A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein.

A crude extract from ginseng root inhibits high-threshold, voltage-dependent Ca2+ channels through an unknown receptor linked to a pertussis toxin-sensitive G protein. We now have found the particular compound that seems responsible for the effect: it is a saponin, called ginsenoside Rf (Rf), that is present in only trace amounts within ginseng. At saturating concentrations, Rf rapidly and reversibly inhibits N-type, and other high-threshold, Ca2+ channels in rat sensory neurons to the same degree as a maximal dose of opioids. The effect is dose-dependent (half-maximal inhibition: 40 microM) and it is virtually eliminated by pretreatment of the neurons with pertussis toxin, an inhibitor of G(o) and Gi GTP-binding proteins. Other ginseng saponins--ginsenosides Rb1, Rc, Re, and Rg1--caused relatively little inhibition of Ca2+ channels, and lipophilic components of ginseng root had no effect. Antagonists of a variety of neurotransmitter receptors that inhibit Ca2+ channels fail to alter the effect of Rf, raising the possibility that Rf acts through another G protein-linked receptor. Rf also inhibits Ca2+ channels in the hybrid F-11 cell line, which might, therefore, be useful for molecular characterization of the putative receptor for Rf. Because it is not a peptide and it shares important cellular and molecular targets with opioids, Rf might be useful in itself or as a template for designing additional modulators of neuronal Ca2+ channels.

Immunophenotyping of melanomas for tyrosinase: implications for vaccine development.

Tyrosinase (EC 1.14.18.1), the key enzyme in melanin synthesis, has been shown to be one of the targets for cytotoxic T-cell recognition in melanoma patients. To develop serological reagents useful for immunophenotyping melanoma for tyrosinase, human tyrosinase cDNA was expressed in an Escherichia coli expression vector. The purified recombinant tyrosinase was used to generate mouse monoclonal and rabbit polyclonal antibodies. The prototype monoclonal antibody, T311, recognized a cluster of protein moieties ranging from 70 to 80 kDa in tyrosinase mRNA-positive melanoma cell lines and melanoma specimens as well as in L cells transfected with tyrosinase cDNA. Untransfected L cells and L cells transfected with tyrosinase-related protein 1, TRP-1(gp75), were nonreactive. Immunohistochemical analysis of melanomas with T311 showed tyrosinase in melanotic and amelanotic variants, and tyrosinase expression correlated with the presence of tyrosinase mRNA. Melanocytes in skin stained with T311, whereas other normal tissues tested were negative. The expression pattern of three melanosome-associated proteins--tyrosinase, TRP-1(gp75), and gp100--in melanoma was also compared. Tyrosinase and gp100 are expressed in a higher percentage of melanomas than TRP-1(gp75), and the expression of these three antigens was discordant. Tyrosinase expression within individual tumor specimen is usually homogenous, distinctly different from the commonly observed heterogeneous pattern of gp100 expression.

Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design.

The epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL) are generated from cytosolic proteins by proteolytic processing. The nature of the influences exerted by the sequences flanking CTL epitopes on these processing events remains controversial. Here we show that each epitope within an artificial polyepitope protein containing nine minimal CD8+ CTL epitopes in sequence was processed and presented to appropriate CTL clones. Natural flanking sequences were thus not required to direct class I proteolytic processing. In addition, unnatural flanking sequences containing other CTL epitopes did not interfere with processing. The ability of every CTL epitope to be effectively processed from a protein containing only CTL epitopes is likely to find application in the construction of recombinant polyepitope CTL vaccines.

Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains.

Recently, two cell surface molecules, CD46 and moesin, have been found to be functionally associated with measles virus (MV) infectivity of cells. We investigated the receptor usage of MV wild-type, subacute sclerosing panencephalitis, and vaccine strains and their effect on the down-regulation of CD46 after infection. We found that the infection of human cell lines with all 19 MV strains tested was inhibitable with antibodies against CD46. In contrast, not all strains of MV led to the downregulation of CD46 following infection. The group of CD46 non-downregulating strains comprised four lymphotropic wild-type isolates designated AB, DF, DL, and WTF. Since the downregulation of CD46 is caused by interaction with newly synthesized MV hemagglutinin (MV-H), we tested the capability of recombinant MV-H proteins to downregulate CD46. Recombinant MV-H proteins of MV strains Edmonston, Halle, and CM led to the down-regulation of CD46, whereas those of DL and WTF did not. This observed differential downregulation by different MV strains has profound consequences, since lack of CD46 on the cell surface leads to susceptibility of cells to complement lysis. These results suggest that lymphotropic wild-type strains of MV which do not downregulate CD46 may have an advantage for replication in vivo. The relatively weak immune response against attenuated vaccine strains of MV compared with wild-type strains might be related to this phenomenon.

Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity.

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2Ld-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8+ T cells.

