RESUMEN
ß-propiolactone (BPL) is an alkylating agent used for inactivation of biological samples such as vaccines. Due to its known carcinogenic properties, complete hydrolysis of BPL is essential, and the detection of trace amounts is crucial. In this study a novel High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) method was developed. Rhodamine B hydrazide (RBH) was synthesized and utilized as a derivatizing reagent to react with BPL. The reaction was optimized in a weak acidic solution, resulting in a high yield. The separation of the RBH-derivatized BPL was achieved on a C8 column and detected by a UV detector at a wavelength of 560 nm. The method's validation demonstrated a high linearity (r2 > 0.99) over a concentration range of 0.5-50 µg/mL, with detection and quantification limits of 0.17 µg/mL and 0.5 µg/mL, respectively. The average recovery of samples was 85.20 % with a relative standard deviation (RSD) of 1.75 %. This method was successfully applied for BPL residue analysis in inactivated COVID-19 vaccines. This novel derivatization method offers a promising solution for monitoring BPL residues in the vaccine production process for quality control purposes and compliance with regulatory standards.
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Vacunas contra la COVID-19 , Límite de Detección , Propiolactona , Rodaminas , Cromatografía Líquida de Alta Presión/métodos , Propiolactona/química , Rodaminas/química , Reproducibilidad de los Resultados , Vacunas contra la COVID-19/química , Vacunas de Productos Inactivados/química , Vacunas de Productos Inactivados/análisis , Modelos Lineales , SARS-CoV-2/química , Humanos , Hidrazinas/química , Hidrazinas/análisisRESUMEN
Virus inactivation is a prerequisite for safe handling of high-risk infectious samples. ß-Propiolactone (BPL) is an established reagent with proven virucidal efficacy. BPL primarily reacts with DNA, RNA, and amino acids. The latter may modify antigenic protein epitopes interfering with binding properties of affinity reagents such as antibodies and aptamers used in affinity proteomic screens. We investigated (i) the impact of BPL treatment on the analysis of protein levels in plasma samples using the aptamer-based affinity proteomic platform SomaScan and (ii) effects on protein detection in conditioned medium samples using the proximity extension assay-based Olink Target platform. In the former setup, BPL-treated and native plasma samples from patients with ovarian cancer (n = 12) and benign diseases (n = 12) were analyzed using the SomaScan platform. In the latter, conditioned media samples collected from cultured T cells with (n = 3) or without (n = 3) anti-CD3 antibody stimulation were analyzed using the Olink Target platform. BPL-related changes in protein detection were evaluated comparing native and BPL-treated states, simulating virus inactivation, and impact on measurable group differences was assessed. While approximately one-third of SomaScan measurements were significantly changed by the BPL treatment, a majority of antigen/aptamer interactions remained unaffected. Interaction effects of BPL treatment and disease state, potentially altering detectability of group differences, were observable for less than one percent of targets (0.6%). BPL effects on protein detection with Olink Target were also limited, affecting 3.6% of detected proteins with no observable interaction effects. Thus, effects of BPL treatment only moderately interfere with affinity proteomic detectability of differential protein expression between different experimental groups. Overall, the results prove high-throughput affinity proteomics well suited for the analysis of high-risk samples inactivated using BPL.
