Enhancing viral vaccine production using engineered knockout vero cell lines - A second look.

The global adoption of vaccines to combat disease is hampered by the high cost of vaccine manufacturing. The work described herein follows two previous publications (van der Sanden et al., 2016; Wu et al., 2017) that report a strategy to enhance poliovirus and rotavirus vaccine production through genetic modification of the Vero cell lines used in large-scale vaccine manufacturing. CRISPR/Cas9 gene editing tools were used to knockout Vero target genes previously shown to play a role in polio- and rotavirus production. Subsequently, small-scale models of current industry manufacturing systems were developed and adopted to assess the increases in polio- and rotavirus output by multiple stable knockout cell lines. Unlike previous studies, the Vero knockout cell lines failed to achieve desired target yield increases. These findings suggest that additional research will be required before implementing the genetically engineered Vero cell lines in the manufacturing process for polio- and rotavirus vaccines to be able to supply vaccines at reduced prices.

Human adenovirus type 35 vector for gene therapy of brain cancer: improved transduction and bypass of pre-existing anti-vector immunity in cancer patients.

Clinical trials in malignant glioma have demonstrated excellent safety of recombinant adenovirus type 5 (Ad5) but lack of convincing efficacy. The overall low expression levels of the Coxsackie and Adenovirus receptor and the presence of high anti-Ad5-neutralizing antibody (NAb) titers in the human population are considered detrimental for consistency of clinical results. To identify an adenoviral vector better suited to infect primary glioma cells, we tested a library of fiber-chimeric Ad5-based adenoviral vectors on 12 fresh human glioma cell suspensions. Significantly improved marker gene expression was obtained with several Ad5-chimeric vectors, predominantly vectors carrying fiber molecules derived from B-group viruses (Ad11, Ad16, Ad35 and Ad50). We next tested Ad35 sero prevalence in sera derived from 90 Dutch cancer patients including 30 glioma patients and investigated the transduction efficiency of this vector in glioma cell suspensions. Our results demonstrate that the sero prevalence and the titers of NAb against Ad35 are significantly lower than against Ad5. Also, recombinant Ad35 has significantly increased ability to transfer a gene to primary glioma cells compared to Ad5. We thus conclude that Ad35 represents an interesting candidate vector for gene therapy of malignant glioma.

Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells.

Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (> 5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.

Mapping of rabbit microsatellite markers using chromosome-specific libraries.

Recently, rabbit microsatellite markers were developed from a chromosome 1-specific library, and seven new markers were incorporated into the genetic map of the rabbit. We have now developed microsatellite markers from chromosomes 3-, 5-, 6-, 7-, 12-, and 19-specific libraries. Linkage analysis was performed with use of these new markers, five recently physically mapped markers (PMP2, TCRB, ALOX15, MT1, and Sol33), microsatellite markers located in the HBA gene cluster, the MHC region and FABP6 gene, and seven biochemical markers (Es-1, Es-3, Est-2, Est-4, Est-6, Est-X, and HP). This analysis enabled us to verify the specificity of the libraries and to determine the position and orientation of the linkage groups on the chromosomes.

Chromosomal localization of genes involved in biosynthesis, metabolism or transport of cholesterol in the rat.

Several genes involved in biosynthesis, transport or metabolism of cholesterol have been localized on rat chromosomes by using a radiation hybrid (RH) panel. The genes, coding for squalene epoxidase (Sqle), mevalonate kinase (Mvk), and farnesyl diphosphate farnesyl transferase 1 (Fdft1) which are involved in cholesterol biosynthesis, have been mapped on chromosome 7, 12, and 15, respectively. The genes coding for phospholipid transfer protein (Pltp), sterol carrier protein-2 (Scp2), ATP binding cassette reporter A7 (Abca7), scavenger receptor class B, type 1 (Cd36l1), steroidogenic acute regulatory protein (Star), and lecithin:cholesterol acyl transferase (Lcat), which are involved in the transfer and/or metabolism of cholesterol, have been mapped on chromosome 3, 5, 7, 12, 16, and 19, respectively. Each of the genes Scp2, Sqle and Fdft1 maps close to a QTL for serum total cholesterol in rat, suggesting that these three genes might represent candidate genes for the previously mapped QTLs.

