Dog Kidney Cells Research Articles

Cationic effect in the formation of toxic and antiviral properties of Keggon heteropoly compounds

The Cytotoxicity indices (IC50) of Keggin’s phosphorus-molybdenum heteropoly acids (HPCAs) and their sodium and potassium salts on dog kidney cells (MDSC) were determined. The antiviral activity of these compounds against topical strains of influenza A (H3N2 and H1N1) was revealed. The dependence of the biological properties of polyoxometalates (POMs) on the elemental composition of their molecules has been confirmed. It has been shown that when some of the molybdenum atoms are replaced by vanadium atoms, HPCA and their salts acquire higher cytotoxicities, which increase monotonically as the number of substitutions increases. For the first time, the dependence of the biological activity of HPCA and their salts on the mass of cations has been established and interpreted. In vivo (on white outbred mice) the values of semi-lethal doses (DL50) of these compounds were established. For aqueous solutions of sodium and potassium salts of GPCA in a wide range of concentrations (from 0.05 μM to 15 μM), the values of the toxicity index (It) were determined on the model of motile cells. It has been established that GPCA and their salts are classified as moderately dangerous toxic substances and have selective antiviral activity, which at low concentrations (less than 15 μM) for influenza A strains is manifested mainly by a decrease in hemagglutination activity (HA).

Efficacy of TurmiZn, a Metallic Complex of Curcuminoids-Tetrahydrocurcumin and Zinc on Bioavailability, Antioxidant, and Cytokine Modulation Capability

Complexes of curcumin with metals have shown much-improved stability, solubility, antioxidant capability, and efficacy when compared to curcumin. The present research investigates the relative bioavailability, antioxidant, and ability to inhibit inflammatory cytokine production of a curcuminoid metal chelation complex of tetrahydrocurcumin-zinc-curcuminoid termed TurmiZn. In vitro uptake assay using pig intestinal epithelial cells showed that TurmiZn has an ~3-fold increase (p ≤ 0.01) in uptake compared to curcumin and a ~2-fold increase (p ≤ 0.01) over tetrahydrocurcumin (THC). In a chicken model, an oral 1-g dose of TurmiZn showed a ~2.5-fold increase of a specific metabolite peak compared to curcumin (p = 0.004) and a ~3-fold increase compared to THC (p = 0.001). Oral doses (5 g/Kg) of TurmiZn in rats also showed the presence of curcumin and THC metabolites in plasma, indicating bioavailability across cell membranes in animals. Determination of the antioxidant activity by a 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging assay indicated that TurmiZn was about 13x better (p ≤ 0.0001) than curcumin and about 4X better (p ≤ 0.0001) than THC, in reducing free radicals. In vitro experiments further showed significant (p ≤ 0.01) reductions of lipopolysaccharide (LPS)-induced proinflammatory cytokines such as interleukin (IL) IL-6, IL-8, IL-15, IL-18, and tumor necrosis factor (TNF)-alpha, while showing a significant (p ≤ 0.01) increase of granulocyte-macrophage colony-stimulating factor (GM-CSF) in dog kidney cells. In vivo cytokine modulations were also observed when TurmiZn was fed for 6 weeks to newborn chickens. TurmiZn reduced IL-1 and IL-6, but significantly reduced (p ≤ 0.01) IL-10 levels while there was a concurrent significant (p = 0.02) increase in interferon gamma compared to controls. Overall, these results indicate that TurmiZn has better bioavailability and antioxidant capability than curcumin or THC and has the ability to significantly modulate cytokine levels. Thus, TurmiZn could be an excellent candidate for a novel ingredient that can be incorporated into food and supplements to help overall health during the aging process.

Knockdown of FLT4, Nup98, and Nup205 Cellular Genes Effectively Suppresses the Reproduction of Influenza Virus Strain A/WSN/1933 (H1N1) In vitro.

