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Journal of Virological Methods Jun 2019Residual host cell DNA (rcDNA) from continuous cell lines used for manufacturing of biological medicinal products has been considered as safety risk. Historically,... (Comparative Study)
Comparative Study
Residual host cell DNA (rcDNA) from continuous cell lines used for manufacturing of biological medicinal products has been considered as safety risk. Historically, several analytical methods have been used for rcDNA quantitation including hybridization assay, Threshold assay and quantitative polymerase chain reaction (qPCR). Sanofi Pasteur has a wealth of experience in the development of methods quantifying rcDNA in vaccines. Here, we compared the performance of our in-house assays for quantifying rcDNA in viral vaccines produced in Vero cells. Vero alpha-satellite sequence qPCR was compared with the hybridization and Threshold assays in terms of specificity, sensitivity and precision. The impact of viral inactivation with β-propiolactone (BPL) on rcDNA, within the vaccine production process, was also assessed. We demonstrate that the quantity of rcDNA measured is influenced by the analytical method used. Vero cell DNA-specific qPCR assay was shown to be robust with a large dynamic range and no matrix interference on a range of products. The qPCR assay demonstrated greater sensitivity and specificity versus the hybridization and Threshold methods. Vero alpha-satellite sequence qPCR is a specific and sensitive method for the assessment of the quantity of Vero rcDNA in the highly purified vaccines.
Topics: Animals; Chlorocebus aethiops; DNA; DNA Contamination; Host Microbial Interactions; Humans; Vero Cells; Viral Vaccines; Virology
PubMed: 30611776
DOI: 10.1016/j.jviromet.2019.01.001 -
Journal of Virological Methods Jan 2021Coronavirus disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome - coronavirus-2 (SARS-CoV-2) continues to affect many countries...
BACKGROUND
Coronavirus disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome - coronavirus-2 (SARS-CoV-2) continues to affect many countries and large populations. Serologic assays for antibody detection aid patient diagnosis and seroepidemiologic investigations.
METHODS
An indirect IgG ELISA was developed indigenously using β-propiolactone (BPL) inactivated SARS-CoV-2. This assay was used for screening 200 healthy donor sera collected prior to COVID-19 emergence (2017-2019), 185 serum/plasma samples of confirmed COVID-19 patients (n = 137) and 57 samples of viral RNA positive asymptomatic contacts (n = 51). The IgG response was studied in relation to duration and severity of illness.
RESULTS
The ELISA demonstrated 97 % specificity and IgG detection in >50 %, 80 %, 93.8 % and 100 % of the patients respectively during the first, second, third and fourth week of illness. IgG detection rate was higher in patients with severe disease (SD, 90.9 %) than those with mild disease (MD, 68.8 %) during the second week of illness (P = 0.027). IgG seropositivity among asymptomatic contacts was 64.7 %. IgG ELISA absorbance values were higher in SD than MD patients during the first 2 weeks of illness (P < 0.05). No significant difference was observed between the absorbance values of asymptomatic subjects and MD patients (P = 0.94).
CONCLUSION
The BPL inactivated virus-based ELISA could detect IgG antibodies early and in a significant proportion of COVID-19 patients suggesting its potential utility as a supplement to the currently used viral RNA detection tests in patient diagnosis and contact screening algorithms.
Topics: Antibodies, Viral; COVID-19; COVID-19 Serological Testing; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin G; Propiolactone; SARS-CoV-2; Sensitivity and Specificity; Seroepidemiologic Studies; Virus Inactivation
PubMed: 33126149
DOI: 10.1016/j.jviromet.2020.113996 -
Journal of Virological Methods Sep 2015In view of the emerging avian influenza (AI) viruses, it is important to study the susceptibility of AI viruses to inactivating agents for preparation of antigens and...
