Convincing data on experimental vaccines against AIDS have been obtained in the simian immunodeficiency virus (SIV) macaque model by preinfection with a virus attenuated by a nef deletion. To investigate the efficacy of a nef deletion mutant of SIVmac32H called pC8 as a live-attenuated vaccine after shorter preinfection periods and to learn more about the nature of the immune protection induced, eight rhesus monkeys were infected intravenously with the pC8 virus. All monkeys became persistently infected, exhibiting low cell-associated viral loads, but strong cellular and, in terms of binding antibodies, strong humoral antiviral responses. Two of eight pC8-infected monkeys developed an immunodeficiency and were not challenged. Sequence analysis of their nef revealed complete replenishment of the deletion. The other six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged with pathogenic spleen-derived SIV. Complete protection was achieved in four vaccinees. Virus was consistently detected in two vaccinees from the 22-week-group challenge, however, they remained clinically healthy over a prolonged period. Protection from challenge virus infection or a delayed disease development seemed to be associated with a sustained SIV-specific T helper cell response after challenge. Thus, a sterilizing immunity against superinfection with pathogenic SIV can be induced even after a relatively short waiting period of 22 weeks. Nevertheless, such a vaccine raises severe safety concerns because of its potential to revert to virulence.
Single-use bioprocessing bags and bioreactors gained significant importance in the industry as they offer a number of advantages over traditional stainless steel solutions. However, there is continued concern that the plastic materials might release potentially toxic substances negatively impacting cell growth and product titers, or even compromise drug safety when using single-use bags for intermediate or drug substance storage. In this study, we have focused on the in vitro detection of potentially cytotoxic leachables originating from the recently developed new polyethylene (PE) multilayer film called S80. This new film was developed to guarantee biocompatibility for multiple bioprocess applications, for example, storage of process fluids, mixing, and cell culture bioreactors. For this purpose, we examined a protein-free cell culture medium that had been used to extract leachables from freshly gamma-irradiated sample bags in a standardized cell culture assay. We investigated sample bags from films generated to establish the operating ranges of the film extrusion process. Further, we studied sample bags of different age after gamma-irradiation and finally, we performed extended media extraction trials at cold room conditions using sample bags. In contrast to a nonoptimized film formulation, our data demonstrate no cytotoxic effect of the S80 polymer film formulation under any of the investigated conditions. The S80 film formulation is based on an optimized PE polymer composition and additive package. Full traceability alongside specifications and controls of all critical raw materials, and process controls of the manufacturing process, that is, film extrusion and gamma-irradiation, have been established to ensure lot-to-lot consistency. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1171–1176, 2014
The inactivation of the simian immunodeficiency viruses SIVmac251 and SIVagm by pressures of 150 and 250 MPa was determined. The extent of inactivation depended on the time that the virus was subjected to compression as well as the level of the pressure and at 150 Mpa reached 5 log1l dilution units after '10 hr. The inactivations, which were uniformly carried out at room temperature, were independent of the concentration of the virus. Possible applications of pressure inactivation for molecular biological and clinical use are discussed.The development of effective methods for eliminating unwanted or harmful components, such as viruses, present in biological samples and preparations is an important scientific endeavor with many possible practical applications. Sterilizing biological preparations and introducing new vaccines are obvious motives; moreover, all procedures that modify the biological activity of viruses can lead to a better understanding of their requirements for successful infectivity. Key considerations in practical, large-scale applications are the simplicity and reproducibility of the procedures. Physical methods are not always highly selective but they are simple, easy to reproduce, universally applicable, and relatively easy to apply on a large scale. High pressure can be applied to almost all biological preparations, is readily implemented routinely and safely in a laboratory environment, and is often selective in its action on macromolecular structures. In this report we present results showing that subjecting virus samples to pressures under 250 MPa (1 MPa = 10 atmospheres) can inactivate the simian immunodeficiency viruses SIVmac251 and SIVagm. Pressure often perturbs selectively the properties of biological molecules and complex biological systems by disrupting noncovalent associations, while leaving unaffected the covalent architecture of the separate components (1-3). This is of particular interest in prospective viral vaccines because the precise relations of the capsid proteins with each other, with the lipid membrane when there is one, and with the nucleic acids must be responsible for the specific infectivity, whereas an intact covalent framework of the proteins is necessary for the proper immune response. Hydrostatic pressures under 300 MPa can disrupt icosahedral viruses (4) and a membrane-enveloped animal virus, causing loss of infectivity with retention of the immunogenic capacity (5, 6). Therefore hydrostatic pressure procedures may by themselves, or in conjunction with other physical, chemical, or biochemical techniques, offer a suitable procedure for the preparation of vaccines. MATERIALS AND METHODSSIVmac251 was isolated from a rhesus monkey (Macaca mulatta) (7) and SIVagm Tyo 7 was isolated from an African green monkey (Cercopithecus aethiops) (8). The simian immunodeficiency viruses were grown in the CEM human T-cell line. The medium for cell growth and for virus inactivation was RPMI 1640 supplemented with 20% fetal bovine serum. The high-pressure inactivation of th...
