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1.
Virus Res ; 305: 198555, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34487766

RESUMO

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.


Assuntos
Antivirais/farmacologia , Vacinas contra COVID-19/química , Propiolactona/farmacologia , SARS-CoV-2/efeitos dos fármacos , Vírion/efeitos dos fármacos , Inativação de Vírus/efeitos dos fármacos , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Chlorocebus aethiops , Floculação/efeitos dos fármacos , Humanos , Soros Imunes/química , RNA Viral/química , RNA Viral/imunologia , SARS-CoV-2/química , SARS-CoV-2/imunologia , Vacinas de Produtos Inativados , Células Vero , Vírion/química , Vírion/imunologia
2.
J Microbiol Methods ; 184: 106208, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33766606

RESUMO

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.


Assuntos
Desinfetantes/farmacologia , Desinfecção/métodos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Desinfecção/instrumentação , Etanol/farmacologia , Formaldeído/farmacologia , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/crescimento & desenvolvimento , Temperatura Alta , Viabilidade Microbiana/efeitos dos fármacos , Propiolactona/farmacologia
3.
J Virol Methods ; 287: 113996, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33126149

RESUMO

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 (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.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Imunoglobulina G/sangue , Propiolactona/farmacologia , SARS-CoV-2/imunologia , Anticorpos Antivirais/sangue , COVID-19/sangue , COVID-19/patologia , Ensaio de Imunoadsorção Enzimática , Humanos , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Inativação de Vírus/efeitos dos fármacos
4.
Structure ; 28(11): 1218-1224.e4, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33058760

RESUMO

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.


Assuntos
Betacoronavirus/ultraestrutura , Desinfetantes/farmacologia , Propiolactona/farmacologia , Vírion/efeitos dos fármacos , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Vacinas contra COVID-19 , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Humanos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Vacinas de Produtos Inativados/imunologia , Células Vero , Vacinas Virais/imunologia , Vírion/ultraestrutura
5.
JAMA ; 324(10): 951-960, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32789505

RESUMO

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.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Imunogenicidade da Vacina , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Adolescente , Adulto , Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/efeitos adversos , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/genética , COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/imunologia , Relação Dose-Resposta Imunológica , Método Duplo-Cego , Feminino , Humanos , Injeções Intramusculares , Masculino , Pneumonia Viral/imunologia , Propiolactona , SARS-CoV-2 , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversos , Adulto Jovem
6.
Viruses ; 12(6)2020 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-32517266

RESUMO

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.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Ensaio de Placa Viral/métodos , Inativação de Vírus , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , Betacoronavirus/patogenicidade , COVID-19 , Celulose , Chlorocebus aethiops , Meios de Cultura/química , Formaldeído , Guanidinas/farmacologia , Células HEK293 , Humanos , Pandemias , Fenóis/farmacologia , Propiolactona/farmacologia , SARS-CoV-2 , Sefarose , Células Vero
7.
Chem Res Toxicol ; 33(3): 769-781, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32056425

RESUMO

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.


Assuntos
Carcinogênese/induzido quimicamente , Propiolactona/química , Propiolactona/toxicidade , Teoria da Densidade Funcional , Humanos , Modelos Moleculares , Estrutura Molecular , Termodinâmica
8.
Fish Shellfish Immunol ; 98: 285-295, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31962149

RESUMO

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.


Assuntos
Doenças dos Peixes/prevenção & controle , Carpa Dourada , Infecções por Herpesviridae/veterinária , Herpesviridae/imunologia , Imunização/veterinária , Alcaloides de Solanáceas/imunologia , Vacinas Virais/imunologia , beta-Glucanas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Aquicultura , Doenças dos Peixes/virologia , Carpa Dourada/imunologia , Carpa Dourada/virologia , Herpesviridae/fisiologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Imunidade Inata , Imunidade nas Mucosas , Imunização/métodos , Propiolactona , Escopolamina/administração & dosagem , Escopolamina/imunologia , Alcaloides de Solanáceas/administração & dosagem , Taxa de Sobrevida , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/administração & dosagem , Replicação Viral , beta-Glucanas/administração & dosagem
9.
Int J Mol Sci ; 21(3)2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31973096

RESUMO

[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.