In view of the emerging avian influenza (AI) viruses, it is important to study the susceptibility of AI viruses to inactivating agents for preparation of antigens and inactivated vaccines. The available information on susceptibility of both the high and low pathogenic AI viruses to different inactivating agents is inadequate and ambiguous. It has been shown that different subtypes of influenza viruses require different physical and chemical conditions for inactivation of infectivity. The present study was undertaken to evaluate the use of beta-propiolactone (BPL), formalin and ether for inactivation and its impact on antigenicity of AI viruses. A total of nine high and low pathogenic AI viruses belonging to four influenza A subtypes were included in the study. The H5N1 viruses were from the clades 2.2, 2.3.2.1 and 2.3.4. The H9N2 virus included in the study was of the G1 genotype, while the H11N1 and H4N6 viruses were from the Eurasian lineage. The viruses were treated with BPL, formalin and with ether. The confirmation of virus inactivation was performed by two serial passages of inactivated viruses in embryonated chicken eggs. The infectivity of all tested AI viruses was eliminated using 0.1% BPL and 0.1% formalin. Ether eliminated infectivity of all tested low pathogenic AI viruses; however, ether with 0.2% or 0.5% Tween-20 was required for inactivation of the highly pathogenic AI H5N1 viruses. Treatment with BPL, ether and formalin retained virus hemagglutination (HA) titers. Interestingly ether treatment resulted in significant rise in HA titers (P<0.05) of all tested AI viruses. This data demonstrated the utility of BPL, formalin and ether for the inactivation of infectivity of AI viruses used in the study for the preparation of inactivated virus antigens for research and diagnosis of AI.
Topics: Animals; Antigens; Chick Embryo; Chickens; Disinfectants; Disinfection; Ether; Formaldehyde; Hemagglutination Inhibition Tests; Hemagglutinin Glycoproteins, Influenza Virus; Influenza A virus; Influenza in Birds; Microbial Viability; Propiolactone; Virus Inactivation
PubMed: 25997377
DOI: 10.1016/j.jviromet.2015.05.004 -
Vaccine Mar 2019The vast majority of commercially available inactivated influenza vaccines are produced from egg-grown or cell-grown live influenza virus. The first step in the...
The vast majority of commercially available inactivated influenza vaccines are produced from egg-grown or cell-grown live influenza virus. The first step in the production process is virus inactivation with β-propiolactone (BPL) or formaldehyde (FA). Recommendations for production of inactivated vaccines merely define the maximal concentration for both reagents, leaving the optimization of the process to the manufacturers. We assessed the effect of inactivation with BPL and FA on 5 different influenza virus strains. The properties of the viral formulation, such as successful inactivation, preservation of hemagglutinin (HA) binding ability, fusion capacity and the potential to stimulate a Toll-like receptor 7 (TLR7) reporter cell line were then assessed and compared to the properties of the untreated virus. Inactivation with BPL resulted in undetectable infectivity levels, while FA-treated virus retained very low infectious titers. Hemagglutination and fusion ability were highly affected by those treatments that conferred higher inactivation, with BPL-treated virus binding and fusing at a lower degree compared to FA-inactivated samples. On the other hand, BPL-inactivated virus induced higher levels of activation of TLR7 than FA-inactivated virus. The alterations caused by BPL or FA treatments were virus strain dependent. This data shows that the inactivation procedures should be tailored on the virus strain, and that many other elements beside the concentration of the inactivating agent, such as incubation time and temperature, buffer and virus concentration, have to be defined to achieve a functional product.
Topics: Animals; Cell Line; Formaldehyde; Hemagglutinin Glycoproteins, Influenza Virus; Humans; Influenza A virus; Influenza Vaccines; Vaccines, Inactivated; Virion; Virus Inactivation
PubMed: 30765167
DOI: 10.1016/j.vaccine.2019.01.086 -
Biochemistry and Biophysics Reports Dec 2015Differential Scanning Calorimetry (DSC) has been used in the past to study the thermal unfolding of many different viruses. Here we present the first DSC analysis of...