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Propiolactona , Proteómica , Humanos , Propiolactona/farmacología , Propiolactona/metabolismo , Propiolactona/química , Femenino , Biomarcadores/sangre , Biomarcadores/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Inactivación de Virus/efectos de los fármacos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Aptámeros de Nucleótidos/farmacologíaRESUMEN
The global impact of emerging viral infections emphasizes the urgent need for effective broad-spectrum antivirals. The cellular organelle, lipid droplet (LD), is utilized by many types of viruses for replication, but its reduction does not affect cell survival. Therefore, LD is a potential target for developing broad-spectrum antivirals. In this study, we found that 2-bromopalmitate (2 BP), a previously defined palmitoylation inhibitor, depletes LD across all studied cell lines and exerts remarkable antiviral effects on different coronaviruses. We comprehensively utilized 2 BP, alongside other palmitoylation inhibitors such as cerulenin and 2-fluoro palmitic acid (2-FPA), as well as the enhancer palmostatin B and evaluated their impact on LD and the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. While cerulenin and 2-FPA exhibited moderate inhibition of viral replication, 2 BP exhibited a much stronger suppressive effect on MHV-A59 replication, although they share similar inhibitory effects on palmitoylation. As expected, palmostatin B significantly enhanced viral replication, it failed to rescue the inhibitory effects of 2 BP, whereas it effectively counteracted the effects of cerulenin and 2-FPA. This suggests that the mechanism that 2 BP used to inhibit viral replication is beyond palmitoylation inhibition. Further investigations unveil that 2 BP uniquely depletes LDs, a phenomenon not exhibited by 2-FPA and cerulenin. Importantly, the depletion of LDs was closely associated with the inhibition of viral replication because the addition of oleic acid to 2 BP significantly rescued LD depletion and its inhibitory effects on MHV-A59. Our findings indicate that the inhibitory effects of 2 BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Intriguingly, fatty acid (FA) assays demonstrated that 2 BP reduces the FA level in mitochondria while concurrently increasing FA levels in the cytoplasm. These results highlight the crucial role of LDs in viral replication and uncover a novel biological activity of 2 BP. These insights contribute to the development of broad-spectrum antiviral strategies. IMPORTANCE: In our study, we conducted a comparative investigation into the antiviral effects of palmitoylation inhibitors including 2-bromopalmitate (2-BP), 2-fluoro palmitic acid (2-FPA), and cerulenin. Surprisingly, we discovered that 2-BP has superior inhibitory effects on viral replication compared to 2-FPA and cerulenin. However, their inhibitory effects on palmitoylation were the same. Intrigued by this finding, we delved deeper into the underlying mechanism of 2-BP's potent antiviral activity, and we unveiled a novel biological activity of 2-BP: depletion of lipid droplets (LDs). Importantly, we also highlighted the crucial role of LDs in viral replication. Our insights shed new light on the antiviral mechanism of LD depletion paving the way for the development of broad-spectrum antiviral strategies by targeting LDs.
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Antivirales , Coronavirus , Virus de la Hepatitis Murina , Palmitatos , Animales , Humanos , Ratones , Antivirales/farmacología , Antivirales/metabolismo , Cerulenina/metabolismo , Cerulenina/farmacología , Coronavirus/efectos de los fármacos , Coronavirus/fisiología , Gotas Lipídicas/efectos de los fármacos , Palmitatos/farmacología , Ácido Palmítico/farmacología , Ácido Palmítico/metabolismo , Propiolactona/análogos & derivados , Replicación Viral/efectos de los fármacos , Virus de la Hepatitis Murina/efectos de los fármacos , Virus de la Hepatitis Murina/fisiologíaRESUMEN
BACKGROUND: Mpox virus (MPXV) infections have increased in many countries since May 2022, increasing demand for diagnostic tests and research on the virus. To ensure personnel safety, appropriate and reliable measures are needed to disinfect and inactivate infectious samples; Methods: We evaluated the stability of infectious MPXV cultures stored at different temperatures and through freeze-thaw cycles. Heat physical treatment (56 °C, 70 °C, 95 °C), chemical treatment (beta-propiolactone (BPL)) and two commercialized disinfectants (Micro-Chem Plus (MCP) and ethanol) were tested against infectious MPXV cultures; Results: The results indicated that MPXV stability increases with lower temperatures. The MPXV titer was stable within three freeze-thaw cycles and only decreased by 1.04 log10 (lg) 50% cell culture infective dose (CCID50) per milliliter (12.44%) after twelve cycles. MPXV could be effectively inactivated at 56 °C for 40 min, 70 °C for 10 min, and 95 °C for 5 min. For BPL inactivation, a 1:1000 volume ratio (BPL:virus) could also effectively inactivate MPXV. A total of 2% or 5% MCP and 75% ethanol treated with MPXV for at least 1 min could reduce >4.25 lg; Conclusions: MPXV shows high stability to temperature and freeze-thaw. Heat and BPL treatments are effective for the inactivation of MPXV, while MCP and ethanol are effective for disinfection, which could help laboratory staff operate the MPXV under safer conditions and improve operational protocols.