Assuntos
Anticarcinógenos/química , Anticarcinógenos/farmacologia , Carcinógenos/química , Carcinógenos/farmacologia , Catecóis/química , Catecóis/farmacologia , Álcoois Graxos/química , Álcoois Graxos/farmacologia , Aflatoxina B1 , Alquilação , Linhagem Celular , Quimioprevenção , Compostos de Epóxi/farmacologia , Óxido de Etileno/análogos & derivados , Gengibre/química , Humanos , Propiolactona , Uretana/análogos & derivados
10.
Biomed Res Int ; 2019: 4518163, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31008105

RESUMO

Inactivation of rabies virus is essential for rabies vaccine preparation where the inactivating compound that is currently recommended for rabies vaccine preparation is ß-propiolactone (ß-PL). This compound is considered better than phenol and formalin but it is expensive and potentially carcinogenic. Data revealed that Ascorbic acid (AA) with cupric ions could yield complete and irreversible inactivation of rabies virus without adversely affecting its antigenicity. Additionally, the results of testing the vaccine potency with the selected inactivating compounds were comparable (P<0.05), and ED50 was higher than the recommended World Health Organization (WHO) limits. The use of HemaGel (plasma substitute) for testing vaccine stabilization was compared with the currently used vaccine stabilizers (human albumin and lactose). HemaGel yielded better stability than the other tested stabilizers. Monitoring of cellular and humoral immune responses indicated that both the total IgG level against rabies vaccine and the IFN and IL5 levels obtained with the HemaGel-stabilized vaccines were higher than those obtained with human albumin- and lactose-stabilized vaccine candidates.


Assuntos
Imunogenicidade da Vacina/efeitos dos fármacos , Propiolactona/farmacologia , Vacinas Antirrábicas/farmacologia , Raiva/prevenção & controle , Albuminas/farmacologia , Animais , Anticorpos Antivirais/efeitos dos fármacos , Anticorpos Antivirais/imunologia , Ácido Ascórbico/farmacologia , Chlorocebus aethiops , Humanos , Imunoglobulina G/imunologia , Interferons/imunologia , Interleucina-5 , Lactose/química , Propiolactona/química , Raiva/imunologia , Raiva/virologia , Vacinas Antirrábicas/química , Vacinas Antirrábicas/genética , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Vírus da Raiva/patogenicidade , Potência de Vacina , Células Vero/virologia
11.
Vet Microbiol ; 226: 23-30, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30389040

RESUMO

In this study, we developed and evaluated the beta-propiolactone inactivated bivalent bluetongue virus (BTV) serotypes 4 and 16 vaccine delivered with Montanide™ ISA-71VG adjuvant. The safety, stability and immunological profile of the fresh and after three years of long-term storage of the vaccine formulation was analyzed. We observed after long-term storage that the vaccine emulsion was stable as indicated by unchanged pH and viscosity. The stored vaccine formulation induced virus neutralizing antibodies (VNA) in sheep against both the bluetongue virus serotypes at 7-10 day post-vaccination (dpv). VNA titers reached the peak by 60 dpv and detectable during the entire study period. Antibodies against bluetongue virus structural protein VP7 were detected by ELISA in all BTV vaccinated experimental animal groups. Partial clinical protection was observed in vaccinates against challenge virulent BTV-4 and BTV-16 serotypes by 10 dpv, while complete protection was observed at 14 dpv. The levels of viremia was decreased in challenged sheep by 10 dpv while the viremia was undetectable by 14 dpv. In summary, our newly formulated bivalent BTV (BTV-4 and BTV-16) vaccine delivered with Montanide™ ISA-71VG adjuvant was found safe and stable for over three years and induced protective response in sheep.


Assuntos
Anticorpos Antivirais/imunologia , Vírus Bluetongue/efeitos dos fármacos , Vírus Bluetongue/imunologia , Bluetongue/prevenção & controle , Propiolactona/farmacologia , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/administração & dosagem , Bluetongue/virologia , Armazenamento de Medicamentos , Sorogrupo , Ovinos/imunologia , Fatores de Tempo , Potência de Vacina , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Carga Viral , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversos , Viremia
12.
Vaccine ; 36(27): 3908-3916, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29853199

