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Molecular Therapy. Methods & Clinical... Dec 2021Because of the relatively limited understanding of coronavirus disease 2019 (COVID-19) pathogenesis, immunological analysis for vaccine development is needed. Mice and...
Because of the relatively limited understanding of coronavirus disease 2019 (COVID-19) pathogenesis, immunological analysis for vaccine development is needed. Mice and macaques were immunized with an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine prepared by two inactivators. Various immunological indexes were tested, and viral challenges were performed on day 7 or 150 after booster immunization in monkeys. This inactivated SARS-CoV-2 vaccine was produced by sequential inactivation with formaldehyde followed by propiolactone. The various antibody responses and specific T cell responses to different viral antigens elicited in immunized animals were maintained for longer than 150 days. This comprehensive immune response could effectively protect vaccinated macaques by inhibiting viral replication in macaques and substantially alleviating immunopathological damage, and no clinical manifestation of immunopathogenicity was observed in immunized individuals during viral challenge. This candidate inactivated vaccine was identified as being effective against SARS-CoV-2 challenge in rhesus macaques.
PubMed: 34462721
DOI: 10.1016/j.omtm.2021.08.005 -
Emerging Microbes & Infections Dec 2021The unprecedented in recent history global COVID-19 pandemic urged the implementation of all existing vaccine platforms to ensure the availability of the vaccines...
The unprecedented in recent history global COVID-19 pandemic urged the implementation of all existing vaccine platforms to ensure the availability of the vaccines against COVID-19 to every country in the world. Despite the multitude of high-quality papers describing clinical trials of different vaccine products, basic detailed data on general toxicity, reproductive toxicity, immunogenicity, protective efficacy and durability of immune response in animal models are scarce. Here, we developed a β-propiolactone-inactivated whole virion vaccine CoviVac and assessed its safety, protective efficacy, immunogenicity and stability of the immune response in rodents and non-human primates. The vaccine showed no signs of acute/chronic, reproductive, embryo- and fetotoxicity, or teratogenic effects, as well as no allergenic properties in studied animal species. The vaccine induced stable and robust humoral immune response both in form of specific anti-SARS-CoV-2 IgG and NAbs in mice, Syrian hamsters, and common marmosets. The NAb levels did not decrease significantly over the course of one year. The course of two immunizations protected Syrian hamsters from severe pneumonia upon intranasal challenge with the live virus. Robustness of the vaccine manufacturing process was demonstrated as well. These data encouraged further evaluation of CoviVac in clinical trials.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; COVID-19; COVID-19 Vaccines; Callithrix; Cricetinae; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Guinea Pigs; Humans; Immunity, Humoral; Immunogenicity, Vaccine; Immunoglobulin G; Male; Mesocricetus; Mice; Mice, Inbred BALB C; Rats; Rats, Wistar; SARS-CoV-2; Time Factors; Vaccines, Inactivated
PubMed: 34427172
DOI: 10.1080/22221751.2021.1971569 -
Materials Advances Mar 2021Cowpea mosaic virus (CPMV) is currently in the development pipeline for multiple biomedical applications, including cancer immunotherapy. In particular the application...
Cowpea mosaic virus (CPMV) is currently in the development pipeline for multiple biomedical applications, including cancer immunotherapy. In particular the application of CPMV as vaccine has shown promise; here the plant viral nanoparticle is used as an adjuvant and is injected directly into a tumor to reverse immunosuppression and prime systemic anti-tumor immunity. Efficacy of this CPMV-based cancer immunotherapy has been demonstrated in multiple tumor mouse models and canine cancer patients. However, while CPMV is non-infectious to mammals, it is infectious to legumes and therefore, from a safety perspective, it is desired to develop non-infectious CPMV formulations. Non-infectious virus-like particles of CPMV devoid of nucleic acids have been produced; nevertheless, efficacy of such empty CPMV nanoparticles does not match efficacy of nucleic acid-laden CPMV. The multivalent capsid activates the innate immune system through pathogen pattern recognition receptors (PRRs) such as toll-like receptors (TLRs); the RNA cargo provides additional signaling through TLR-7/8, which boosts the efficacy of this adjuvant. Therefore, in this study, we set out to develop RNA-laden, but non-infectious CPMV. We report inactivation of CPMV using UV light and chemical inactivation using β-propiolactone (βPL) or formalin. 7.5 J cm UV, 50 mM βPL or 1 mM formalin was determined to be sufficient to inactivate CPMV and prevented plant infection. We compared the immunogenicity of native CPMV and inactivated CPMV formulations and using RAW-Blue™ reporter cells and a murine syngeneic, orthotropic melanoma model (using B16F10 cells and C57BL6 mice). While the assay indicated activation of the RAW-Blue™ reporter cells by formaldehyde and UV-inactivated CPMV at levels comparable to native CPMV; βPL-inactivated CPMV appeared to have diminished activity. Tumor mouse model experiments indicate potent efficacy of the chemically-inactivated CPMV (UV-treated CPMV was not tested) leading to tumor regression and increased survival; efficacy was somewhat reduced when compared to CPMV, however these samples outperformed the empty CPMV nanoparticles. These results will facilitate the translational development of safe and potent CPMV-based cancer immunotherapies.
