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Vaccines Jan 2021Tilapia lake virus (TiLV) is a newly emerging pathogen responsible for high mortality and economic losses in the global tilapia industry. Currently, no antiviral therapy...
Tilapia lake virus (TiLV) is a newly emerging pathogen responsible for high mortality and economic losses in the global tilapia industry. Currently, no antiviral therapy or vaccines are available for the control of this disease. The goal of the present study was to evaluate the immunological effects and protective efficacy of formaldehyde- and β-propiolactone-inactivated vaccines against TiLV in the presence and absence of the Montanide IMS 1312 VG adjuvant in tilapia. We found that β-propiolactone inactivation of viral particles generated a vaccine with a higher protection efficacy against virus challenge than did formaldehyde. The relative percent survivals of vaccinated fish at doses of 10, 10, and 10 50% tissue culture infectious dose (TCID)/mL were 42.9%, 28.5%, and 14.3% in the absence of the adjuvant and 85.7%, 64.3%, and 32.1% in its presence, respectively. The vaccine generated specific IgM and neutralizing antibodies against TiLV at 3 weeks following immunization that were significantly increased after a second booster immunization. The steady state mRNA levels of the genes tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon γ (IFN-γ), cluster of differentiation 4 (CD4), major histocompatibility complex (MHC)-Ia, and MHC-II were all increased and indicated successful immune stimulation against TiLV. The vaccine also significantly lowered the viral loads and resulted in significant increases in survival, indicating that the vaccine may also inhibit viral proliferation as well as stimulate a protective antibody response. The β-propiolactone-inactivated TiLV vaccine coupled with the adjuvant Montanide IMS 1312 VG and booster immunizations can provide a high level of protection from virus challenge in tilapia.
PubMed: 33503930
DOI: 10.3390/vaccines9020086 -
International Journal of Infectious... Mar 2021We evaluated molecular-based point-of-care influenza virus detection systems in a laboratory prior to a field evaluation of on-site specimen testing.
BACKGROUND
We evaluated molecular-based point-of-care influenza virus detection systems in a laboratory prior to a field evaluation of on-site specimen testing.
METHODS
The performance characteristics of 1) insulated isothermal polymerase chain reaction (PCR) on a POCKIT™ device and 2) real-time reverse transcription-PCR (rRT-PCR) on a MyGo Mini™ device were evaluated using human clinical specimens, beta-propiolactone-inactivated influenza viruses, and RNA controls. The rRT-PCR carried out on a CXF-96™ real-time detection system was used as a gold standard for comparison.
RESULTS
Both systems demonstrated 100% sensitivity and specificity and test results were in 100% agreement with the gold standard. POCKIT™ only correctly identified influenza A (M gene) in clinical specimens due to the unavailability of typing and subtyping reagents for human influenza viruses, while MyGo Mini™ had either a one log higher or the same sensitivity in detecting influenza viruses in clinical specimens compared to the gold standard. For inactivated viruses and/or viral RNA, the analytic sensitivity of POCKIT™ was shown to be comparable to, or more sensitive, than the gold standard. The analytic sensitivity of MyGo Mini™ had mixed results depending on the types and subtypes of influenza viruses.
CONCLUSIONS
The performance of the two systems in a laboratory is promising and supports further evaluation in field settings.
Topics: Early Diagnosis; Humans; Influenza, Human; Laboratories; Laos; Orthomyxoviridae; Point-of-Care Systems; RNA, Viral; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 33359952
DOI: 10.1016/j.ijid.2020.12.059 -
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 Jan 2021Zika virus (ZIKV) infection remains a public health concern necessitating demand for long-term virus production for diagnostic assays and R&D activities. Inactivated...
Zika virus (ZIKV) infection remains a public health concern necessitating demand for long-term virus production for diagnostic assays and R&D activities. Inactivated virus constitutes an important component of the Trioplex rRT-PCR assay and serological IgM assay (MAC-ELISA). The aim of our study is to establish standard methods of ZIKV inactivation while maintaining antigenicity and RNA integrity. We tested viral supernatants by four different inactivation methods: 1. Heat inactivation at 56 °C and 60 °C; 2. Gamma-Irradiation; 3. Chemical inactivation by Beta-propiolactone (BPL) and 4. Fast-acting commercial disinfecting agents. Effectivity was measured by cytopathic effect (CPE) and plaque assay. RNA stability and antigenicity were measured by RT-PCR and MAC-ELISA, respectively. Results: Heat inactivation: Low titer samples, incubated at 56 °C for 2 h, showed neither CPE or plaques compared to high titer supernatants that required 2.5 h. Inactivation occurred at 60 °C for 60 min with all virus titers. Gamma irradiation: Samples irradiated at ≥3 Mrad for low virus concentrations and ≥5Mrad for high virus titer completely inactivated virus. Chemical Inactivation: Neither CPE nor plaques were observed with ≥0.045 % BPL inactivation of ZIKV. Disinfectant: Treatment of viral supernatants with Micro-Chem Plus™, inactivated virus in 2 min, whereas, Ethanol (70 %) and STERIS Coverage® Spray TB inactivated the virus in 5 min.