A rat linkage map based on BC x LEW intercross.

A genetic linkage map consisting of 258 polymorphic loci has been constructed on the basis of an F2 intercross between the BC/CpbU and LEW/OlaHsd inbred rat strains. When compared to previously published maps a discrepancy was found for rat chromosome 7. The map spans a sex-averaged genetic length of 1790 cM and has an average marker spacing of 7.7 cM. It was estimated that this genetic map is linked to about 90% of the DNA in the rat genome. Because LEW/OlaHsd and BC/CpbU strains differ for dietary cholesterol susceptibility and hepatic copper content, the map is considered to be a valuable tool for studying the genetic background of these complex traits.

Mapping of rabbit chromosome 1 markers generated from a microsatellite-enriched chromosome-specific library.

A genomic DNA library was produced from flow-sorted rabbit chromosome 1 and enriched for fragments containing CA-repeats. Clones containing CA-repeats were identified and primers for amplification of the microsatellite were developed after sequencing the clone. The degree of polymorphism was tested in rabbits from different breeds. This approach identified 12 microsatellite markers which could be used for studying linkage relationships in the progeny of an F(2)-intercross: (AX/JUxIIIVO/JU) F(2), and two backcrosses: (OS/JxX/J)X/J and (WH/JxX/J)X/J. Seven of these markers were mapped on chromosome 1.

Mapping of a QTL for serum HDL cholesterol in the rabbit using AFLP technology.

The amplified fragment length polymorphism (AFLP) technique is a DNA technology that generates the so-called AFLP markers. These markers are genomic restriction fragments detected after two rounds of polymerase chain reaction (PCR) without prior knowledge of nucleotide sequence. Here we describe the first application of the AFLP technique in the rabbit. We have tested two primer combinations. The results obtained with the DNA from rabbits of different breeds justify the conclusion that AFLP analysis is an effective tool for genetic studies in the rabbit. In addition, we contribute to the linkage map of the rabbit by localizing two AFLP markers on rabbit linkage group VI (LG VI). For this purpose the progeny of a IIIVO/JU x [IIIVO/JU x AX/JU]F(1) backcross were genotyped for 12 AFLP markers and 3 LG VI classical markers [one coat color marker (e) and two biochemical markers (Es-1 and Est-2)]. AX/JU is a dietary cholesterol-susceptible (hyperresponding) inbred strain and IIIVO/JU is a dietary cholesterol resistant (hyporesponding) inbred strain. Moreover, it is possible to evoke dietary cholesterol-induced aorta atherosclerosis in a relatively short time period in AX/JU rabbits, in contrast to IIIVO/JU rabbits. A significant cosegregation was found between basal serum HDL cholesterol level (i.e., the level on a low-cholesterol, control diet) and an AFLP marker on LG VI. It is concluded that one or more genes of LG VI are regulating the basal serum HDL cholesterol level in rabbits. Thus the present study with rabbits clearly illustrates the value of AFLP markers for the construction of linkage maps and mapping of quantitative trait loci (QTL).

Mapping of microsatellite loci and association of aorta atherosclerosis with LG VI markers in the rabbit.

Twenty-three rabbit microsatellites were extracted from the EMBL nucleotide database. Nine of these markers, together with nine earlier published microsatellite markers, were found to be polymorphic between the AX/JU and IIIVO/JU inbred strains. By using an F(2) intercross we could integrate five markers into the rabbit linkage map. One anonymous microsatellite marker could be assigned to chromosome 1, and one microsatellite marker, located within the metallothionein-1 gene, could be added to linkage group VI (LG VI). Three microsatellite markers (one anonymous, one located within the PMP2 gene, and one located within the FABP6 gene) constitute a new linkage group (LG XI). We also measured the degree of dietary cholesterol-induced aorta atherosclerosis in the F(2) animals. A significant cosegregation was found between the degree of aorta atherosclerosis and the allelic variation of the biochemical marker Est-2 on LG VI in male rabbits. This association was not found in female rabbits.

"Inverse" effects of cortisone in experimental infection of mice.