Differential Scanning Calorimetry (DSC) has been used in the past to study the thermal unfolding of many different viruses. Here we present the first DSC analysis of rabies virus. We show that non-inactivated, purified rabies virus unfolds cooperatively in two events centered at approximately 62 and 73 °C. Beta-propiolactone (BPL) treatment does not alter significantly viral unfolding behavior, indicating that viral inactivation does not alter protein structure significantly. The first unfolding event was absent in bromelain treated samples, causing an elimination of the G-protein ectodomain, suggesting that this event corresponds to G-protein unfolding. This hypothesis was confirmed by the observation that this first event was shifted to higher temperatures in the presence of three monoclonal, G-protein specific antibodies. We show that dithiothreitol treatment of the virus abolishes the first unfolding event, indicating that the reduction of G-protein disulfide bonds causes dramatic alterations to protein structure. Inactivated virus samples heated up to 70 °C also showed abolished recognition of conformational G-protein specific antibodies by Surface Plasmon Resonance analysis. The sharpness of unfolding transitions and the low standard deviations of the Tm values as derived from multiple analysis offers the possibility of using this analytical tool for efficient monitoring of the vaccine production process and lot to lot consistency.
PubMed: 29124221
DOI: 10.1016/j.bbrep.2015.10.010 -
Veterinary Microbiology Apr 2023Duck tembusu virus (DTMUV), belonging to the Flavivirus genus, Flaviviridae family, has caused huge economic losses in the duck industry. However, the inactivated DTMUV...
Duck tembusu virus (DTMUV), belonging to the Flavivirus genus, Flaviviridae family, has caused huge economic losses in the duck industry. However, the inactivated DTMUV vaccine requires multiple immunizations and has incomplete effectiveness. The humoral immune response is a key factor in the control of DTMUV infection. IL-7 derived from mammals has the ability to enhance antibody production. Whether duck IL-7 (duIL-7) possesses the ability to improve the humoral immunity of inactivated DTMUV vaccine has not yet been declared. Here, a beta-propiolactone (BPL)-inactivated DTMUV vaccine was employed to characterize the adjuvant property of duIL-7 in humoral immune responses. Intramuscular injection of DTMUV inactivated vaccine with or without duIL-7 was administered twice to the ducks. The results showed that duIL-7 was able to promote rapid antibody responses and enhance DTMUV-specific IgG and neutralizing antibody production to the vaccine. T follicular helper (Tfh) cells play a key role in assisting long humoral immunity. It was found that duIL-7 upregulated duIl-6 and duIl-21 gene expression at 3 w post first vaccination, which encode Tfh cell differentiation-related cytokines duIL-6 and duIL-21, respectively. This may be the reason that duIL-7 could prolong the humoral immune response to the inactivated DTMUV vaccine. Next, the ability of duIL-7 to simplify the immunization procedure of the inactivated DTMUV vaccine was tested. When ducks were immunized once, the titers of neutralizing antibodies in ducks from the inactivated DTMUV vaccine supplemented with duIL-7 group were significantly higher than those of ducks from the inactivated DTMUV vaccine group (P < 0.05). In addition, duIL-7 could assist the inactivated DTMUV vaccine in maintaining neutralizing antibodies at high levels during the whole experimental period. The viral titers in the ducks immunized with the inactivated DTMUV vaccine and duIL-7 were lower than those in the ducks immunized with the inactivated DTMUV vaccine alone at 3 days post infection (3 dpi, P < 0.05). Overall, duIL-7 possessed the ability to promote and prolong humoral immune responses to the inactivated DTMUV vaccine, even at one dose. This study provides a new efficient adjuvant for inactivated DTMUV vaccine development.
Topics: Animals; Ducks; Immunity, Humoral; Flavivirus Infections; Vaccines, Inactivated; Interleukin-7; Flavivirus; Antibodies, Neutralizing; Adjuvants, Immunologic; Poultry Diseases; Mammals
PubMed: 36716633
DOI: 10.1016/j.vetmic.2023.109665 -
Pathogens (Basel, Switzerland) May 2021The development of a safe and effective vaccine to protect against COVID-19 is a global priority due to the current high SARS-CoV-2 infection rate. Currently, there are...