Topics: Disinfection; Humans; Indicators and Reagents; Virus Inactivation; Zika Virus; Zika Virus Infection
PubMed: 33098957
DOI: 10.1016/j.jviromet.2020.114004 -
Virology Journal Oct 2020Transmissible gastroenteritis virus (TGEV) causes enteric infection in piglets, characterized by vomiting, severe diarrhea and dehydration, and the mortality in suckling...
BACKGROUND
Transmissible gastroenteritis virus (TGEV) causes enteric infection in piglets, characterized by vomiting, severe diarrhea and dehydration, and the mortality in suckling piglets is often high up to 100%. Vaccination is an effective measure to control the disease caused by TGEV.
METHODS
In this study, cell-cultured TGEV HN-2012 strain was inactivated by formaldehyde (FA), β-propiolactone (BPL) or binaryethylenimine (BEI), respectively. Then the inactivated TGEV vaccine was prepared with freund's adjuvant, and the immunization effects were evaluated in mice. The TGEV-specific IgG level was detected by ELISA. The positive rates of CD4, CD8, CD4IFN-γ, CD4IL-4 T lymphocytes were detected by flow cytometry assay. Lymphocyte proliferation assay and gross pathology and histopathology examination were also performed to assess the three different inactivating reagents in formulating TGEV vaccine.
RESULTS
The results showed that the TGEV-specific IgG level in FA group (n = 17) was earlier and stronger, while the BEI group produced much longer-term IgG level. The lymphocyte proliferation test demonstrated that the BEI group had a stronger ability to induce spleen lymphocyte proliferation. The positive rates of CD4 and CD8 T lymphocyte subsets of peripheral blood lymphocyte in BEI group was higher than that in FA group and BPL groups by flow cytometry assay. The positive rate of CD4IFN-γ T lymphocyte subset was the highest in the BPL group, and the positive rate of CD4IL-4 T lymphocyte subset was the highest in the FA group. There were no obvious pathological changes in the vaccinated mice and the control group after the macroscopic and histopathological examination.
CONCLUSIONS
These results indicated that all the three experimental groups could induce cellular and humoral immunity, and the FA group had the best humoral immunity effect, while the BEI group showed its excellent cellular immunity effect.
Topics: Animals; Antibodies, Viral; Female; Gastroenteritis, Transmissible, of Swine; Immunity, Cellular; Immunity, Humoral; Immunoglobulin G; Indicators and Reagents; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Swine; T-Lymphocytes; Transmissible gastroenteritis virus; Vaccines, Inactivated; Viral Vaccines; Virus Inactivation
PubMed: 33097081
DOI: 10.1186/s12985-020-01433-8 -
Structure (London, England : 1993) Nov 2020The 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...
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 β-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.
Topics: Animals; Betacoronavirus; COVID-19 Vaccines; Chlorocebus aethiops; Coronavirus Infections; Cryoelectron Microscopy; Disinfectants; Electron Microscope Tomography; Humans; Propiolactone; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Vaccines, Inactivated; Vero Cells; Viral Vaccines; Virion
PubMed: 33058760
DOI: 10.1016/j.str.2020.10.001 -
Archives of Medical Research Jan 2021The Coronavirus disease 2019 (COVID-19) pandemic has spread to almost all nooks and corners of the world. There are numerous potential approaches to pharmacologically... (Review)
Review
The Coronavirus disease 2019 (COVID-19) pandemic has spread to almost all nooks and corners of the world. There are numerous potential approaches to pharmacologically fight COVID-19: small-molecule drugs, interferon therapies, vaccines, oligonucleotides, peptides, and monoclonal antibodies. Medications are being developed to target the spike, membrane, nucleocapsid or envelope proteins. The spike protein is also a critical target for vaccine development. Immunoinformatic approaches are being used for the identification of B cell and cytotoxic T lymphocyte (CTL) epitopes in the SARS-CoV-2 spike protein. Different vaccine vectors are also being developed. Chemical and physical methods such as formaldehyde, UV light or β-propiolactone are being deployed for the preparation of inactivated virus vaccine. Currently, there are many vaccines undergoing clinical trials. Even though mRNA and DNA vaccines are being designed and moved into clinical trials, these types of vaccines are yet to be approved by regulatory bodies for human use. This review focuses on the drugs and vaccines being developed against the COVID-19.