Influenza is one of the most common infectious diseases, which affects the lower respiratory tract, and can lead to serious complications, including death. It is known that currently available therapeutic agents and vaccines do not provide 100% protection against influenza viruses. The development of drugs based on the RNA interference mechanism in the context of this problem is a promising area. This paper aims to assess the effect of FLT4, Nup98, and Nup205 cellular gene knockdown on the reproduction of the influenza A virus in human lung cell culture. Influenza virus strain A/WSN/1933 (St. Jude's Children's Research Hospital, USA) was used in this work as well as A549 cell culture (human lung adenocarcinoma, ATCC® CCL- 185, USA) and MDCK cell culture (dog kidney cells, Institut Pasteur, France). Small interfering RNAs (siRNAs) (Syntol, Russia) were synthesized for targeting the FLT4, Nup98, and Nup205 genes. Lipofectamin 2000 (Invitrogen, USA) was used for transfection. After 4 hours, the transfected cells were infected with the influenza virus at MOI = 0.1. Virus-containing fluid was collected within three days from the moment of transfection, and the intensity of viral reproduction was assessed by CPE titration and hemagglutination reactions. Viral RNA concentration was determined by RT-PCR. Mann- Whitney U test was used for statistical analysis. In cells treated with siRNA for FLT4, Nup98, and Nup205 genes, there was a significant decrease in the expression of target genes and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) at MOI = 0.1, although the cell survival rate did not decrease significantly. On the first day, the viral titer in cells treated with declared siRNA was lower, on average, by 1 Lg, and on the second and third days, by 2.2-2.3 Lg, compared to cells treated with nonspecific siRNA. During RT-PCR, a significant decrease in the concentration of viral RNA with Nup98.1 and Nup205 siRNA was detected: up to 190 times and 30 times on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies has been decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained while determining the hemagglutinating activity of the virus. The hemagglutinating activity decreased mostly (by 16 times) in cells treated with Nup205 and FLT4.2 siRNAs on the third day. In cells treated with FLT4.1, Nup98.1, and Nup98.2 siRNAs, the hemagglutinating activity decreased by 8 times. We identified a number of genes, such as FLT4, Nup98, and Nup205, whose expression can efficiently suppress viral reproduction when their expression is decreased. The original siRNA sequences were also obtained. These results are important for the creation of therapeutic and prophylactic agents, whose action is based on the RNA interference mechanism.

In Vitro Nephrotoxicity and Permeation of Vancomycin Hydrochloride Loaded Liposomes.

Drugs can be toxic to the fetus depending on the amount that permeates across the maternal–fetal barrier. One way to limit the amount which penetrates this barrier is to increase the molecular size of the drug. In this study, we have achieved this by encapsulating our model antibiotic (vancomycin hydrochloride, a known nephrotoxic agent) in liposomes. PEGylated and non-PEGylated liposomes encapsulating vancomycin hydrochloride were prepared using two different methods: thin-film hydration followed by the freeze–thaw method and the reverse-phase evaporation method. These liposomes were characterized by their hydrodynamic size and zeta potential measurements, CryoTEM microscopy, loading and encapsulation efficiency studies, in vitro release measurements and in vitro cytotoxicity assays using NRK-52 E rat kidney cells. We also determined the in vitro permeability of these liposomes across the human placental cell and dog kidney cell barriers. Vancomycin hydrochloride-loaded PEGylated liposomes (VHCL-lipo) of a size less than 200 nm were prepared. The VHCL-lipo were found to have the faster release of vancomycin hydrochloride and resulted in greater viability of NRK-52E cells. In vitro, the VHCL-lipo permeated the human placental cell and dog kidney cell barriers to a lesser extent than the free vancomycin hydrochloride. The data suggest a reduction in nephrotoxicity and permeability of vancomycin hydrochloride after encapsulation in PEGylated liposomes.

A Small-Plaque Isolate of the Zika Virus with Envelope Domain III Mutations Affect Viral Entry and Replication in Mammalian but Not Mosquito Cells.