The development of a safe and effective vaccine to protect against COVID-19 is a global priority due to the current high SARS-CoV-2 infection rate. Currently, there are over 160 SARS-CoV-2 vaccine candidates at the clinical or pre-clinical stages of development. Of these, there are only three whole-virus vaccine candidates produced using β-propiolactone or formalin inactivation. Here, we prepared a whole-virus SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) using a novel psoralen inactivation method and evaluated its immunogenicity in mice using two different adjuvants, alum and Advax-2. We compared the immunogenicity of SARS-CoV-2 PsIV against SARS-CoV-2 DNA vaccines expressing either full-length or truncated spike proteins. We also compared the psoralen-inactivated vaccine against a DNA prime, psoralen-inactivated vaccine boost regimen. After two doses, the psoralen-inactivated vaccine, when administered with alum or Advax-2 adjuvants, generated a dose-dependent neutralizing antibody responses in mice. Overall, the pattern of cytokine ELISPOT responses to antigen-stimulation observed in this study indicates that SARS-CoV-2 PsIV with the alum adjuvant promotes a Th2-type response, while SARS-CoV-2 PsIV with the Advax-2 adjuvant promotes a Th1-type response.
PubMed: 34069575
DOI: 10.3390/pathogens10050626 -
Oncotarget Feb 2016Oncogenic NRAS mutations are frequent in melanoma and lead to increased downstream signaling and uncontrolled cell proliferation. Since the direct inhibition of NRAS is...
Oncogenic NRAS mutations are frequent in melanoma and lead to increased downstream signaling and uncontrolled cell proliferation. Since the direct inhibition of NRAS is not possible yet, modulators of NRAS posttranslational modifications have become an area of interest. Specifically, interfering with NRAS posttranslational palmitoylation/depalmitoylation cycle could disturb proper NRAS localization, and therefore decrease cell proliferation and downstream signaling. Here, we investigate the expression and function of NRAS depalmitoylating acyl protein thioesterases 1 and 2 (APT-1, APT-2) in a panel of NRAS mutant melanoma cells. First, we show that all melanoma cell lines examined express APT-1 and APT-2. Next, we show that siRNA mediated APT-1 and APT-2 knock down and that the specific APT-1 and -2 inhibitors ML348 and ML349 have no biologically significant effects in NRAS mutant melanoma cells. Finally, we test the dual APT-1 and APT-2 inhibitor palmostatin B and conclude that palmostatin B has effects on NRAS downstream signaling and cell viability in NRAS mutant melanoma cells, offering an interesting starting point for future studies.
Topics: Apoptosis; Blotting, Western; Cell Proliferation; Enzyme Inhibitors; GTP Phosphohydrolases; Humans; Melanoma; Membrane Proteins; Molecular Targeted Therapy; Mutation; Propiolactone; RNA, Small Interfering; Thiolester Hydrolases; Tumor Cells, Cultured
PubMed: 26771141
DOI: 10.18632/oncotarget.6907 -
Vaccines Mar 2023Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole...
Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 μg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.
PubMed: 36992191
DOI: 10.3390/vaccines11030607 -
BMC Research Notes Jul 2018Infection of chickens with low pathogenic avian influenza virus, such as H9N2 virus, culminates in decreased egg production and increased mortality and morbidity if...
OBJECTIVE
Infection of chickens with low pathogenic avian influenza virus, such as H9N2 virus, culminates in decreased egg production and increased mortality and morbidity if co-infection with other respiratory pathogens occurs. We have previously observed the induction of antibody- and cell-mediated immune responses after intramuscular administration of an H9N2 beta-propiolactone inactivated virus vaccine to chickens. Given the fact that in ovo vaccination represents a practical option for vaccination against H9N2 AIV in chickens, in the current study, we set out to characterize immune responses in chickens against a beta-propiolactone inactivated H9N2 virus vaccine after primary vaccination in ovo on embryonic day 18, and secondary intramuscular vaccination on day 14 post-hatch. We also included the Toll-like receptor 21 ligand, CpG ODN 2007, and an oil emulsion adjuvant, AddaVax™, as adjuvants for the vaccines.
RESULTS
Antibody-mediated immune responses were observed after administering the secondary intramuscular vaccine. Cell-mediated immune responses were observed in chickens that received the beta-propiolactone inactivated H9N2 virus combined with AddaVax™. Our results demonstrate that adaptive immune responses can be induced in chickens after a primary in ovo vaccination and secondary intramuscular vaccination.
Topics: Animals; Antibodies, Viral; Antibody Formation; Chickens; Influenza A Virus, H9N2 Subtype; Influenza Vaccines; Influenza in Birds
PubMed: 29970157
DOI: 10.1186/s13104-018-3537-9