Topics: COVID-19; COVID-19 Vaccines; Drug Development; Epitopes, T-Lymphocyte; Humans; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 32950264
DOI: 10.1016/j.arcmed.2020.09.010 -
JAMA Sep 2020A vaccine against coronavirus disease 2019 (COVID-19) is urgently needed. (Randomized Controlled Trial)
Randomized Controlled Trial
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.
Topics: Adjuvants, Immunologic; Adolescent; Adult; Aluminum Hydroxide; Antibodies, Neutralizing; Antibodies, Viral; Betacoronavirus; COVID-19; COVID-19 Vaccines; Coronavirus Infections; Dose-Response Relationship, Immunologic; Double-Blind Method; Female; Humans; Immunogenicity, Vaccine; Injections, Intramuscular; Male; Pandemics; Pneumonia, Viral; Propiolactone; SARS-CoV-2; Vaccines, Inactivated; Viral Vaccines; Young Adult
PubMed: 32789505
DOI: 10.1001/jama.2020.15543 -
Biomacromolecules Jul 2020The use of safe natural catalyst such as enzymes for ring opening polymerization (ROP) of β-substituted β-lactones such as benzyl malolactonate (MLABe) is an important...
The use of safe natural catalyst such as enzymes for ring opening polymerization (ROP) of β-substituted β-lactones such as benzyl malolactonate (MLABe) is an important objective considering the biomedical applications of the resulting (co)polymers. However, the preparation of well-defined polymeric materials using such systems requires an understanding of enzyme-substrate interactions. In this context, we investigated the mechanism of lipase-catalyzed ROP of MLABe, because it appears that it is probably not the same as the one widely described for other lactones such ε-caprolactone, propiolactone. and lactide. Enzymatic-catalyzed ROPs of MLABe in the presence of the lipase/acyltransferase CpLip2 and its serine knockout (serine KO) mutant (CpLip2_180A) have led to poly(benzyl malate) (PMLABe) terminated by a monobenzyl fumarate group with monomer conversion higher than 70% and weight-average molar mass of about 3600 g/mol ( = 1.42). On the other hand, only less than 7% of MLABe conversion and no polymer formation were observed when the polymerization reaction was conducted in the presence of inactivated CpLip2 (heated at 100 °C). Moreover, the ROP of MLABe in the presence of imidazole, a synthetic mimic of the catalytic histidine, led to a PMLABe terminated by a monobenzyl fumarate group. On the contrary, neither the enzymatic-catalyzed ROP of benzyl dimethylmalolactonate (diMeMLABe), a MLABe with two methyl groups instead of the two "acidic" protons on the lactone's ring, in the presence of CpLip2 and CpLip2_180A nor its chemical ROP in the presence of imidazole were successful. Together, all these results suggested that the lipase-catalyzed polymerization of malolactonates occurred through the abstraction of one of the two "acidic" protons of the lactone's ring by the histidine of the catalytic triad leading to the corresponding monobenzyl fumarate responsible for the polymerization of the remaining monomer. Finally, molecular modeling of the positioning of the monomer into the catalytic site of the CpLip2 and DFT quantum-chemical calculations highlighted an interaction of ()- and ()-MLABe with the catalytic histidine of the enzyme preferentially to serine, in the form of a strong hydrogen bond with one of the "acidic" protons of MLABe, thus, supporting the important role of the catalytic histidine in the polymerization of such cyclic lactones.
Topics: Catalysis; Lactones; Lipase; Molecular Weight; Polymerization; Polymers
PubMed: 32551525
DOI: 10.1021/acs.biomac.0c00593 -
Viruses Jun 2020In 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,...
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.
Topics: Animals; Betacoronavirus; COVID-19; Cellulose; Chlorocebus aethiops; Coronavirus Infections; Culture Media; Formaldehyde; Guanidines; HEK293 Cells; Humans; Pandemics; Phenols; Pneumonia, Viral; Propiolactone; SARS-CoV-2; Sepharose; Vero Cells; Viral Plaque Assay; Virus Inactivation
PubMed: 32517266
DOI: 10.3390/v12060622