PubMed: 34368764
DOI: 10.1039/D0MA00752H -
BMC Infectious Diseases Jul 2021The main strategy to contain the current SARS-CoV-2 pandemic remains to implement a comprehensive testing, tracing and quarantining strategy until vaccination of the... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
The main strategy to contain the current SARS-CoV-2 pandemic remains to implement a comprehensive testing, tracing and quarantining strategy until vaccination of the population is adequate. Scent dogs could support current testing strategies.
METHODS
Ten dogs were trained for 8 days to detect SARS-CoV-2 infections in beta-propiolactone inactivated saliva samples. The subsequent cognitive transfer performance for the recognition of non-inactivated samples were tested on three different body fluids (saliva, urine, and sweat) in a randomised, double-blind controlled study.
RESULTS
Dogs were tested on a total of 5242 randomised sample presentations. Dogs detected non-inactivated saliva samples with a diagnostic sensitivity of 84% (95% CI: 62.5-94.44%) and specificity of 95% (95% CI: 93.4-96%). In a subsequent experiment to compare the scent recognition between the three non-inactivated body fluids, diagnostic sensitivity and specificity were 95% (95% CI: 66.67-100%) and 98% (95% CI: 94.87-100%) for urine, 91% (95% CI: 71.43-100%) and 94% (95% CI: 90.91-97.78%) for sweat, 82% (95% CI: 64.29-95.24%), and 96% (95% CI: 94.95-98.9%) for saliva respectively.
CONCLUSIONS
The scent cognitive transfer performance between inactivated and non-inactivated samples as well as between different sample materials indicates that global, specific SARS-CoV-2-associated volatile compounds are released across different body secretions, independently from the patient's symptoms. All tested body fluids appear to be similarly suited for reliable detection of SARS-CoV-2 infected individuals.
Topics: Animals; Body Fluids; COVID-19; Dogs; Humans; Odorants; Pandemics; SARS-CoV-2; Saliva
PubMed: 34315418
DOI: 10.1186/s12879-021-06411-1 -
Biochemistry. Biokhimiia Jul 2021COVID-19, a new human respiratory disease that has killed nearly 3 million people in a year since the start of the pandemic, is a global public health challenge. Its... (Review)
Review
COVID-19, a new human respiratory disease that has killed nearly 3 million people in a year since the start of the pandemic, is a global public health challenge. Its infectious agent, SARS-CoV-2, differs from other coronaviruses in a number of structural features that make this virus more pathogenic and transmissible. In this review, we discuss some important characteristics of the main SARS-CoV-2 surface antigen, the spike (S) protein, such as (i) ability of the receptor-binding domain (RBD) to switch between the "standing-up" position (open pre-fusion conformation) for receptor binding and the "lying-down" position (closed pre-fusion conformation) for immune system evasion; (ii) advantage of a high binding affinity of the RBD open conformation to the human angiotensin-converting enzyme 2 (ACE2) receptor for efficient cell entry; and (iii) S protein preliminary activation by the intracellular furin-like proteases for facilitation of the virus spreading across different cell types. We describe interactions between the S protein and cellular receptors, co-receptors, and antagonists, as well as a hypothetical mechanism of the homotrimeric spike structure destabilization that triggers the fusion of the viral envelope with the cell membrane at physiological pH and mediates the viral nucleocapsid entry into the cytoplasm. The transition of the S protein pre-fusion conformation to the post-fusion one on the surface of virions after their treatment with some reagents, such as β-propiolactone, is essential, especially in relation to the vaccine production. We also compare the COVID-19 pathogenesis with that of severe outbreaks of "avian" influenza caused by the A/H5 and A/H7 highly pathogenic viruses and discuss the structural similarities between the SARS-CoV-2 S protein and hemagglutinins of those highly pathogenic strains. Finally, we touch on the prospective and currently used COVID-19 antiviral and anti-pathogenetic therapeutics, as well as recently approved conventional and innovative COVID-19 vaccines and their molecular and immunological features.