An Asian Zika virus (ZIKV) isolated from a Thai patient that was serially passaged in Primary Dog Kidney (PDK) cells for attenuation displayed both big and small plaque-forming viruses by the 7th passage. Two small-plaque isolates were selected and purified for characterization as attenuated ZIKV candidates. In vitro growth kinetics showed significantly reduced titers for small-plaque isolates in Vero cells early post-infection compared to the parental ZIKV and a big-plaque isolate, but no significant difference was observed in C6/36 cells. Viral entry experiments elucidate that titer reduction likely occurred due to the diminished entry capabilities of a small-plaque isolate. Additionally, a small-plaque isolate displayed lowered neurovirulence in newborn mice compared to 100% lethality from infection with the parental ZIKV. Genomic analysis revealed the same three unique non-synonymous mutations for both small-plaque isolates: two on the envelope (E) protein at residues 310, alanine to glutamic acid (A310E), and 393, glutamic acid to lysine (E393K), and one on residue 355 of NS3, histidine to tyrosine (H355Y). Three-dimensional (3D) mapping suggests that the E protein mutations located on the receptor-binding and fusion domain III likely affect cell entry, tropism, and virulence. These ZIKV isolates and genotypic markers will be beneficial for vaccine development.

The positive effect of selective prostaglandin E2 receptor EP2 and EP4 blockade on cystogenesis in vitro is counteracted by increased kidney inflammation in vivo

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major cause of end-stage kidney disease in man. The central role of cyclic adenosine monophosphate (cAMP) in ADPKD pathogenesis has been confirmed by numerous studies including positive clinical trial data. Here, we investigated the potential role of another major regulator of renal cAMP, prostaglandin E2 (PGE2), in modifying disease progression in ADPKD models using selective receptor modulators to all four PGE2 receptor subtypes (EP1-4). In 3D-culture model systems utilizing dog (MDCK) and patient-derived (UCL93, OX161-C1) kidney cell lines, PGE2 strikingly promoted cystogenesis and inhibited tubulogenesis by stimulating proliferation while reducing apoptosis. The effect of PGE2 on tubulogenesis and cystogenesis in 3D-culture was mimicked or abolished by selective EP2 and EP4 agonists or antagonists but not those specific to EP1 or EP3. In a Pkd1 mouse model (Pkd1nl/nl), kidney PGE2 and COX-2 expression were increased by two-fold at the peak of disease (week four). However, Pkd1nl/nl mice treated with selective EP2 (PF-04418948) or EP4 (ONO-AE3-208) antagonists from birth for three weeks had more severe cystic disease and fibrosis associated with increased cell proliferation and macrophage infiltration. A similar effect was observed for the EP4 antagonist ONO-AE3-208 in a second Pkd1 model (Pax8rtTA-TetO-Cre-Pkd1f/f). Thus, despite the positive effects of slowing cyst growth invitro, the more complex effects of inhibiting EP2 or EP4 invivo resulted in a worse outcome, possibly related to unexpected pro-inflammatory effects.

