-
Poultry Science Jun 2023An immunization experiment was conducted in specific pathogen-free chickens with the inactivated Newcastle disease virus (NDV) vaccine encapsulated in the...
An immunization experiment was conducted in specific pathogen-free chickens with the inactivated Newcastle disease virus (NDV) vaccine encapsulated in the poly-(lactic-co-glycolic) acid (PLGA) nanoparticles (NP) to evaluate its immunogenicity and protective efficacy. The NDV vaccine was prepared by inactivating one virulent Indian strain of NDV belonging to Genotype VII by using beta-propiolactone. PLGA nanoparticles encapsulating inactivated NDV were prepared by the solvent evaporation method. Scanning electron microscopy and zeta sizer analysis revealed that the (PLGA+NDV) NP were spherical, with an average size of 300 nm, having a zeta potential of -6 mV. The encapsulation efficiency and loading efficiency were 72% and 2.4%, respectively. On immunization trial in chicken, the (PLGA+NDV) NP induced significantly (P < 0.0001) higher levels of HI and IgY antibodies with the peak HI titer of 2 and higher expression of IL-4 mRNA. The consistency of higher antibody levels suggests slow and pulsatile release of the antigens from the (PLGA+NDV) NP. The nano-NDV vaccine also induced cell mediated immunity with higher expression of IFN-γ indicating strong Th1 mediated immune responses in contrast to the commercial oil adjuvanted inactivated NDV vaccine. Further, the (PLGA+NDV) NP afforded 100% protection against the virulent NDV challenge. Our results suggested that PLGA NP have adjuvant potential on induction of humoral as well as Th1 biased cell mediated immune responses and also enhanced protective efficacy of the inactivated NDV vaccine. This study provides an insight for development of PLGA NP based inactivated NDV vaccine using the same genotype circulating in the field as well as for other avian diseases at exigencies.
Topics: Animals; Newcastle disease virus; Newcastle Disease; Chickens; Vaccines, Inactivated; Glycols; Adjuvants, Immunologic; Immunity, Cellular; Nanoparticles; Viral Vaccines
PubMed: 37116285
DOI: 10.1016/j.psj.2023.102679 -
Journal of Virological Methods Jul 2023β-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... (Review)
Review
β-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 NaHCO (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 TCID/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.
Topics: Dogs; Animals; Propiolactone; Saliva; Odorants; COVID-19; Virus Inactivation; SARS-CoV-2; Viruses
PubMed: 37068591
DOI: 10.1016/j.jviromet.2023.114733 -
Vaccines Mar 2023Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole...
Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 μg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.
PubMed: 36992191
DOI: 10.3390/vaccines11030607 -
Viruses Feb 2023Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by β-propiolactone, formaldehyde,...
Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by β-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 β-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 β-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 β-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.
Topics: Animals; Humans; Spike Glycoprotein, Coronavirus; Vaccines, Inactivated; COVID-19; COVID-19 Serotherapy; COVID-19 Vaccines; Pandemics; Propiolactone; SARS-CoV-2; Formaldehyde
PubMed: 36851694
DOI: 10.3390/v15020480 -
Development of an Antigen Detection Kit Capable of Discriminating the Omicron Mutants of SARS-CoV-2.Vaccines Jan 2023Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread around the world, caused millions of deaths and a severe illness which poses a serious threat to...
INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread around the world, caused millions of deaths and a severe illness which poses a serious threat to human health.
OBJECTIVE
To develop an antigen detection kit that can identify Omicron novel coronavirus mutants.
METHODS
BALB/c mice were immunized with the nucleocapsid protein of SARS-CoV-2 Omicron mutant treated with β-propiolactone. After fusion of myeloma cells with immune cells, Elisa was used to screen the cell lines capable of producing monoclonal antibodies. The detection kit was prepared by colloidal gold immunochromatography. Finally, the sensitivity, specificity and anti-interference of the kit were evaluated by simulating positive samples.
RESULTS
The sensitivity of the SARS-CoV-2 antigen detection kit can reach 62.5 TCID/mL, and it has good inclusiveness for different SARS-CoV-2 strains. The kit had no cross-reaction with common respiratory pathogens, and its sensitivity was still not affected under the action of different concentrations of interferences, indicating that it had good specificity and stability.