RESUMO

Several types of avian influenza virus (AIV) vaccines exist, including live-attenuated, vectored, and whole inactivated virus (WIV) vaccines. Inactivated vaccines offer some advantages compared to other types of vaccines, including ease of production and lack of ability to revert to a virulent state. However, WIV are poorly immunogenic, especially when these vaccines are delivered to mucosal surfaces. There are several factors that contribute to the immunogenicity of vaccines, one of which is the method used to inactivate viruses. Several methods exist for producing influenza WIVs, including formaldehyde, a chemical that affects protein structures leading to virus inactivation. Other methods include treatment with beta-propiolactone (BPL) and the application of gamma radiation, both of which have less effects on protein structures compared to formaldehyde, and instead alter nucleic acids in the virion. Here, we sought to determine the effect of the above inactivation methods on immunogenicity of AIV vaccines. To this end, chickens were vaccinated with three different H9N2 WIVs using formaldehyde, BPL, and gamma radiation for inactivation. In addition to administering these three WIVs alone as vaccines, we also included CpG ODN 2007, a synthetic ligand recognized by Toll-like receptor (TLR)21 in chickens, as an adjuvant for each WIV. Subsequently, antibody- and cell-mediated immune responses were measured following vaccination. Antibody-mediated immune responses were increased in chickens that received the BPL and Gamma WIVs compared to the formaldehyde WIV. CpG ODN 2007 was found to significantly increase antibody responses for each WIV compared to WIV alone. Furthermore, we observed the presence of cell-mediated immune responses in chickens that received the BPL WIV combined with CpG ODN 2007. Based on these results, the BPL WIV + CpG ODN 2007 combination was the most effective vaccine at inducing adaptive immune responses against H9N2 AIV. Future studies should characterize mucosal adaptive immune responses to these vaccines.


Assuntos
Imunidade Celular/imunologia , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H9N2/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Doenças das Aves Domésticas/prevenção & controle , Inativação de Vírus , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Galinhas , Formaldeído , Raios gama , Vacinas contra Influenza/administração & dosagem , Influenza Aviária/imunologia , Influenza Aviária/terapia , Oligodesoxirribonucleotídeos/administração & dosagem , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/terapia , Propiolactona , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia
13.
Vaccine ; 36(29): 4346-4353, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29885769

RESUMO

During the past decade, H5N1 highly pathogenic avian influenza (HPAI) viruses have diversified genetically and antigenically, suggesting the need for multiple H5N1 vaccines. However, preparation of multiple vaccines from live H5N1 HPAI viruses is difficult and economically not feasible representing a challenge for pandemic preparedness. Here we evaluated a novel multi-clade recombinant H5N1 virus-like particle (VLP) design, in which H5 hemagglutinins (HA) and N1 neuraminidase (NA) derived from four distinct clades of H5N1 virus were co-localized within the VLP structure. The multi-clade H5N1 VLPs were prepared by using a recombinant baculovirus expression system and evaluated for functional hemagglutination and neuraminidase enzyme activities, particle size and morphology, as well as for the presence of baculovirus in the purified VLP preparations. To remove residual baculovirus, VLP preparations were treated with beta-propiolactone (BPL). Immunogenicity and efficacy of multi-clade H5N1 VLPs were determined in an experimental ferret H5N1 HPAI challenge model, to ascertain the effect of BPL on immunogenicity and protective efficacy against lethal challenge. Although treatment with BPL reduced immunogenicity of VLPs, all vaccinated ferrets were protected from lethal challenge with influenza A/VietNam/1203/2004 (H5N1) HPAI virus, indicating that multi-clade VLP preparations treated with BPL represent a potential approach for pandemic preparedness vaccines.


Assuntos
Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Propiolactona/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Desinfetantes/metabolismo , Furões , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Masculino , Infecções por Orthomyxoviridae/prevenção & controle , Análise de Sobrevida , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética
14.
Vaccine ; 36(24): 3445-3452, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29739716

RESUMO

Coxsackievirus belongs to the Enterovirus genus of the Picornaviridae family and is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD). Historically, outbreaks of HFMD have mainly been caused by enterovirus 71 and coxsackievirus A16. Recently, coxsackieviruses A6 and A10 have been associated with increased occurrences of sporadic HFMD cases and outbreak events globally. In this study, the immunogenicity of coxsackieviruses A6, A10, and A16 (CA6, CA10, and CA16), which were inactivated by formalin or ß-propiolactone (BPL) under different conditions, was evaluated as multivalent vaccine candidates. CA6 induced similar immune responses with both inactivation methods, and the immune efficacy of CA10 and CA16 was better following inactivation with BPL than with formalin. There was no sufficient cross-reactivity or cross-protectivity against heterologous strains in groups vaccinated with the BPL-inactivated (BI) monovalent vaccine. Sufficient neutralizing antibody and cell-mediated immune responses were induced in the BI-trivalent vaccinated group. These findings suggest that BI-CA6, CA10, and CA16 are potential multivalent vaccine candidates and that a multivalent vaccine is needed to control HFMD. The coxsackievirus multivalent vaccine could be useful for the development of effective HFMD vaccines.


Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Enterovirus Humano A/efeitos dos fármacos , Doença de Mão, Pé e Boca/prevenção & controle , Imunogenicidade da Vacina , Vacinas Virais/administração & dosagem , Animais , Proteção Cruzada , Enterovirus Humano A/imunologia , Enterovirus Humano A/patogenicidade , Feminino , Formaldeído/química , Doença de Mão, Pé e Boca/imunologia , Doença de Mão, Pé e Boca/mortalidade , Doença de Mão, Pé e Boca/virologia , Humanos , Imunidade Celular/efeitos dos fármacos , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Propiolactona/química , Análise de Sobrevida , Potência de Vacina , Vacinas de Produtos Inativados , Vacinas de Subunidades , Vacinas Virais/imunologia
15.
Biochemistry ; 57(2): 221-225, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29023093

RESUMO

S-Palmitoylation is an abundant lipid post-translational modification that is dynamically installed on and removed from target proteins to regulate their activity and cellular localization. A dearth of tools for studying the activities and regulation of protein S-depalmitoylases, thioesterase "erasers" of protein cysteine S-palmitoylation, has contributed to an incomplete understanding of the role of dynamic S-palmitoylation in regulating proteome lipidation. Recently, we developed "depalmitoylation probes" (DPPs), small molecule probes that become fluorescent upon S-depalmitoylase enzymatic activity. To be suitable for application in live cells, the first-generation DPPs relied on a shorter lipid substrate (C8 vs naturally occurring C16), which enhanced solubility and cell permeability. However, the use of an unnatural lipid substrate on the probes potentially limits the utility of the approach. Herein, we present a new member of the DPP family, DPP-5, which features an anionic carboxylate functional group that increases the probe water solubility. The enhanced water solubility of DPP-5 permits the use of a natural, palmitoylated substrate (C16), rather than a surrogate lipid. We show that DPP-5 is capable of monitoring endogenous S-depalmitoylases in live mammalian cells and that it can reveal changes in S-depalmitoylation levels due to lipid stress. DPP-5 should prove to be a useful new tool for probing the regulation of proteome lipidation through dynamic S-depalmitoylation.


Assuntos
Carbamatos/análise , Corantes Fluorescentes/análise , Microscopia Intravital/métodos , Piperazinas/análise , Processamento de Proteína Pós-Traducional , Ativação Metabólica , Animais , Carbamatos/química , Cisteína/metabolismo , Corantes Fluorescentes/química , Células HEK293 , Humanos , Lipoilação , Mamíferos/metabolismo , Microscopia de Fluorescência , Palmitatos/metabolismo , Piperazinas/química , Propiolactona/análogos & derivados , Propiolactona/farmacologia , Solubilidade , Relação Estrutura-Atividade , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/metabolismo , Água , Xantonas/química
16.
Int J Mol Sci ; 18(6)2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28632154

RESUMO

Although the copolymerizations of l-lactide (LA) with seven- or six-membered ring lactones have been extensively studied, the copolymerizations of LA with four-membered ring lactones have scarcely been reported. In this work, we studied the copolymerization of LA with ß-propiolactone (PL) and the properties of the obtained copolymers. The copolymerization of LA with PL was carried out using trifluoromethanesulfonic acid as a catalyst and methanol as an initiator to produce poly(LA-co-PL) with Mn of ~50,000 and PL-content of 6-67 mol %. The Tg values of the copolymers were rapidly lowered with increasing PL-contents. The Tm and ΔHm of the copolymers gradually decreased with increasing PL-contents, indicating their decreased crystallinity. Biodegradation test of the copolymers in compost demonstrated their improved biodegradability in comparison with the homopolymer of LA.