ВИВЧЕННЯ ПРОТИВІРУСНОЇ АКТИВНОСТІ ТАБЛЕТОК АЛЬТАБОР

<p><strong>STUDY OF ANTIVIRAL ACTIVITY OF TABLETS ALTABOR </strong></p><p><strong>S.L. Rybalko<sup>1</sup>, </strong><strong>Т</strong><strong>.V. </strong><strong>К</strong><strong>rutskikh<sup>2</sup>, </strong><strong>А</strong><strong>.S. Shalamay<sup>3</sup></strong></p><p><sup>1</sup>L.V.Gromashevsky Institute of epidemiology and infectious diseases Academy of Medical Science of Ukraine, Kyiv</p><p><sup>2</sup>National Pharmaceutical University, Kharkiv</p><p><sup>3</sup>JSC SIC "Borshchahivka CPP", Kyiv</p><p><strong>Summary:</strong> studied antiviral activity of tablets Altabor. Determined that the drug has antiviral activity and inhibits the reproduction of Epstein-Barr virus.</p><p><strong>Key words:</strong> altabor, antiviral activity, influenza, Epstein-Barr virus.</p><p><strong>Introduction.</strong> Among all infectious diseases flu and SARS took first place in frequency and the number of cases in the world and account for 95%. According to WHO influenza and flu-like illness in the world is sick 5 - 30 % of the population is 500 million people. For 400 years the world's recorded over 100 epidemics of influenza pandemics and 18, which bring it with them tens of millions of lives. Today, global losses from influenza epidemics is much inferior to the scale of past pandemics due to the possibilities of modern diagnosis of the virus , the availability of antiviral and antibacterial drugs and improving health services. But the problem of treatment of disease really matter remains relevant.</p><p>Against the background of acute viral infections of herpes viruses can be said not, but no less threatening. Herpes was first fixed in a thousand years BC, but the problem that attracts attention, was only in the 20th century after the emergence of HIV. Today open 80 more members of the family of herpes viruses, of which 8 types pathogenic to man. One of the pathogenic types are Epstein-Barr virus, which, according to research antibodies detected in 95% of the population. At this time this virus play an important role in the formation of cancer cells in the body that causes the need for increasing attention from the medical and pharmaceutical community.</p><p>Create antiviral agents is one of the most difficult tasks chemotherapy infections. The reason is that viruses containing RNA and DNA components are obligate intracellular parasites. In the process of viral replication is mainly used machine biosynthesis microorganism cells, modifying its outstanding manner. In this regard, it is very difficult to find selective operating means which would killed viruses without damaging the cells are "master". However, in recent years, certain antiviral drugs that possess outstanding selective action against the infected cells and suppress the virus replicative cycle. One of these drugs is original domestic product - Altabor tablets.</p><p><strong>Methods.</strong> Antiviral activity of Altabor tablets studied in models in vitro. To determine influenza activity using dog kidney cell culture .The influence of tablets Altabor on virus Epstein-Barr conducted in the system Raji cells infected with the said virus.</p><p><strong>Results and discussion</strong>. Altabor tablets influenza activity based on the inhibition of viral neuraminidase, an enzyme responsible for viral virion ability to penetrate the host cell and leave it after reproduction. Analyzing these data, it should be noted that tablets Altabor actively inhibit the reproduction of the influenza virus in doses from 100 mg/ml to 6.75 ug/ml 7.5-3.0 lg ID50, which indicates a strong flu drug activity. As for the study of the effect of tablets on Altabor Epstein-Barr virus, the maximum tolerated concentration was 750 mg/ml. Determined that the minimum active concentration for the drug was 0.1 mg/ml. Based on the value of the minimum and maximum tolerated concentration of active therapeutic index of tablets Altabor established at 5000, indicating a pronounced effects on reproduction of the virus.</p><p><strong>Conclusions.</strong> Result of studies on the antiviral activity of tablets Altabor found that the preparation has flu activity. Also, the drug inhibits the reproduction of Epstein-Barr virus, as evidenced therapeutic index of tablets Altabor set at 5000.</p>

Trials for Preparation of Dog Kidney Cell Line

Trials for Preparation of Dog Kidney Cell Line

Overview of measles and mumps vaccine: origin, present, and future of vaccine production

Measles and mumps are common viral childhood diseases that can cause serious complications. Vaccination remains the most efficient way to control the spread of these viruses. The manufacturing capability for viral vaccines produced in embryonated hen eggs and conventional/classical cell substrates, such as chicken embryo fibroblast or primary dog kidney cell substrates, is no longer sufficient. This limitation can be overcome by utilizing other recognized cell substrates such as Madin Darby Canine Kidney (MDCK), Chinese Hamster Ovary (CHO), Vero (monkey origin) cells, MRC-5 (human diploid) or as an alternative, introducing new cell substrates of human or avian origin. A very important factor in vaccine production is the safety and immunogenicity of the final vaccine, where the proper choice of cell substrate used for virus propagation is made. All substrates used in vaccine production must be fully characterized to avoid the contamination of hidden unknown pathogens which is difficult to achieve in primary cell substrates.