CONCLUSION
In this study, monoclonal antibodies with high specificity to the N protein of the Omicron mutant strain were obtained by monoclonal antibody screening technology. Colloidal gold immunochromatography technology was used to prepare an antigen detection kit with high sensitivity to detect and identify the mutant Omicron strain.
PubMed: 36851181
DOI: 10.3390/vaccines11020303 -
Life (Basel, Switzerland) Jan 2023This study investigates inclusion behavior of amylose towards, poly(β-propiolactone) (PPL), that is a hydrophobic polyester, via the vine-twining process in glucan...
This study investigates inclusion behavior of amylose towards, poly(β-propiolactone) (PPL), that is a hydrophobic polyester, via the vine-twining process in glucan phosphorylase (GP, isolated from thermophilic bacteria, VF5)-catalyzed enzymatic polymerization. As a result of poor dispersibility of PPL in sodium acetate buffer, the enzymatically produced amylose by GP catalysis incompletely included PPL in the buffer media under the general vine-twining polymerization conditions. Alternatively, we employed an ethyl acetate-sodium acetate buffer emulsion system with dispersing PPL as the media for vine-twining polymerization. Accordingly, the GP (from thermophilic bacteria)-catalyzed enzymatic polymerization of an α-d-glucose 1-phosphate monomer from a maltoheptaose primer was performed at 50 °C for 48 h in the prepared emulsion to efficiently form the inclusion complex. The powder X-ray diffraction profile of the precipitated product suggested that the amylose-PPL inclusion complex was mostly produced in the above system. The H NMR spectrum of the product also supported the inclusion complex structure, where a calculation based on an integrated ratio of signals indicated an almost perfect inclusion of PPL in the amylosic cavity. The prevention of crystallization of PPL in the product was suggested by IR analysis, because it was surrounded by the amylosic chains due to the inclusion complex structure.
PubMed: 36836651
DOI: 10.3390/life13020294 -
Veterinary Microbiology Apr 2023Duck tembusu virus (DTMUV), belonging to the Flavivirus genus, Flaviviridae family, has caused huge economic losses in the duck industry. However, the inactivated DTMUV...
Duck tembusu virus (DTMUV), belonging to the Flavivirus genus, Flaviviridae family, has caused huge economic losses in the duck industry. However, the inactivated DTMUV vaccine requires multiple immunizations and has incomplete effectiveness. The humoral immune response is a key factor in the control of DTMUV infection. IL-7 derived from mammals has the ability to enhance antibody production. Whether duck IL-7 (duIL-7) possesses the ability to improve the humoral immunity of inactivated DTMUV vaccine has not yet been declared. Here, a beta-propiolactone (BPL)-inactivated DTMUV vaccine was employed to characterize the adjuvant property of duIL-7 in humoral immune responses. Intramuscular injection of DTMUV inactivated vaccine with or without duIL-7 was administered twice to the ducks. The results showed that duIL-7 was able to promote rapid antibody responses and enhance DTMUV-specific IgG and neutralizing antibody production to the vaccine. T follicular helper (Tfh) cells play a key role in assisting long humoral immunity. It was found that duIL-7 upregulated duIl-6 and duIl-21 gene expression at 3 w post first vaccination, which encode Tfh cell differentiation-related cytokines duIL-6 and duIL-21, respectively. This may be the reason that duIL-7 could prolong the humoral immune response to the inactivated DTMUV vaccine. Next, the ability of duIL-7 to simplify the immunization procedure of the inactivated DTMUV vaccine was tested. When ducks were immunized once, the titers of neutralizing antibodies in ducks from the inactivated DTMUV vaccine supplemented with duIL-7 group were significantly higher than those of ducks from the inactivated DTMUV vaccine group (P < 0.05). In addition, duIL-7 could assist the inactivated DTMUV vaccine in maintaining neutralizing antibodies at high levels during the whole experimental period. The viral titers in the ducks immunized with the inactivated DTMUV vaccine and duIL-7 were lower than those in the ducks immunized with the inactivated DTMUV vaccine alone at 3 days post infection (3 dpi, P < 0.05). Overall, duIL-7 possessed the ability to promote and prolong humoral immune responses to the inactivated DTMUV vaccine, even at one dose. This study provides a new efficient adjuvant for inactivated DTMUV vaccine development.