Purpose: We conducted a phase I/II randomized trial to evaluate the clinical and immunologic effect of chemotherapy combined with vaccination in primary metastatic colorectal cancer patients with a carcinoembryonic antigen^derived peptide in the setting of adjuvants granulocyte macrophage colony-stimulating factor, CpG-containing DNA molecules (dSLIM), and dendritic cells. Experimental Design: HLA-A2^positive patients with confirmed newly diagnosed metastatic colorectal cancer and elevated serum carcinoembryonic antigen (CEA) were randomized toreceive three cycles of standard chemotherapy (irinotecan/high-dose 5-fluorouracil/leucovorin) and vaccinations with CEA-derived CAP-1peptide admixed with different adjuvants [CAP-1/granulocyte macrophage colony-stimulating factor/interleukin-2 (IL-2), CAP-1/dSLIM/IL-2, and CAP-1/IL-2]. After completion of chemotherapy, patients received weekly vaccinations until progression of disease. Immune assessment was done at baseline and after three cycles of combined chemoimmunotherapy. HLA-A2 tetramers complexed with the peptides CAP-1, humanT-cell lymphotrophic virus type I TAX, cytomegalovirus (CMV) pp65, and EBV BMLF-1 were used for phenotypic immune assessment. IFN-g intracellular cytokine assays were done to evaluate CTL reactivity. Results: Seventeen metastatic patients were recruited, of whom 12 completed three cycles. Therapy resulted in five complete response, one partial response, five stable disease, and six progressive disease. Six grade 1local skin reactions and one mild systemic reaction to vaccination treatment were observed. Overall survival after a median observation time of 29 months was 17 months with a survival rate of 35% (6 of17) at that time. Eight patients (47%) showed elevation of CAP-1^specific CTLs. Neither of the adjuvants provided superiority in eliciting CAP-1^specific immune responses. During three cycles of chemotherapy, EBV/CMV recall antigen^specific CD8+ cells decreased by an average 14%. Conclusions: The presented chemoimmunotherapy is a feasible and safe combination therapy with clinical and immunologic efficacy. Despite concurrent chemotherapy, increases in CAP-1ŝ pecificTcells were observed in 47% of patients after vaccination.Despite advances in occult blood screening (1), colonoscopy (2), surgery (3), radiotherapy, and adjuvant (4 -6) chemotherapy, colorectal cancer is still the second leading cause for cancer-related mortality in industrialized countries in men and the third leading cause in women. Palliative chemotherapy regimens have been improved by adding topoisomerase-I inhibitors and platin-based cytotoxic drugs. Currently, the majority of patients with newly diagnosed metastatic colorectal cancer are treated with irinotecan or oxaliplatin together with high-dose 5-fluorouracil (5-FU) and leucovorin (7,8). However, even with therapy, patients with Unio Internationale Contra Cancrum stage IV disease reach an average overall survival time of 15 to 20 months (9, 10). In this setting, immunotherapy offers a potential complement...
Antibiotics from Gliding Bacteria, 45"'. -Phenalamides, New HIV-1 Inhibitors from Myxococcus stipitatus Mx s40We report on the isolation and structure elucidation of the phenalamides A, (l), Az (2), A3 (3), B (4), and C (5), amides from alaninol and a-phenyl-substituted unsaturated carboxylic acids (a pentaene carboxylic acid in 1 -4, and a tetraene carboxylic acid chromophore in 5). Compounds 1 -3 are EIZ isomers with the configuration: 62 for 1, 4 represents 8-methyl-1, and for 5 we determined the l0,ll-double bond in 2 to be epoxidated. C-16 in 1 has the S configuration, as revealed by ozonisation and isolation of the (2s)-( +)-2-methyl-4-phenylbutyric acid. By comparison of their respective CD spectra all the other centers of chirality in 1 should have the same stereochemistry as in the related compound myxalamid B. 1 seems to be identical to stipiamide. First results of enrichment experiments show that the biogenesis of 1 starts with phenylalanine, which after transamination and decarboxylation is connected with propionate and acetate units to generate the highly unsaturated fatty acid. The second amino acid unit alanine terminates the biosynthesis.
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