A public-professional web-bridge for vaccines and vaccination: User concerns about vaccine safety

Vacunas.org (http://www.vacunas.org), a website founded by the Spanish Association of Vaccinology offers a personalized service called Ask the Expert, which answers any questions posed by the public or health professionals about vaccines and vaccination. The aim of this study was to analyze the factors associated with questions on vaccination safety and determine the characteristics of questioners and the type of question asked during the period 2008–2010. A total of 1341 questions were finally included in the analysis. Of those, 30% were related to vaccine safety. Questions about pregnant women had 5.01 higher odds of asking about safety (95% CI 2.82–8.93) than people not belonging to any risk group. Older questioners (>50 years) were less likely to ask about vaccine safety compared to younger questioners (OR: 0.44, 95% CI 0.25–0.76). Questions made after vaccination or related to influenza (including H1N1) or travel vaccines were also associated with a higher likelihood of asking about vaccine safety. These results identify risk groups (pregnant women), population groups (older people) and some vaccines (travel and influenza vaccines, including H1N1) where greater efforts to provide improved, more-tailored vaccine information in general and on the Internet are required.

Estimates of pandemic influenza vaccine effectiveness in Europe, 2009-2010: results of Influenza Monitoring Vaccine Effectiveness in Europe (I-MOVE) multicentre case-control study

A multicentre case-control study based on sentinel practitioner surveillance networks from seven European countries was undertaken to estimate the effectiveness of 2009-2010 pandemic and seasonal influenza vaccines against medically attended influenza-like illness (ILI) laboratory-confirmed as pandemic influenza A (H1N1) (pH1N1).

Monitoring influenza vaccine effectiveness using the national influenza surveillance system

Background: Flu vaccine composition is reformulated on a yearly basis. As such, the vaccine effectiveness (VE) from previous seasons cannot be considered for subsequent years, and it is necessary to monitor the VE for each season. This study (MonitorEVA- monitoring vaccine effectiveness) intends to evaluate the feasibility of using the national influenza surveillance system (NISS) for monitoring the influenza VE. Material and methods: Data was collected within NISS during 2004 to 2014 seasons. We used a case-control design where laboratory confirmed incident influenza like illness (ILI) patients (cases) were compared to controls (ILI influenza negative). Eligible individuals consisted on all aged individuals that consult a general practitioner or emergency room with ILI symptoms with a swab collected within seven days of symptoms onset. VE was estimated as 1- odds ratio of being vaccinated in cases versus controls adjusted for age and month of onset by logistic regression. Sensitivity analyses were conducted to test possible effect of assumptions on vaccination status, ILI definition and timing of swabs (<3 days after onset). Results: During the 2004-2014 period, a total of 5302 ILI patients were collected but 798 ILI were excluded for not complying with inclusion criteria. After data restriction the sample size in both groups was higher than 148 individuals/ season; minimum sample size needed to detect a VE of at least 50% considering a level of significance of 5% and 80% power. Crude VE point estimates were under 45% in 2004/05, 2005/06, 2011/12 and 2013/14 season; between 50%-70% in 2006/07, 2008/09 and 2010/11 seasons, and above 70% in 2007/08 and 2012/13 season. From season 2006/07 to 2013/14, all crude VE estimates were statistically significant. After adjustment for age group and month of onset, the VE point estimates decreased and only 2008/09, 2012/13 and 2013/14 seasons were significant. Discussion and Conclusions: MonitorEVA was able to provide VE estimates for all seasons, including the pandemic, indicating if the VE was higher than 70% and less than 50%. When comparing with other observational studies, MonitorEVA estimates were comparable but less precise and VE estimates were in accordance with the antigenic match of the circulating virus/ vaccine strains. Given the sensitivity results, we propose a MonitorEVA based on: a) Vaccination status defined independently of number of days between vaccination and symptoms onset; b) use of all ILI data independent of the definition; c) stratification of VE according to time between onset and swab (< 3 and ≥3 days).

Short Peptide Vaccine Induces CD4+ T Helper Cells in Patients with Different Solid Cancers

Previous cancer vaccination trials often aimed to activate CD8(+) cytotoxic T-cell (CTL) responses with short (8-10mer) peptides and targeted CD4(+) helper T cells (TH) with HLA class II-binding longer peptides (12-16 mer) that were derived from tumor antigens. Accordingly, a study of immunomonitoring focused on the detection of CTL responses to the short, and TH responses to the long, peptides. The possible induction of concurrent TH responses to short peptides was widely neglected. In a recent phase I vaccination trial, 53 patients with different solid cancers were vaccinated with EMD640744, a cocktail of five survivin-derived short (9- or 10-mer) peptides in Montanide ISA 51VG. We monitored 49 patients and found strong CD8(+) T-cell responses in 63% of the patients. In addition, we unexpectedly found CD4(+) TH cell responses against at least two of the five short peptides in 61% (23/38) of the patients analyzed. The two peptides were recognized by HLA-DP4- and HLA-DR-restricted TH1 cells. Some short peptide-reactive (sp)CD4 T cells showed high functional avidity. Here, we show that a short peptide vaccine is able to activate a specific CD4(+) T-cell repertoire in many patients, facilitating a strong combined CD4(+)/CD8(+) T-cell response. Cancer Immunol Res; 4(1); 18-25. ©2015 AACR.

Tabulation work at the Vaccine Evaluation Center

verso: IBM Tabulation

State Department : serious problems in the anthrax vaccine immunization program : report to congressional requesters /

"GAO-01-21."