Telomerase peptide vaccination: a phase I/II study in patients with non-small cell lung cancer.

PURPOSE: A phase I/II study was conducted to investigate the safety, tolerability and clinical response to vaccination with a combination of telomerase peptides GV1001 (hTERT: 611-626) and HR2822 (hTERT: 540-548) in patients with non-small cell lung cancer. EXPERIMENTAL DESIGN: Twenty-six patients with non-small cell lung cancer received intradermal administrations of either 60 nmole (112 microg) or 300 nmole (560 microg) GV1001 in combination with 60 nM (68.4 microg) HR2822 and granulocyte macrophage-colony stimulating factor. The treatment period was 10 weeks. Booster vaccinations with 300 nM GV1001 were offered as follow-up. Monitoring of blood samples, clinical examination and radiological staging were performed regularly. Immune responses were measured as delayed-type hypersensitivity skin reaction and in vitro T cell proliferation. Bone marrow function was monitored in long time survivors. RESULTS: The treatment was well tolerated with minor side effects. No bone marrow toxicities were observed in long time survivors with immune responses. Immune responses against GV1001 were detected in 11 of 24 evaluable patients during the primary regimen and in additional two patients following booster injections. Two patients responded to HR2822. Cloned GV1001-specific CD4+ T cells displayed a Th1 cytokine profile and recognized autologous antigen presenting cells pulsed with recombinant telomerase protein. A complete tumor response was observed in one patient who developed GV1001-specific cytotoxic T cells that could be cloned from peripheral blood. CONCLUSION: The results demonstrate that GV1001 and HR2822 are immunogenic and safe to use in patients with NSCLC. Induction of GV1001-specific immune responses may result in objective tumor responses. Based on these initial encouraging results, further clinical studies of GV1001 in NSCLC patients are warranted.

Infection with chicken anaemia virus impairs the generation of pathogen-specific cytotoxic T lymphocytes.

Infection with chicken anaemia virus (CAV), a circovirus, can result in immunosuppression and subsequent increased susceptibility to secondary infections. This is the first report of impairment of pathogen-specific cytotoxic T lymphocytes (CTL) after natural and experimental infection of chickens with CAV and Marek's disease virus (MDV) or reticuloendotheliosis virus (REV). MDV- and REV-specific CTL were generated at 7 days post infection by 9-30-day-old-chickens that were positive for maternal antibodies to CAV at 9-17 days of age. Replication of CAV could not be demonstrated in these chickens using quantitative real-time polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR assays. In contrast, REV-specific CTL failed to develop when chickens negative for maternal antibodies at 9-17 days of age were infected. Infection with CAV at 45 days of age after CAV maternal antibodies had waned also caused a decreased REV-specific CTL response. In these chickens increased levels of CAV DNA of up to 107 copy numbers per micro g DNA and increased relative transcript levels of CAV by up to a factor of 106 were detected by quantitative real-time PCR and RT-PCR. Interleukin (IL)-1beta and IL-2 mRNA levels were not significantly affected by CAV infection at 7 or 14 days p.i. Similar assays for interferon-gamma (IFN-gamma) transcripts demonstrated a 10-fold increase in IFN-gamma mRNA levels at 7 days post infection following REV or REV + CAV infection, while CAV alone caused a two- to fourfold increase. These results show a strong link between CAV antibody status, CAV replication, and the ability to generate REV-specific CTL. It is likely that the immunosuppressive effects of subclinical infection have previously been underestimated.

Cytotoxic T lymphocyte responses to Marek's disease herpesvirus-encoded glycoproteins.

Cell-mediated immune responses are important for protective immunity to Marek's disease (MD), especially because MD herpesvirus (MDV) infection is strictly cell-associated in chickens with the exception of the feather follicle epithelium. A system previously developed using reticuloendotheliosis (REV)-transformed cell lines stably expressing individual MDV genes allows the determination of relevant MDV proteins for the induction of cytotoxic T lymphocyte (CTL) responses. To examine the importance of glycoproteins for the induction of CTL, the MDV genes coding for glycoproteins (g) C, D, E, H, I, K, L, and M were stably transfected into the REV-transformed chicken cell lines RECC-CU205 (major histocompatibility complex (MHC): B(21)B(21)) and RECC-CU91 (MHC: B(19)B(19)). All transfected cell lines were lysed by REV-sensitized, syngeneic splenocytes obtained from MD-resistant N2a (MHC: B(21)B(21)) and MD-susceptible P2a (MHC: B(19)B(19)) chickens, indicating that the expression of individual MDV glycoproteins did not interfere with antigen processing pathways. Only cell lines expressing gI were recognized by CTL from both N2a and P2a MDV-infected chickens. Cell lines expressing glycoproteins gC and gK, and to a lesser extent, gH, gL, and gM were lysed by syngeneic MDV-sensitized splenocytes from N2a birds but not P2a birds. In contrast, gE was recognized by MDV-sensitized effector cells from the P2a line and not the N2a line. Glycoprotein D was not recognized by either line, with the exception of one marginally significant P2a assay. These results indicate that late viral glycoproteins are relevant for the induction of cell-mediated immunity during MDV infection.

Development of strain-specific real-time PCR and RT-PCR assays for quantitation of chicken anemia virus.

Chicken anemia virus (CAV) is a ubiquitous pathogen of poultry. A CAV specific TaqMan-based PCR and RT-PCR assay for real-time quantitation of viral load and relative quantitation of virus-specific transcript levels was developed. Detection of viral DNA copy number from infected MDCC-CU147 cells was determined by extrapolation from a CAV plasmid-based standard curve. Viral load increased proportionally with increasing cell number harvested, increasing from 4x10(2) copies in 250 cells with 38% virus positive cells in an indirect immunofluorescence assay to 8x10(5) copies in 250,000 cells with 64% infected cells. The estimated average viral copy number per infected cell ranged from 5 to 14. Strain-specific primers were developed to distinguish between the Cux-1 and CIA-1 strains of CAV. These primers exhibited a 3 to 4 log differential in amplification comparing homologous versus heterologous virus-primer combinations. The sensitivity of the real-time assay was found to be comparable to a nested PCR assay using DNA samples from a SPF poultry flock exposed to the SH-1 strain of CAV. The real-time PCR detected from 1.7 to 4.2 target molecules in three out of four samples that were positive by nested PCR using 50% of the DNA used in the nested PCR. Relative viral transcript levels for Cux-1 and CIA-1 infected cell cultures increased proportionally with increasing cell numbers harvested for RNA extraction. This assay will be important for both diagnosis and in understanding the complex pathogenesis of CAV infection.