Hydrocortisone (HC) ameliorated the course of experimental infection with E. coli given intraperitoneally. In experiments with Staphylococcus aureus and treatment with endotoxin on the day of infection, HC had also a beneficial effect, however only when LPS was injected intraperitoneally and bacteria intravenously. With the footpad swelling reaction as a model for cellular immunity it could be shown that HC alone reduced the reactivity by 75 per cent. Under endotoxin treatment, however, HC induced further inhibition when LPS was given intraperitoneally the day of sensitization but changing to a stimulatory effect when LPS was given two days later. In case of intravenous application of LPS, HC had no effect.

Modulation of antimicrobial effects of beta-lactams by amino acids in vitro.

Glycine as well as 11 and 10, respectively, out of a total of 12 D-amino-acids tested increased the antimicrobial efficacy of imipenem (IMI) and of ampicillin (AMP) using the serosensitive strain E. coli ATCC 8739. D-proline was ineffective in assays with IMI as well as D-proline and D-leucine in assays with AMP. - In contrast, L-amino-acids behaved differently: In assays with IMI, 9 out of 13 isomers were ineffective whereas 3 were antagonistic (L-phenylalanine, L-serine, L-tryptophan). In combination with AMP, however, 10 L-amino acids had an antagonistic effect and 2 (L-leucine, L-methionine) were ineffective. L-alanine was an exception and showed a synergism with both antibiotics which was assumed to have been due to a racemase activity of cells. - Seroresistance of E. coli apparently reduced the synergistic effect of glycine and beta-lactams. - Glycine, alanine and tryptophan lost their typical synergistic or antagonistic effect with AMP when tested as di- or tri-amino-acid compounds. This was not the case with di-L-alanine - It is supposed that the synergistic effect of glycine or of D-amino-acids with beta-lactams can be explained mainly by an inhibition of carboxypeptidases.

[Importance of the antimicrobial mode of action of antibiotics for their combined effect with glycine in subinhibitory concentrations on microbial growth]. / Importance du mode d'action d'un antibiotique pour son effet combiné avec la glycine à concentrations sub-inhibitrices sur la croissance microbienne.

Following previous observations of an increase in microbial sensitivity to the bactericidal beta-lactams ampicillin and imipenem in the presence of glycine, the aim of the presented study was to examine if such an effect is due to the antimicrobial mode of action of an antibiotic and/or to its bactericidal or bacteriostatic capacity. Using growth curves as an experimental parameter the same synergistic glycine effect could be shown if tested concomitantly with a number of other antibiotics acting equally on bacterial cell wall formation as cefaclor, cefadroxil, or fosfomycin. This glycine effect is, therefore, associated with the antibiotic mode of action, but is independent of wether the antibiotics are beta-lactams or not (fosfomycin). In contrast, glycine had no particular effect in combination with antibiotics inhibiting protein synthesis (sisomicin, kanamycin, chloramphenicol, oxy-tetracycline) or nucleic acid polymerase activity (ciprofloxacin, cinoxacin; rifampicin being a certain exception) as well as with those acting on cytoplasmic and external membrane as polymyxin B. The synergistic effect of glycine and cell wall active antibiotics was interpreted predominantly by an action on carboxypeptidases.

[Interaction of bactericidal serum effect and antibiotics in subminimal inhibitory concentrations on E. coli strains]. / Interaction entre l'effet bactéricide du sérum et des antibiotiques à concentrations sous-inhibitrices sur les souches d'E. coli.

Imipenem (IMI) forming round cells in Gram negative rods reduces in subinhibitory concentrations (subMIC) the seroresistance of E. coli. This effect is distinctly more pronounced in a moderately seroresistant strain of E. coli than in a high seroresistant one. Conversely, human serum (HS) increases the sensitivity of E. coli strains to IMI dependent on their original seroresistance. In contrast, ampicillin (AMP), a filament inducer in E. coli, reduces equally seroresistance but only to a minimal degree and that only in a moderately seroresistant strain; the high seroresistant strain was nonreactive in this respect. It was concluded, that a synergism of antibiotics and bactericidal serum effect is predominantly produced with round cell forming antibiotics, whereas filament forming ones show only minimal effects. Moreover, the original seroresistance of strains in apparently important for the degree tho which these phenomena are expressed.