<i>In vitro </i>anti-viral effect of fructopyrano-(1→4)-glucopyranose from <i>Radix isatidis</i> on infuenza virus A

To investigate the in vitro anti-viral effect of fructopyrano-(1→4)-glucopyranose (FG) from traditional Chinese medicine Radix isatidis, dog kidney cells (MDCK) were with treated with either FG and then with virus (Group A); virus and then with FG (Group B); or FG and virus simultaneously (Group C). Results showed the in vitro cell protection rate and virus inhibition rate increased significantly with the increase of FG concentration when compared with cells without FG treatment (p<0.01). The anti-viral effect was the best in Group C and that in Group A was superior to that in Group B. FG could exert significant inhibited effect on neuraminidase activity with the IC50 of 0.07 mmol/mL and on hemagglutinin of avian influenza virus with minimum inhibitory concentration of 3.13 mg/mL. Our findings indicate FG can confer obvious antiviral effects on influenza A virus in vitro.

Bioprocess development for canine adenovirus type 2 vectors

Canine adenovirus type 2 (CAV-2) vectors overcome many of the clinical immunogenic concerns related to vectors derived from human adenoviruses (AdVs). In addition, CAV-2 vectors preferentially transduce neurons with an efficient traffic via axons to afferent regions when injected into the brain. To meet the need for preclinical and possibly clinical uses, scalable and robust production processes are required. CAV-2 vectors are currently produced in E1-transcomplementing dog kidney (DK) cells, which might raise obstacles in regulatory approval for clinical grade material production. In this study, a GMP-compliant bioprocess was developed. An MDCK-E1 cell line, developed by our group, was grown in scalable stirred tank bioreactors, using serum-free medium, and used to produce CAV-2 vectors that were afterwards purified using column chromatographic steps. Vectors produced in MDCK-E1 cells were identical to those produced in DK cells as assessed by SDS-PAGE and dynamic light scatering measurements (diameter and Zeta potential). Productivities of ∼10(9) infectious particles (IP) ml(-1) and 2 × 10(3) IP per cell were possible. A downstream process using technologies transferable to process scales was developed, yielding 63% global recovery. The total particles to IP ratio in the purified product (<20:1) was within the limits specified by the regulatory authorities for AdV vectors. These results constitute a step toward a scalable process for CAV-2 vector production compliant with clinical material specifications.

In Vitro Assessment of the Allergenicity of Novel H1N1 Influenza Vaccine Produced in Dog Kidney Cells in Subjects with Dog Allergy

A licensed inactivated seasonal influenza vaccine produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and did not trigger degranulation in rat basophilic leukemia (RBL) cells passively sensitized with human anti-dog IgE, supporting its safe use in dog-allergic individuals. The canine kidney cell-derived H1N1 pandemic vaccine, however, was adjuvanted with the emulsion adjuvant MF59®, and support for its similar safe use was sought. We evaluated allergenicity of the adjuvanted canine kidney cell-derived H1N1 vaccine in subjects with dog allergy, with utilization of an in vitro mediator release assay.

Choice for host-specific high-adhesive &amp;lt;i&amp;gt;Lactobacillus&amp;lt;/i&amp;gt; strains

Adhesive ability was tested in seven Lactobacillus solated from the chicken digestive tract after cultivation with CaCo-2 cells (intestinal epithelial cells), MDCK (dog kidney) cells and CEF (chicken embryo fiber) cells. We noted the following important observations regarding the adhesive ability between different Lactobacillus strains and three cell types: the adhesive interaction between the SDnB7, SDnE1 and SDnA3 lactobacillus strains and CaCo-2cells was greater compared to controls, the adhesive effect between SDnB1 and CEF cells was also greater than controls and Lactobacillus showed only minimal adherence to MDCK cells. Incubation time also affected Lactobacillus adherence to CaCo-2cells: adhesive ability was optimal at 37°C when incubated for 2 days and this was confirmed by a local increase in the concentration of Lactobacillus around CaCo-2 cells when incubated for 24 h as opposed to 3 h. Adherence ability in lactobacillus was also tested at various concentrations (108, 107, 106, 105 and 104 ). The number of Lactobacillus that adhered around the cells was significantly increased in the treatment with 108 bacterial cells. Transmission electron microscopy revealed that the cellularity of the junction between CaCo-2cells and Lactobacillus was not compromised. Transmission electron microscopy also revealed that the thalli fabric structure remained intact.