Assuntos
Biodegradação Ambiental , Dioxanos/síntese química , Dioxanos/metabolismo , Polímeros/síntese química , Polímeros/metabolismo , Propiolactona/síntese química , Propiolactona/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Catálise , Lactonas/metabolismo , Teste de Materiais , Peso Molecular , Polimerização , Polímeros/química , Temperatura
17.
Vaccine ; 35(26): 3401-3408, 2017 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-28511853

RESUMO

Avian influenza viruses (AIV) are a threat to poultry production worldwide. Vaccination is utilized as a component of control programs for both high pathogenicity (HP) and low pathogenicity (LP) AIV. Over 95% of all AIV vaccine used in poultry are inactivated, adjuvanted products. To identify the best formulations for chickens, vaccines were prepared with beta-propiolactone (BPL) inactivated A/British Columbia/314514-1/2004 H7N3 LP AIV using ten commercially available or experimental adjuvants. Each vaccine formulation was evaluated for immunogenicity in chickens. Challenge studies with an antigenically homologous strain of HPAIV were conducted to compare protection against mortality and measure reductions in virus levels in oral swabs. The four best adjuvants from the studies with BPL inactivated antigen were selected and tested identically, but with vaccines prepared from formalin inactivated virus. Mineral and vegetable oil based adjuvants generally induced the highest antibody titers with 100% seroconversion by 3weeks post vaccination. Chitosan induced positive antibody titers in 100% of the chickens, but the titers were significantly lower than those of most of the oil based adjuvants. Antibody levels from calcium phosphate and alginate adjuvanted groups were similar to those of non-adjuvanted virus. All groups that received adjuvanted vaccines induced similar levels of protection against mortality (0-20%) except the groups vaccinated with calcium phosphate adjuvanted vaccines, where mortality was similar (70%) to groups that received non-adjuvanted inactivated virus or no vaccine (60-100% mortality). Virus shedding in oral swabs was variable among the treatment groups. Formalin inactivated vaccine induced similar antibody titers and protection against challenge compared to BPL inactivated vaccine groups. These studies support the use of oil adjuvanted vaccines for use in the poultry industry for control for AIV.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacinas contra Influenza/uso terapêutico , Influenza Aviária/prevenção & controle , Animais , Anticorpos Antivirais , Galinhas/imunologia , Formaldeído/administração & dosagem , Vírus da Influenza A Subtipo H7N3 , Propiolactona/administração & dosagem , Vacinação/veterinária , Vacinas de Produtos Inativados/uso terapêutico , Eliminação de Partículas Virais
18.
J Virol ; 91(8)2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28148783

RESUMO

Beta-propiolactone (BPL) is an inactivating agent that is widely used in the vaccine industry. However, its effects on vaccine protein antigens and its mechanisms of action remain poorly understood. Here we present cryo-electron microscopy (cryo-EM) structures of BPL-treated coxsackievirus A16 (CVA16) mature virions and procapsids at resolutions of 3.9 Å and 6.5 Å, respectively. Notably, both particles were found to adopt an expanded conformation resembling the 135S-like uncoating intermediate, with characteristic features including an opened 2-fold channel, the externalization of the N terminus of VP1 capsid protein, and the absence of pocket factor. However, major neutralizing epitopes are very well preserved on these particles. Further biochemical analyses revealed that BPL treatment impairs the abilities of CVA16 particles to bind to the attachment receptor heparan sulfate and to a conformation-dependent monoclonal antibody in a BPL dose-dependent manner, indicating that BPL is able to modify surface-exposed amino acid residues. Taken together, our results demonstrate that BPL treatment may induce alteration of the overall structure and surface properties of a nonenveloped viral capsid, thus revealing a novel mode of action of BPL.IMPORTANCE Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. It is recognized that BPL inactivates viral infectivity through modification of viral nucleic acids. However, its effect on viral proteins remains largely unknown. Here, we present high-resolution cryo-EM structures of BPL-treated coxsackievirus A16 (CVA16) mature virions and procapsids, which reveals an expanded overall conformation and characteristic features that are typical for the 135S-like uncoating intermediate. We further show that the BPL concentration affects the binding of inactivated CVA16 particles to their receptor/antibody. Thus, BPL treatment can alter the overall structure and surface properties of viral capsids, which may lead to antigenic and immunogenic variations. Our findings provide important information for future development of BPL-inactivated vaccines.