[Interpretation of the influence of immunoglobulins and glycine on bacterial sensitivity to antibiotics in vitro]. / Interprétation de l'influence des immunoglobulines et de la glycine sur la sensibilité des bactéries aux antibiotiques in vitro.

The effect of human immunoglobulins (Ig) and of glycine, often added to commercial preparations as a stabilizer, have been examined in vitro on the growth of E. coli strains in the absence or presence of antibiotics in subminimal inhibitory concentrations (subMic). The Ig's (= 7S or 5S = F(ab')2 as well as glycine had no effect by themselves on bacterial growth at concentrations up to 32 mg and 4.5 mg per ml respectively. In contrast, in the presence of ampicillin, glycine induced a concentration dependent increase of bacterial sensitivity to antibiotics. This is apparently more pronounced in serosensitive than in seroresistant E. coli strains. Such a synergism could equally be shown with cefadroxil and fosfomycin, i.e. with other antibiotics interacting with cell wall synthesis, but not with those of another mode of action, as ciprofloxacin, polymyxin B or chloramphenicol.

Side effects of antibiotics on immune response parameters and their possible implications in antimicrobial chemotherapy.

Antibiotics may influence immune response by quite different ways. By screening the multitude of publications on this subject, the aim of this overview was to arrive at a basic generalizing statement on the relationship between chemical structure or mode of action of antibiotics and the effect on immune response and to get an indication on whether certain in vitro and/or ex vivo parameters could represent comparable effects under clinical conditions. - The influence of antibiotics on immune response may arise by direct effects on immunocompetent cells, i.e. in the absence of microorganisms, or indirectly by changes in structure or metabolic products of germs induced by subminimal inhibitory concentrations (subMIC's). In the former case, stimulatory and inhibitory effects have been observed on phagocytosis and intracellular killing activity, on antibody production including IgE, on different parameters of cellular immunity (e.g. foodpad swelling reaction, MIF-production, mitogen/antigen induced lymphocyte proliferation and delayed type hypersensitivity skin reaction), on mediator production as interleukins or prostaglandins and the expression of corresponding receptors on immunocompetent cells as well as on the course of experimental infections with primary resistant microorganisms. - Indirect effects are related to the influence of subMIC's of antibiotics on the morphology and structure of microorganisms, on their antigenicity/immunogenicity or on their serosensitivity and enzyme and toxin production. - This overview shows that - according to the actual knowledge - antibiotics may exhibit immunological side effects which, however, can not strictly be attributed to certain chemical structures or to a certain mode of action. - It has to be considered that a literary study comparing the results of different authors is rendered difficult by the often nonhomogeneity of experimental procedures and the fact that little is known yet about immunological side effects of antibiotics in man, i.e. under clinical conditions.

[Effect of fosfomycin on cellular immune reactions as affected by cyclophosphamide or corticosteroids]. / L'effet de la fosfomycine sur l'immunoréaction cellulaire sous l'influence de la cyclophosphamide ou des corticostéroïdes.

The effect of fosfomycin (FM) has been examined on the "footpad swelling reaction" (FPSR) as a model for cellular immunity and on the course of experimental infection with FM resistant E. coli using normal and immunocompromised mice. FM given the day of immunization improved FPSR response progressively up to a dose of 30 mg/kg, higher doses had no effect. If FM was injected 1 or 3 days after immunization doses of 30 mg/kg induced a considerable stimulation, whereas higher doses were inhibitory. In contrast to the high dose of 50 mg of FM per kg, 30 mg/kg increased the stimulatory effect induced by small doses of CY and reduced the inhibitory effect of high CY doses. The depressing effect of hydrocortisone (HC) was also reduced by 30 mg/kg of FM, but not by the higher one. In experimental infection with E. coli, FM in doses of 30 mg/kg was more effective than the higher ones in reducing mortality rate. Comparable results were obtained with CY pretreated animals.

[Clavulanic acid. Its effect on Staphylococcus aureus other than its inhibitory effect on beta-lactamases]. / L'acide clavulanique. Son effet sur le staphylocoque doré outre l'effet inhibiteur sur les bêtalactamases.