Evolution of attenuating mutations in dengue-2 strain S16803 PDK50 vaccine and comparison of growth kinetics with parent virus

A live-attenuated dengue-2 virus strain S16803 vaccine candidate that is immunogenic and safe in humans was derived by 50 passages in primary dog kidney (PDK) cells. To identify mutations associated with attenuation of the dengue-2 PDK50 vaccine strain, we determined the nucleotide changes that arose during PDK passage of the dengue-2 virus. Thirteen mutations distinguished the PDK50 virus from low-passage parent resulting in amino acid substitutions in the premembrane (E89G), envelope (E202K, N203D), nonstructural proteins NS1 (A43T), NS2A (L181F), NS2B (I26V), and NS4B (I/T108T, L112F). In addition, the PDK50 virus contained a C to T change of nucleotide 57 in the 5' non-coding region and four silent mutations of nucleotides 591, 987, 6471, and 8907. An infectious PDK50 cDNA clone virus was produced and characterized for growth kinetics in monkey (LLC-MK(2), Vero) and mosquito (C6/36) cells. Identification of mutations in the vaccine strain and availability of an infectious clone will permit systematic analysis of the importance of individual or collective mutations on attenuation of dengue virus.

Two microscopes are better than one

Researchers reveal way to sharpen vision of double-microscope technique.

In vitro assessment of the allergenicity of a novel influenza vaccine produced in dog kidney cells in individuals with dog allergy

An inactivated influenza vaccine produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and may be used to immunize egg-allergic patients. Although no major dog allergens were identified in MDCK 33016-PF cells, minor dog allergens might be present and cause reactions in dog-allergic individuals. To evaluate the allergenicity of the inactivated influenza vaccine produced in cell culture in a mediator release assay. Rat basophil leukemia (RBL) cells transfected with human IgE receptor-1 were sensitized with sera from dog-allergic adults with positive skin prick test reactions to dog extract and detectable dog dander IgE and were stimulated with serial dilutions of vaccine and dog dander extract. N-hexosaminidase release (NHR) was used as a marker of RBL cell degranulation. Western blots were performed, and UniCAP was used to measure dog-specific IgE antibody levels. The median (interquartile range) level of dog dander IgE was 8.31 kU(A)/L (1.895-14.5 kU(A)/L) and of dog epithelium IgE was 3.19 kU(A)/L (0.835-6.27 kU(A)L). Median (range) maximum NHR (at the first 10-fold dilution) was 0% (0%-1.4%) to vaccine and 10.2% (0%-35.9%) to dog dander (P < .001). In an egg-allergic control subject, the maximum NHR to a vaccine cultured in chick embryo and containing egg protein was 10.2%. IgE antibodies in pooled sera did not bind to vaccine on immunoblots but produced strong binding to dog dander and epithelium extracts. Serum from an egg-allergic control subject strongly bound embryonated egg-derived vaccine. An influenza vaccine produced in continuous canine kidney cells did not trigger degranulation in RBL cells passively sensitized with human anti-dog IgE.