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Desinfectantes , Desinfección , Humanos , Monkeypox virus , Desinfectantes/farmacología , Técnicas de Cultivo de Célula , Etanol/farmacología , PropiolactonaRESUMEN
Traditional antiviral vaccines are currently created by inactivating the virus chemically, most often using formaldehyde or ß-propiolactone. These approaches are not optimal since they negatively affect the safety of the antigenic determinants of the inactivated particles and require additional purification stages. The most promising platforms for creating vaccines are based on pseudoviruses, i.e., viruses that have completely preserved the outer shell (capsid), while losing the ability to reproduce owing to the destruction of the genome. The irradiation of viruses with electron beam is the optimal way to create pseudoviral particles. In this review, with the example of the poliovirus, the main algorithms that can be applied to characterize pseudoviral particles functionally and structurally in the process of creating a vaccine preparation are presented. These algorithms are, namely, the analysis of the degree of genome destruction and coimmunogenicity. The structure of the poliovirus and methods of its inactivation are considered. Methods for assessing residual infectivity and immunogenicity are proposed for the functional characterization of pseudoviruses. Genome integrity analysis approaches, atomic force and electron microscopy, surface plasmon resonance, and bioelectrochemical methods are crucial to structural characterization of the pseudovirus particles.
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Poliomielitis , Poliovirus , Vacunas , Humanos , Poliovirus/genética , Formaldehído , Propiolactona , Poliomielitis/prevención & controlRESUMEN
Two air-stable organic radicals derived from oxalyl chloride and cAAC were synthesized, resulting in the unexpected formation of a known (amino)(carboxy) radical cation ([2]BF4) and the oxidative formation of a 1,2-dicarbonyl radical cation ([3]BF4) from a neutral 3-oxetanone compound (4). The highly strained and newly discovered 4 was obtained by a single-electron reduction of [3]BF4 with a mild reducing agent. This result differs from the generation of NHC-based 1,2-dicarbonyl radicals, indicating the uniqueness of cAAC.
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Electrones , Metano , PropiolactonaRESUMEN
ß-Propiolactone (BPL) is an organic compound widely used as an inactivating agent in vaccine development and production, for example for SARS-CoV, SARS-CoV-2 and Influenza viruses. Inactivation of pathogens by BPL is based on an irreversible alkylation of nucleic acids but also on acetylation and cross-linking between proteins, DNA or RNA. However, the protocols for BPL inactivation of viruses vary widely. Handling of infectious, enriched SARS-CoV-2 specimens and diagnostic samples from COVID-19 patients is recommended in biosafety level (BSL)- 3 or BSL-2 laboratories, respectively. We validated BPL inactivation of SARS-CoV-2 in saliva samples with the objective to use saliva from COVID-19 patients for training of scent dogs for the detection of SARS-CoV-2 positive individuals. Therefore, saliva samples and cell culture medium buffered with NaHCO3 (pH 8.3) were comparatively spiked with SARS-CoV-2 and inactivated with 0.1 % BPL for 1 h (h) or 71 h ( ± 1 h) at 2-8 °C, followed by hydrolysis of BPL at 37 °C for 1 or 2 h, converting BPL into non-toxic beta-hydroxy-propionic acid. SARS-CoV-2 inactivation was demonstrated by a titre reduction of up to 10^4 TCID50/ml in the spiked samples for both inactivation periods using virus titration and virus isolation, respectively. The validated method was confirmed by successful inactivation of pathogens in saliva samples from COVID-19 patients. Furthermore, we reviewed the currently available literature on SARS-CoV-2 inactivation by BPL. Accordingly, BPL-inactivated, hydrolysed samples can be handled in a non-laboratory setting. Furthermore, our BPL inactivation protocols can be adapted to validation experiments with other pathogens.