Topics: Animals; Ducks; Immunity, Humoral; Flavivirus Infections; Vaccines, Inactivated; Interleukin-7; Flavivirus; Antibodies, Neutralizing; Adjuvants, Immunologic; Poultry Diseases; Mammals
PubMed: 36716633
DOI: 10.1016/j.vetmic.2023.109665 -
IScience Feb 2023Vaccines have relieved the public health burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and globally inactivated vaccines are most widely used....
Vaccines have relieved the public health burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and globally inactivated vaccines are most widely used. However, poor vaccination accessibility and waning immunity maintain the pandemic, driving emergence of variants. We developed an inactivated SARS-CoV-2 (I-SARS-CoV-2) vaccine based on a viral isolate with the Spike mutation D614G, produced in Vero cells in a scalable bioreactor, inactivated with β-propiolactone, purified by membrane-based steric exclusion chromatography, and adjuvanted with MF59-like adjuvant AddaVax. I-SARS-CoV-2 and a derived split vaccine induced persisting neutralizing antibodies in mice; moreover, lyophilized antigen was immunogenic. Following homologous challenge, I-SARS-CoV-2 immunized hamsters were protected against disease and lung pathology. In contrast with reports for widely used vaccines, hamster plasma similarly neutralized the homologous and the Delta (B.1.617.2) variant viruses, whereas the Omicron (B.1.1.529) variant was neutralized less efficiently. Applied bioprocessing approaches offer advantages regarding scalability and production, potentially benefitting worldwide vaccine coverage.
PubMed: 36644321
DOI: 10.1016/j.isci.2023.105949 -
Journal of Extracellular Vesicles Dec 2022The interaction of SARS-CoV-2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS-CoV-2 contaminated-EV isolates in...
The interaction of SARS-CoV-2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS-CoV-2 contaminated-EV isolates in instruments located outside of the biosafety level-3 (BSL-3) environment requires knowing how viral inactivation methods affect the structure and function of extracellular vesicles (EVs). Therefore, three common viral inactivation methods, ultraviolet-C (UVC; 1350 mJ/cm ), β-propiolactone (BPL; 0.005%), heat (56°C, 45 min) were performed on defined EV particles and their proteins, RNAs, and function. Small EVs were isolated from the supernatant of SARS-CoV-2-infected human lung epithelial Calu-3 cells by stepwise centrifugation, ultrafiltration and qEV size-exclusion chromatography. The EV isolates contained SARS-CoV-2. UVC, BPL and heat completely abolished SARS-CoV-2 infectivity of the contaminated EVs. Particle detection by electron microscopy and nanoparticle tracking was less affected by UVC and BPL than heat treatment. Western blot analysis of EV markers was not affected by any of these three methods. UVC reduced SARS-CoV-2 spike detectability by quantitative RT-PCR and slightly altered EV-derived β-actin detection. Fibroblast migration-wound healing activity of the SARS-CoV-2 contaminated-EV isolate was only retained after UVC treatment. In conclusion, specific viral inactivation methods are compatible with specific measures in SARS-CoV-2 contaminated-EV isolates. UVC treatment seems preferable for studying functions of EVs released from SARS-CoV-2 infected cells.
Topics: Humans; SARS-CoV-2; Virus Inactivation; COVID-19; Extracellular Vesicles; Lung; Epithelial Cells
PubMed: 36468940
DOI: 10.1002/jev2.12291 -
Viruses Aug 2022Severe 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...
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 β-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 β-sheet, parallel β-sheet, β-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.
Topics: Antibodies, Viral; COVID-19; COVID-19 Vaccines; Formaldehyde; Humans; Propiolactone; SARS-CoV-2; Vaccines, Inactivated
PubMed: 36146745
DOI: 10.3390/v14091938