Exposure to antiepileptic drugs does not alter the functionality of P-glycoprotein in brain capillary endothelial and kidney cell lines

Several major antiepileptic drugs, including carbamazepine, phenytoin and phenobarbital, induce xenobiotic metabolizing enzymes via activation of nuclear receptors, including pregnane X receptor (NR1I2) and constitutive androstane receptor (NR1I3). Via activation of these xenobiotic sensors, antiepileptic drugs may also induce the expression of efflux transporters such as P-glycoprotein (Pgp) in different tissues, including intestine, liver, kidney and brain. Increased expression of Pgp in brain capillary endothelial cells, which form the blood–brain barrier, could limit the penetration of antiepileptic drugs into the brain and therefore decrease their therapeutic efficacy. As a consequence, it is important to know whether antiepileptic drugs alter the expression or functionality of Pgp in endothelial cells. In the present study, we studied the effects of exposure to phenobarbital, phenytoin and carbamazepine on Pgp expression and functionality in the rat brain endothelial cell line GPNT. For comparison with drug effects on endothelial cells, a dog kidney cell line (MDCK II) was used. Furthermore, several known Pgp inducers (dexamethasone, doxorubicin, and rifampicin) were included in the study. Functionality of Pgp was determined by uptake assays, using known Pgp substrates (digoxin and vinblastine) and transport inhibitors (tariquidar, MK571). In GPNT cells, exposure to dexamethasone increased Pgp functionality, while antiepileptic drug exposure at clinically relevant concentrations did not exert any significant induction of Pgp expression or function. Similarly, antiepileptic drug exposure did not affect Pgp in MDCK cells. The lack of antiepileptic drugs to induce Pgp in brain capillary endothelial cells and kidney cells is in contrast to their known effect on Pgp expression in hepatic and intestinal cells, substantiating tissue differences in the regulation of Pgp.

Identification of mutations in a candidate dengue 4 vaccine strain 341750 PDK20 and construction of a full-length cDNA clone of the PDK20 vaccine candidate

Dengue 4 virus strain 341750 serially passaged 20 times in primary dog kidney (PDK) cells was shown to have reduced infectivity for rhesus monkeys but was immunogenic and protected the monkeys from challenge with low passage parent dengue 4 virus. The dengue 4 PDK20 virus was also shown to be attenuated for human volunteers. We compared the genomic nucleotide sequences of low passage parent and PDK20 attenuated vaccine strains and identified 11 nucleotide (nt) substitutions in the PDK20 genome. Five mutations caused amino acid changes in viral proteins E (N366N/S), NS1 (E146Q), NS4B (S/L112L and A240V), and NS5 (F/L790L). Silent mutations occurred in genes encoding NS1 (nt 2609), NS3 (nt 6113, 6230 and 6239) and NS5 (nt 8081 and 8588). A full-length cDNA clone of the dengue 4 strain 341750 PDK20 was constructed and RNA transcripts of the clone were infectious in monkey kidney (LLC-MK 2) and Aedes albopictus (C6/36) cells. The sequence analysis and availability of an infectious clone provide molecular tools to investigate the basis for the attenuation of dengue 4 virus.

In vitro Assessment of the Allergenicity of Novel Influenza Vaccine Produced in Dog Kidney Cells in Subjects with Dog Allergy