Assuntos
Capsídeo/efeitos dos fármacos , Capsídeo/ultraestrutura , Desinfetantes/farmacologia , Enterovirus/efeitos dos fármacos , Enterovirus/ultraestrutura , Propiolactona/farmacologia , Inativação de Vírus , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Capsídeo/imunologia , Microscopia Crioeletrônica , Enterovirus/imunologia
19.
Int J Pharm ; 511(2): 1098-111, 2016 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-27523619

RESUMO

Spray dried vaccine formulations might be an alternative to traditional lyophilized vaccines. Compared to lyophilization, spray drying is a fast and cheap process extensively used for drying biologicals. The current study provides an approach that utilizes Design of Experiments for spray drying process to stabilize whole inactivated influenza virus (WIV) vaccine. The approach included systematically screening and optimizing the spray drying process variables, determining the desired process parameters and predicting product quality parameters. The process parameters inlet air temperature, nozzle gas flow rate and feed flow rate and their effect on WIV vaccine powder characteristics such as particle size, residual moisture content (RMC) and powder yield were investigated. Vaccine powders with a broad range of physical characteristics (RMC 1.2-4.9%, particle size 2.4-8.5µm and powder yield 42-82%) were obtained. WIV showed no significant loss in antigenicity as revealed by hemagglutination test. Furthermore, descriptive models generated by DoE software could be used to determine and select (set) spray drying process parameter. This was used to generate a dried WIV powder with predefined (predicted) characteristics. Moreover, the spray dried vaccine powders retained their antigenic stability even after storage for 3 months at 60°C. The approach used here enabled the generation of a thermostable, antigenic WIV vaccine powder with desired physical characteristics that could be potentially used for pulmonary administration.


Assuntos
Química Farmacêutica/métodos , Vírus da Influenza A , Vacinas contra Influenza/síntese química , Propiolactona/síntese química , Previsões , Vacinas de Produtos Inativados/síntese química
20.
Viral Immunol ; 29(7): 430-5, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27548006

RESUMO

Infectious hematopoietic necrosis virus (IHNV) infects salmonid fish, resulting in high mortality and serious economic losses to salmonid aquaculture. Therefore, an effective IHNV vaccine is urgently needed. To select an inactivation agent for the preparation of an effective IHNV vaccine, rainbow trout were immunized with mineral oil emulsions of IHNV vaccines inactivated by formaldehyde, binary ethylenimine (BEI), or ß-propiolactone (BPL). The fish were challenged 8 weeks after vaccination, and their IgM antibody response and relative percent survival (RPS) were evaluated. The results show that formaldehyde, BEI, and BPL abolished IHNV HLJ-09 infectivity within 24, 48, and 24 h at final concentrations of 0.2%, 0.02%, and 0.01%, respectively. The mean levels of specific IgM, both in serum and mucus (collected from the skin surface and gills), for the three immunized groups (from high to low) ranked as follows: the BPL group, BEI group, and formaldehyde group. From weeks 5 to 9, the mean log2 serum titers of IgM in the BPL group were significantly higher compared with those of the other groups (p < 0.05) during the 9 weeks of observation after vaccination (immunized at weeks 0 and6). Mucus OD490 values of the BPL group were significantly higher compared with those of the other groups (p < 0.05) when reaching their peak at weeks 5 and 8, but the difference between the formaldehyde and BEI groups was not significant (p > 0.05). The BPL-inactivated whole-virus vaccine had the greatest protective effect on the rainbow trout after challenge by an intraperitoneal injection of live IHNV, with an RPS rate of 91.67%, which was significantly higher compared with the BEI (83.33%) and formaldehyde (79.17%) groups. These results indicate that the BPL-inactivated IHNV oil-adjuvant vaccine was more effective than the formaldehyde- or BEI-inactivated vaccines. The results of this study provide an important foundation for further studies on inactivated IHNV vaccines.


Assuntos
Anticorpos Antivirais/análise , Desinfetantes/farmacologia , Doenças dos Peixes/prevenção & controle , Vírus da Necrose Hematopoética Infecciosa/efeitos dos fármacos , Vírus da Necrose Hematopoética Infecciosa/imunologia , Infecções por Rhabdoviridae/veterinária , Vacinas Virais/administração & dosagem , Animais , Formação de Anticorpos , Aziridinas/farmacologia , Sangue/imunologia , Formaldeído/farmacologia , Imunoglobulina M/análise , Muco/imunologia , Oncorhynchus mykiss , Propiolactona/farmacologia , Infecções por Rhabdoviridae/prevenção & controle , Análise de Sobrevida , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Inativação de Vírus
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