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COVID-19 , Virus , Perros , Animales , Propiolactona , Saliva , Odorantes , COVID-19/diagnóstico , Inactivación de Virus , SARS-CoV-2RESUMEN
Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by ß-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein's pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the ß-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while ß-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of ß-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Animales , Humanos , Vacunas de Productos Inactivados , COVID-19/prevención & control , Sueroterapia para COVID-19 , Vacunas contra la COVID-19 , Pandemias , Propiolactona/farmacología , SARS-CoV-2 , FormaldehídoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused huge social and economic distress. Given its rapid spread and the lack of specific treatment options, SARS-CoV-2 needs to be inactivated according to strict biosafety measures during laboratory diagnostics and vaccine development. The inactivation method for SARS-CoV-2 affects research related to the natural virus and its immune activity as an antigen in vaccines. In this study, we used size exclusion chromatography, western blotting, ELISA, an electron microscope, dynamic light scattering, circular dichroism, and surface plasmon resonance to evaluate the effects of four different chemical inactivation methods on the physical and biochemical characterization of SARS-CoV-2. Formaldehyde and ß-propiolactone (BPL) treatment can completely inactivate the virus and have no significant effects on the morphology of the virus. None of the four tested inactivation methods affected the secondary structure of the virus, including the α-helix, antiparallel ß-sheet, parallel ß-sheet, ß-turn, and random coil. However, formaldehyde and long-term BPL treatment (48 h) resulted in decreased viral S protein content and increased viral particle aggregation, respectively. The BPL treatment for 24 h can completely inactivate SARS-CoV-2 with the maximum retention of the morphology, physical properties, and the biochemical properties of the potential antigens of the virus. In summary, we have established a characterization system for the comprehensive evaluation of virus inactivation technology, which has important guiding significance for the development of vaccines against SARS-CoV-2 variants and research on natural SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Anticuerpos Antivirales , COVID-19/prevención & control , Vacunas contra la COVID-19 , Formaldehído , Humanos , Propiolactona/farmacología , Vacunas de Productos InactivadosRESUMEN
The severe COVID-19 pandemic drives the research toward the SARS-CoV-2 virion structure and the possible therapies against it. Here, we characterized the ß-propiolactone inactivated SARS-CoV-2 virions using transmission electron microscopy (TEM) and atomic force microscopy (AFM). We compared the SARS-CoV-2 samples purified by two consecutive chromatographic procedures (size exclusion chromatography [SEC], followed by ion-exchange chromatography [IEC]) with samples purified by ultracentrifugation. The samples prepared using SEC and IEC retained more spikes on the surface than the ones prepared using ultracentrifugation, as confirmed by TEM and AFM. TEM showed that the spike (S) proteins were in the pre-fusion conformation. Notably, the S proteins could be recognized by specific monoclonal antibodies. Analytical TEM showed that the inactivated virions retained nucleic acid. Altogether, we demonstrated that the inactivated SARS-CoV-2 virions retain the structural features of native viruses and provide a prospective vaccine candidate.
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COVID-19 , Propiolactona , Animales , Chlorocebus aethiops , Humanos , Pandemias , SARS-CoV-2 , Vacunas de Productos Inactivados , Células VeroRESUMEN
S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics.
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Lactonas/análisis , Propiolactona/análogos & derivados , Proteómica , Sulfonas/análisis , Proteínas ras/metabolismo , Humanos , Lactonas/metabolismo , Lactonas/farmacología , Estructura Molecular , Propiolactona/análisis , Propiolactona/metabolismo , Propiolactona/farmacología , Sulfonas/metabolismo , Sulfonas/farmacología , Proteínas ras/antagonistas & inhibidores , Proteínas ras/químicaRESUMEN
Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. ß-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly resulting in insufficient inactivation, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.