RATIONALE: An inactivated influenza vaccine (Optaflu® is manufactured by Novartis, registered in the EU) produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and may be used for immunizing egg-allergic patients. Although no major dog allergens were identified in MDCK 33016-PF cells, minor dog allergens might be present and cause reactions in dog-allergic individuals. We sought to evaluate allergenicity of Optaflu in a passive sensitization assay.METHODS: RBL cells transfected with human IgE receptor-1 were sensitized with sera from adult dog-allergic subjects with positive skin prick tests to dog extract and detectable dog dander -IgE, and stimulated with serial dilutions of Optaflu and dog dander extract. N-hexosaminidase release% (NHR) was used as a marker of RBL degranulation. Western blots were performed and UniCAP was used to measure dog-specific IgE antibody levels.RESULTS: Median dog dander-IgE was 8.31 kUA/L (0.62; 67.9); median dog epithelium-IgE was 3.2 kUA/L, (0.4; 38.2). Median (range) maximum NHR (at 1st 10-fold dilution) was: 1). Optaflu- 0% (0; 1.4); 2). Dog dander-10.2% (0; 35.9), P < 0.001; and 3). Anti-IgE positive control-17.7% (1.2; 45.4). In a control egg-allergic subject, maximum NHR to a vaccine cultured in chick embryo and containing egg protein was 10.2%. IgE antibodies in pooled sera did not bind to Optaflu on immunoblots but produced strong binding to dog dander and epithelium extracts. Serum from a control egg-allergic subject strongly bound embryonated egg-derived vaccine.CONCLUSIONS: Optaflu, an influenza vaccine produced in continuous canine kidney cell line did not trigger degranulation in RBL cells passively sensitized with human anti-dog IgE. RATIONALE: An inactivated influenza vaccine (Optaflu® is manufactured by Novartis, registered in the EU) produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and may be used for immunizing egg-allergic patients. Although no major dog allergens were identified in MDCK 33016-PF cells, minor dog allergens might be present and cause reactions in dog-allergic individuals. We sought to evaluate allergenicity of Optaflu in a passive sensitization assay. METHODS: RBL cells transfected with human IgE receptor-1 were sensitized with sera from adult dog-allergic subjects with positive skin prick tests to dog extract and detectable dog dander -IgE, and stimulated with serial dilutions of Optaflu and dog dander extract. N-hexosaminidase release% (NHR) was used as a marker of RBL degranulation. Western blots were performed and UniCAP was used to measure dog-specific IgE antibody levels. RESULTS: Median dog dander-IgE was 8.31 kUA/L (0.62; 67.9); median dog epithelium-IgE was 3.2 kUA/L, (0.4; 38.2). Median (range) maximum NHR (at 1st 10-fold dilution) was: 1). Optaflu- 0% (0; 1.4); 2). Dog dander-10.2% (0; 35.9), P < 0.001; and 3). Anti-IgE positive control-17.7% (1.2; 45.4). In a control egg-allergic subject, maximum NHR to a vaccine cultured in chick embryo and containing egg protein was 10.2%. IgE antibodies in pooled sera did not bind to Optaflu on immunoblots but produced strong binding to dog dander and epithelium extracts. Serum from a control egg-allergic subject strongly bound embryonated egg-derived vaccine. CONCLUSIONS: Optaflu, an influenza vaccine produced in continuous canine kidney cell line did not trigger degranulation in RBL cells passively sensitized with human anti-dog IgE.

Phase 2 clinical trial of three formulations of tetravalent live-attenuated dengue vaccine in flavivirus-naïve adults

Sixteen dose formulations of our live-attenuated tetravalent dengue virus vaccines (TDV) were previously evaluated for safety and immunogenicity. Two of the sixteen candidate TDV formulations (Formulations 13 and 14) were selected for further evaluation. A new TDV formulation, Formulation 17, using a higher primary dog kidney (PDK) cell passage Dengue-1 virus (DENV-1) and a lower PDK cell passage DENV-4, was developed to optimize the neutralizing antibody response. All three formulations consist of combinations of 10exp3-5 pfu/dose of the four dengue vaccine virus serotypes. This double-blind, randomized trial in 71 healthy adult subjects evaluated vaccine safety, reactogenicity and immunogenicity. TDV’s were given subcutaneously in the deltoid on Day 0 and 180 (6 months). Subjects were seen in clinic on Study Days 0, 10, 28, 180, 190 and 208 and filled out daily symptom diaries for 21 days after each vaccination. Formulation 13 was the most reactogenic, while both Formulations 14 and 17 were similar in reported reactions. Seventy-five percent, 31% and 31% of subjects were viremic on Day 10 after primary vaccination with Formulations 13, 14 and 17 respectively. Viremia was not detected in any subject following the second dose of vaccine. The immunogenicity endpoint was neutralizing antibody titer one month after the second vaccination. Thirty-six percent, 40% and 63% of vaccinated subjects developed tetravalent neutralizing antibodies after two doses of Formulations 13, 14 and 17, respectively. Formulation 17 was selected for further clinical evaluation based on this study.