The influence of clavulanic acid (CA) on growth kinetics of Staph. aureus strains with MIC values (minimal inhibitory concentration) of penicillin (Pc) between 0,06 and 1 024 IU/ml was examined. Furthermore, it was tried to select mutants of the parent strains with increased resistance to CA and, in the affirmative case, to find out a relationship between Pc and CA resistance. The experiments showed that CA in subinhibitory concentrations induced a considerable increase of the latent phase of Pc sensitive strains but less of the Pc resistant ones. CA delayed also the generation time but this effect was apparently independent of Pc resistance. Successive subculturing of strains in gradually increasing CA concentrations resulted in an increase of CA resistance by a factor of 4 to 8 compared with that of the parent strains. In parallel, the Pc resistance increased by a factor of 16 to 64, however only in case of strains originally sensitive to Pc. A synergistic or additive effect of CA and Pc evaluated by the index of the fractional inhibitory concentration (IFIC) was not only shown in Pc resistant but also in Pc sensitive strains. This effect was equally demonstrable in strains with increased CA resistance. In this case, however, the MIC of Pc in the presence of CA was increased by a factor of 32 to 64 in originally Pc sensitive strains and by a factor of 2 to 4 in the originally Pc resistant ones. These results indicate a possible selection of CA resistant strains with an increased Pc resistance.

Increased resistance towards two systemic experimental infections by tetrachlorodecaoxygen anion complex. Possible implications of cellular and humoral immunity.

The tetrachlorodecaoxygen anion complex (TCDO, Oxoferin) given intravenously 1 h after intravenous infection of mice with Candida albicans increased the host's resistance gradually in doses up to 3.1 mumol/kg body weight (b.w.). Above this level, the stimulatory effect decreased, turning to an inhibition at a dose of 18.6 mumol/kg b.w. TCDO. Repeated applications of the optimum single dose had no cumulative effect. In experimental infections with the strictly anaerobic Peptostreptococcus intermedius, TCDO ameliorated the course of the infection not only at a dose of 3.1 mumol/kg b.w. but also at a higher dose of 12.4 mumol/kg b.w. TCDO, which in the Candida sepsis model fell into the range of defence inhibition. A single dose of 3.1 mumol/kg b.w. TCDO also revealed to be the optimum dose to increase the humoral immune response evaluated by the number of immunoglobulin (Ig)M and IgG forming spleen cells after sensitization with sheep red blood cells (SRBC). The same results were obtained, regardless of whether TCDO had been given at the time of immunization or thereafter. The higher single dose of 18.6 mumol/kg b.w. TCDO did not show this effect but was not inhibitory either. Cellular immune reactions evaluated by the footpad swelling test and SRBC as antigen were found considerably enhanced whether TCDO was given once or repeatedly at a dose of 3.1 mumol/kg b.w., whereas single doses of 18.6 mumol/kg or repeated doses of 9.3 mumol/kg induced a certain inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of theophylline treatment on PGE2 production, PGE2 sensitivity and the distribution pattern of lymphocyte classes relative to the immune status.

In mice, theophylline reduced immune response mechanisms in case of primed cells but stimulated the reaction if priming and treatment were performed concomitantly. The reason for these bivalent reactions is unclear. We studied the effect of theophylline on prostaglandin E2 (PGE2) production by resident macrophages (M phi), on PGE2 sensitivity of thymocytes and spleen cells and on the distribution pattern of lymphocyte classes in thymus and spleen dependent on whether animals were immunized with sheep red blood cells (SRBC) or not. We found a theophylline induced decrease of PGE2 production of M phi in normal but an increase in immunized animals which was less pronounced if given concomitantly with a booster injection. The PGE2 resistance of thymocytes and spleen cells defined by the concentration of PGE2 needed for a 50% inhibition of PHA-P induced transformation was considerably increased by theophylline in normal animals. If given concomitantly with primary sensitization, theophylline equally increased PGE2 resistance but diminished it if applied together with a booster injection. This increase or decrease of PGE2 resistance ran parallel with a decrease or increase of the relative number of Lyt 2+ cells (suppressor T cells = Ts) in the thymus and in the spleen of theophylline treated animals. These results demonstrated that the bivalent effect of theophylline on immune response dependent on the immune status could predominantly be explained by its modulating effect on PGE2 sensitivity of thymocytes and spleen cells together with a corresponding shift in the number of Ts-cells, whereas the influence on PGE2 production by M phi seemed less important.