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Antivirales/farmacología , Vacunas contra la COVID-19/química , Propiolactona/farmacología , SARS-CoV-2/efectos de los fármacos , Virión/efectos de los fármacos , Inactivación de Virus/efectos de los fármacos , Animales , Antígenos Virales/química , Antígenos Virales/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Vacunas contra la COVID-19/inmunología , Chlorocebus aethiops , Floculación/efectos de los fármacos , Humanos , Sueros Inmunes/química , ARN Viral/química , ARN Viral/inmunología , SARS-CoV-2/química , SARS-CoV-2/inmunología , Vacunas de Productos Inactivados , Células Vero , Virión/química , Virión/inmunologíaRESUMEN
There are many approaches available to produce inactive bacteria by termination of growth, each with a different efficacy, impact on cell integrity, and potential for application in standardized inactivation protocols. The aim of this study was to compare these approaches and develop a standardized protocol for generation of inactivated Gram-positive and Gram-negative bacteria, yielding cells that are metabolically dead with retained cellular integrity i.e., preserving the surface and limited leakage of intracellular proteins and DNA. These inactivated bacteria are required for various applications, for instance, when investigating receptor-triggered signaling or bacterial contact-dependent analysis of cell lines requiring long incubation times. We inactivated eight different bacterial strains of different species by treatment with beta-propiolactone, ethanol, formalin, sodium hydroxide, and pasteurization. Inactivation efficacy was determined by culturing, and cell wall integrity assessed by quantifying released DNA, bacterial membrane and intracellular DNA staining, and visualization by scanning electron microscopy. Based on these results, we discuss the bacterial inactivation methods, and their advantages and disadvantages to study host-microbe interactions with inactivated bacteria.
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Desinfectantes/farmacología , Desinfección/métodos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/genética , Desinfección/instrumentación , Etanol/farmacología , Formaldehído/farmacología , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/genética , Bacterias Grampositivas/crecimiento & desarrollo , Calor , Viabilidad Microbiana/efectos de los fármacos , Propiolactona/farmacologíaRESUMEN
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 ß-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 (nâ¯=â¯137) and 57 samples of viral RNA positive asymptomatic contacts (nâ¯=â¯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 (Pâ¯=â¯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 (Pâ¯<â¯0.05). No significant difference was observed between the absorbance values of asymptomatic subjects and MD patients (Pâ¯=â¯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.
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Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , Inmunoglobulina G/sangre , Propiolactona/farmacología , SARS-CoV-2/inmunología , Anticuerpos Antivirales/sangre , COVID-19/sangre , COVID-19/patología , Ensayo de Inmunoadsorción Enzimática , Humanos , Sensibilidad y Especificidad , Estudios Seroepidemiológicos , Inactivación de Virus/efectos de los fármacosRESUMEN
The ongoing global pandemic of coronavirus disease 2019 (COVID-19) resulted from the outbreak of SARS-CoV-2 in December 2019. Currently, multiple efforts are being made to rapidly develop vaccines and treatments to fight COVID-19. Current vaccine candidates use inactivated SARS-CoV-2 viruses; therefore, it is important to understand the architecture of inactivated SARS-CoV-2. We have genetically and structurally characterized ß-propiolactone-inactivated viruses from a propagated and purified clinical strain of SARS-CoV-2. We observed that the virus particles are roughly spherical or moderately pleiomorphic. Although a small fraction of prefusion spikes are found, most spikes appear nail shaped, thus resembling a postfusion state, where the S1 protein of the spike has disassociated from S2. Cryoelectron tomography and subtomogram averaging of these spikes yielded a density map that closely matches the overall structure of the SARS-CoV postfusion spike and its corresponding glycosylation site. Our findings have major implications for SARS-CoV-2 vaccine design, especially those using inactivated viruses.
