while IFN-gamma mRNA was even more strongly inducible. This difference in gene inducibility is caused by a lack of T cell function in HD subjects. The defect in IL-2 gene expression in HD subjects, occurring most likely at transcription, may underly their impaired immune function.

Regulation of human interleukin-2 and interferon-gamma gene expression by suppressor T lymphocytes.

Concomitant with induction of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) gene expression in human tonsil cells, mitogenic stimulation induces a transient activation of cells able to effectively suppress expression of these genes. Induction of IL-2 and IFN-gamma genes largely precedes appearance of suppressor cell activity, allowing expression of both genes to occur before strong down-regulation is exerted by activated suppressor cells. Suppressive activity induced in one cell population can inhibit IL-2 and IFN-gamma gene expression in another population from the same donor. The distinct nature of suppressor cells is supported by the absence of down-regulation of IL-2 gene expression in a helper cell line, MLA-144; yet, in these cells, negative control can be expressed when active suppressor cells are introduced. Our findings support the concept that actual levels of IL-2 and IFN-gamma gene activity are regulated to a large extent by the differential kinetics of activation of suppressor cells on one hand and of cells expressing the IL-2 and IFN-gamma genes on the other.

Aberrant regulation of interleukin-2 but not of interferon-gamma gene expression in Down syndrome (trisomy 21).

The regulated expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) genes was analyzed in peripheral blood mononuclear cells derived from 29 noninstitutionalized Down syndrome individuals and compared to that of 32 normal donors. Culture conditions were chosen that measure the transient, phytohemagglutinin-induced expression of IL-2 and IFN-gamma mRNA, as well as the intactness of post-transcriptional and suppressor T cell-dependent mechanisms that control this expression. The latter was achieved by analyzing, respectively, the superinduction of IL-2 and IFN-gamma mRNA occurring upon culture with cycloheximide or after low-dose gamma-irradiation. A convenient, sensitive, and quantitative assay for specific mRNA was devised, suitable for measuring mRNA levels expressed in cells from 1 ml of peripheral blood. Analysis of individuals with Down syndrome revealed a pronounced decrease in inducibility of the IL-2 gene. By contrast, induction of IFN-gamma mRNA was as vigorous as that observed for normal donors. In cells from trisomic subjects, superinduction of IFN-gamma mRNA by cycloheximide was at least as extensive as for normal donors, while in the case of IL-2 mRNA, it was weaker. These abnormal patterns of IL-2 gene expression were seen irrespective of age. Our findings demonstrate a selective impairment of IL-2 gene expression in Down syndrome, rather than a general deficiency in helper T cells.

The potential to express or suppress human interleukin-2 and interferon-gamma genes is not restricted to distinct cell subsets.

Cell surface markers CD4, CD8, Leu8 and Leu15 (CD11) were used to separate human lymphoid cell subsets with monoclonal antibody-coated immunomagnetic beads. We show that each of these subsets is able to suppress the induction of IL-2 and IFN-gamma genes effectively. This is manifested by a pronounced superinduction of IL-2 and IFN-gamma mRNA, as well as IFN-gamma protein, in cell populations depleted of one of these subsets. Co-culture of cell subsets with total cell populations or depleted ones, on the other hand, leads to severe inhibition of expression of these genes. In these experiments, cells in suppressor subsets exhibit little, if any, expression of IL-2 and IFN-gamma genes. By contrast, depending on donor and lymphoid tissue examined (tonsils or peripheral blood mononuclear cells), CD4, CD8, Leu8, and Leu15 cell subsets are also able to express IL-2 or IFN-gamma genes to high levels. Moreover, in Leu8+ cells that do not express the IFN-gamma gene, extensive expression of both mRNA and protein can be elicited by inhibiting the activation of suppressor cells with gamma-irradiation before induction. These results support the concept that the potential to express or suppress human IL-2 and IFN-gamma genes is not restricted to distinct cell subsets. Suppression or expression can be elicited in cells carrying a given surface marker, depending on the state of the immune system in a lymphoid tissue.

Expression of the human IL-2 receptor on lymphocytes involves rapid turnover of its p55 alpha-subunit (Tac).

Upon mitogenic stimulation, both mRNA encoding the p55 alpha-subunit (Tac) of the human IL-2R alpha and IL-2R alpha protein are induced and expressed in tonsil lymphocyte populations over several days. Using a quantitative dot-blot immunoassay for the IL-2R alpha subunit, a rapid disappearance of this polypeptide from cells is demonstrated in the presence of the translation inhibitor, cycloheximide. The half-life of IL-2R alpha subunit protein is 2 to 3 h. This decline in cell-associated IL-2R alpha subunit is matched by a rapid decline in IL-2R alpha on the cell surface and is not accompanied by any increase in soluble IL-2R alpha protein. Long term expression of the IL-2R on the cell surface is thus the result of continual synthesis and rapid breakdown of IL-2R alpha chains in the cell. Steady state expression of the IL-2R after an immune stimulus hence depends upon continuous expression of the IL-2R alpha subunit gene. Rapid turnover of the unstable alpha-subunit on the cell surface provides a novel mechanism for sensitive control of functional IL-2R expression.