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Betacoronavirus/ultraestructura , Desinfectantes/farmacología , Propiolactona/farmacología , Virión/efectos de los fármacos , Animales , Betacoronavirus/efectos de los fármacos , Betacoronavirus/inmunología , Vacunas contra la COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Humanos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Vacunas de Productos Inactivados/inmunología , Células Vero , Vacunas Virales/inmunología , Virión/ultraestructuraRESUMEN
Importance: A vaccine against coronavirus disease 2019 (COVID-19) is urgently needed. Objective: To evaluate the safety and immunogenicity of an investigational inactivated whole-virus COVID-19 vaccine in China. Interventions: In the phase 1 trial, 96 participants were assigned to 1 of the 3 dose groups (2.5, 5, and 10 µg/dose) and an aluminum hydroxide (alum) adjuvant-only group (n = 24 in each group), and received 3 intramuscular injections at days 0, 28, and 56. In the phase 2 trial, 224 adults were randomized to 5 µg/dose in 2 schedule groups (injections on days 0 and 14 [n = 84] vs alum only [n = 28], and days 0 and 21 [n = 84] vs alum only [n = 28]). Design, Setting, and Participants: Interim analysis of ongoing randomized, double-blind, placebo-controlled, phase 1 and 2 clinical trials to assess an inactivated COVID-19 vaccine. The trials were conducted in Henan Province, China, among 96 (phase 1) and 224 (phase 2) healthy adults aged between 18 and 59 years. Study enrollment began on April 12, 2020. The interim analysis was conducted on June 16, 2020, and updated on July 27, 2020. Main Outcomes and Measures: The primary safety outcome was the combined adverse reactions 7 days after each injection, and the primary immunogenicity outcome was neutralizing antibody response 14 days after the whole-course vaccination, which was measured by a 50% plaque reduction neutralization test against live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Results: Among 320 patients who were randomized (mean age, 42.8 years; 200 women [62.5%]), all completed the trial up to 28 days after the whole-course vaccination. The 7-day adverse reactions occurred in 3 (12.5%), 5 (20.8%), 4 (16.7%), and 6 (25.0%) patients in the alum only, low-dose, medium-dose, and high-dose groups, respectively, in the phase 1 trial; and in 5 (6.0%) and 4 (14.3%) patients who received injections on days 0 and 14 for vaccine and alum only, and 16 (19.0%) and 5 (17.9%) patients who received injections on days 0 and 21 for vaccine and alum only, respectively, in the phase 2 trial. The most common adverse reaction was injection site pain, followed by fever, which were mild and self-limiting; no serious adverse reactions were noted. The geometric mean titers of neutralizing antibodies in the low-, medium-, and high-dose groups at day 14 after 3 injections were 316 (95% CI, 218-457), 206 (95% CI, 123-343), and 297 (95% CI, 208-424), respectively, in the phase 1 trial, and were 121 (95% CI, 95-154) and 247 (95% CI, 176-345) at day 14 after 2 injections in participants receiving vaccine on days 0 and 14 and on days 0 and 21, respectively, in the phase 2 trial. There were no detectable antibody responses in all alum-only groups. Conclusions and Relevance: In this interim report of the phase 1 and phase 2 trials of an inactivated COVID-19 vaccine, patients had a low rate of adverse reactions and demonstrated immunogenicity; the study is ongoing. Efficacy and longer-term adverse event assessment will require phase 3 trials. Trial Registration: Chinese Clinical Trial Registry Identifier: ChiCTR2000031809.
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Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Inmunogenicidad Vacunal , Pandemias/prevención & control , Neumonía Viral/prevención & control , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/efectos adversos , Adolescente , Adulto , Hidróxido de Aluminio/administración & dosificación , Hidróxido de Aluminio/efectos adversos , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Betacoronavirus/genética , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/inmunología , Relación Dosis-Respuesta Inmunológica , Método Doble Ciego , Femenino , Humanos , Inyecciones Intramusculares , Masculino , Neumonía Viral/inmunología , Propiolactona , SARS-CoV-2 , Vacunas de Productos Inactivados/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/efectos adversos , Adulto JovenRESUMEN
In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.
Asunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Ensayo de Placa Viral/métodos , Inactivación de Virus , Animales , Betacoronavirus/efectos de los fármacos , Betacoronavirus/crecimiento & desarrollo , Betacoronavirus/patogenicidad , COVID-19 , Celulosa , Chlorocebus aethiops , Medios de Cultivo/química , Formaldehído , Guanidinas/farmacología , Células HEK293 , Humanos , Pandemias , Fenoles/farmacología , Propiolactona/farmacología , SARS-CoV-2 , Sefarosa , Células VeroRESUMEN
The discovery that ß-propiolactone (BPL), once a commercially important chemical, causes various tumors in experimental animals has led to a significant decrease in its use. However, owing to its efficacy this possible human carcinogen remains to be utilized in vaccines for inactivation of viruses. The focus of the current study was to uncover the mechanisms of ß-propiolactone reactions with both nucleobases and glutathione (GSH) through computer simulations based on quantum chemical methods. Our results, in accordance with in vitro studies, show that among all nucleobases guanine most readily forms adducts with BPL through SN2 reaction mechanism. Acquired activation energies with incorporated solvent effects reveal that alkylation represents an energetically more favorable reaction than acylation for all nucleobases. Comparison of activation free energies of glutathione and guanine reactions with BPL suggest that glutathione may represent an efficient natural scavenger of BPL. Therefore, glutathione present in the organism may provide protection to the DNA and thus prevent BPL's genotoxicity, mutagenicity, and possibly even carcinogenicity.
Asunto(s)
Carcinogénesis/inducido químicamente , Propiolactona/química , Propiolactona/toxicidad , Teoría Funcional de la Densidad , Humanos , Modelos Moleculares , Estructura Molecular , TermodinámicaRESUMEN
[6]-Gingerol from ginger has received considerable attention as a potential cancer therapeutic agent because of its chemopreventive and chemotherapeutic effects, as well as its safety. In the current study, we examined [6]-gingerol as a natural scavenger of nine ultimate chemical carcinogens to which we are frequently exposed: glycidamide, styrene oxide, aflatoxin B1 exo-8,9-epoxide, ß-propiolactone, ethylene oxide, propylene oxide, 2-cyanoethylene oxide, chloroethylene oxide, and vinyl carbamate epoxide. To evaluate [6]-gingerol efficacy, we expanded our research with the examination of glutathione-the strongest natural scavenger in human cells. The corresponding activation free energies were calculated using Hartree-Fock method with three flexible basis sets and two implicit solvation models. According to our results, [6]-gingerol proves to be an extremely effective scavenger of chemical carcinogens of the epoxy type. On the other hand, with the exception of aflatoxin B1 exo-8,9-epoxide, glutathione represents a relatively poor scavenger, whose efficacy could be augmented by [6]-gingerol. Moreover, our quantum mechanical study of the alkylation reactions of chemical carcinogens with [6]-gingerol and glutathione provide valuable insights in the reaction mechanisms and the geometries of the corresponding transition states. Therefore, we strongly believe that our research forms a solid basis for further computational, experimental and clinical studies of anticarcinogenic properties of [6]-gingerol as well as for the development of novel chemoprophylactic dietary supplements. Finally, the obtained results also point to the applicability of quantum chemical methods to studies of alkylation reactions related to chemical carcinogenesis.
Asunto(s)
Anticarcinógenos/química , Anticarcinógenos/farmacología , Carcinógenos/química , Carcinógenos/farmacología , Catecoles/química , Catecoles/farmacología , Alcoholes Grasos/química , Alcoholes Grasos/farmacología , Aflatoxina B1 , Alquilación , Línea Celular , Quimioprevención , Compuestos Epoxi/farmacología , Óxido de Etileno/análogos & derivados , Zingiber officinale/química , Humanos , Propiolactona , Uretano/análogos & derivadosRESUMEN
As one of the most important fish in freshwater aquaculture, gibel carp (Carassius auratus gibelio) is easily susceptible to Cyprinid herpesvirus 2 (CyHV-2). Immersion vaccination has attracted many researchers due to its simple operation in preventing infectious diseases. However, the unavoidable disadvantage is that the immersion vaccine must be used with adjuvants to get a better performance. In this study, gibel carps were vaccinated by a 60 min bath in a ß-propiolactone-inactivated Cyprinid herpesvirus 2, mixed with DTT, ß-glucan, anisodamine and scopolamine, respectively. After immunization, the fishs were challenged by CyHV-2 in 2 weeks. By analyzing pathological section, we found that ß-glucan, anisodamine and scopolamine groups protected the gibel carp compared to the control group, which was consistent with the trend of survival rate. Specifically, ß-glucan group in serum appeared best on lysozyme, TSOD and complement C3. Real time quantitative RT-PCR results demonstrated that in both spleen and head kidney tissues, mRNA expressions of typical Th1 immune response cytokines IL-2 and IFN-γ2 in ß-glucan group and anisodamine group were significantly higher than other groups and the level of immunoglobulins related to systemic immunity (IgM) and mucosal immunity (IgZ) were also enhanced in the immune period. DTT group slightly affected immune gene and serum enzyme activity, while did not show an adjuvant effect on survival rate. In addition, four adjuvant groups could obviously inhibit CyHV-2 replication. This study explored and proved the good efficiency of ß-glucan or anisodamine as immersion immune adjuvant and also provided reference for improving the efficiency of immersion immunity.