Topics: Angiotensin-Converting Enzyme 2; COVID-19; Humans; Influenza A virus; Influenza, Human; Pandemics; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 34284707
DOI: 10.1134/S0006297921070026 -
The Indian Journal of Medical Research May 2021Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) continues to be a devastating pandemic. This study was aimed...
BACKGROUND & OBJECTIVES
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) continues to be a devastating pandemic. This study was aimed at performance assessment of SARS-CoV-2 IgM and IgG ELISAs, and investigation of their utility for patient diagnosis and sero-epidemiologic investigations.
METHODS
Serum/plasma samples from COVID-19 patients or asymptomatic contacts (n=180) and healthy donors (n=90) were tested in parallel using two commercial IgM ELISAs (Erbalisa and Inbios), and four IgG ELISAs (Kavach, Euroimmun, Erbalisa and Inbios) along with an indigenous β-propiolactone inactivated virus-based ELISA (IRSHA-IgG-ELISA). Plaque reduction neutralization test (PRNT) was used as reference test.
RESULTS
Among 180 COVID-19 patients, 125 tested positive by PRNT. Inbios-IgM-ELISA showed sensitivity (Se)/specificity (Sp)/positive predictive value (PPV)/negative predictive value (NPV) of 93.6/97.8/98.4/94.4 per cent in relation to PRNT, and performed better than Erbalisa-IgM-ELISA (Se: 48%, Sp: 95.6%, PPV: 95.2%, NPV: 65.2%). During the first week of disease, only 47.4 per cent of the COVID-19 patients tested IgM positive by Inbios-IgM-ELISA, detection improving at two weeks and beyond (~86-100%). Among IgG tests, Inbios-IgG-ELISA ranked first in terms of sensitivity (83.2%), followed by IRSHA (64.8%), Euroimmun (64%), Erbalisa (57.6%) and Kavach (56%) tests. For all IgG tests, sensitivity improved during the third (73.9-95.7%) and fourth week (100%) of illness. The specificity (96.7-100%) and PPV (96.2-100%) of all IgG tests were high; NPV ranged between 71.9 and 87.1 per cent with Inbios-IgG-ELISA scoring highest.
INTERPRETATION & CONCLUSIONS
Our results show that IgM detection by the current, most sensitive ELISAs cannot replace molecular diagnosis, but may aid as a supplement test. The available IgG tests are suitable for serosurveys for the assessment of previous virus exposure.
Topics: Antibodies, Viral; COVID-19; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin G; Immunoglobulin M; Neutralization Tests; SARS-CoV-2; Sensitivity and Specificity
PubMed: 34145085
DOI: 10.4103/ijmr.IJMR_3806_20 -
Pathogens (Basel, Switzerland) May 2021The development of a safe and effective vaccine to protect against COVID-19 is a global priority due to the current high SARS-CoV-2 infection rate. Currently, there are...
The development of a safe and effective vaccine to protect against COVID-19 is a global priority due to the current high SARS-CoV-2 infection rate. Currently, there are over 160 SARS-CoV-2 vaccine candidates at the clinical or pre-clinical stages of development. Of these, there are only three whole-virus vaccine candidates produced using β-propiolactone or formalin inactivation. Here, we prepared a whole-virus SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) using a novel psoralen inactivation method and evaluated its immunogenicity in mice using two different adjuvants, alum and Advax-2. We compared the immunogenicity of SARS-CoV-2 PsIV against SARS-CoV-2 DNA vaccines expressing either full-length or truncated spike proteins. We also compared the psoralen-inactivated vaccine against a DNA prime, psoralen-inactivated vaccine boost regimen. After two doses, the psoralen-inactivated vaccine, when administered with alum or Advax-2 adjuvants, generated a dose-dependent neutralizing antibody responses in mice. Overall, the pattern of cytokine ELISPOT responses to antigen-stimulation observed in this study indicates that SARS-CoV-2 PsIV with the alum adjuvant promotes a Th2-type response, while SARS-CoV-2 PsIV with the Advax-2 adjuvant promotes a Th1-type response.
PubMed: 34069575
DOI: 10.3390/pathogens10050626 -
Macromolecules Jun 2020We report here the synthesis of poly(4-ketovalerolactone) () via ring-opening transesterification polymerization (ROTEP) of the monomer 4-ketovalerolactone (, two steps...
We report here the synthesis of poly(4-ketovalerolactone) () via ring-opening transesterification polymerization (ROTEP) of the monomer 4-ketovalerolactone (, two steps from levulinic acid). The polymerization of proceeds to high equilibrium monomer conversion (up to 96% in the melt) to give the semicrystalline polyketoester with low dispersity. displays glass transition temperatures of 7 °C and two melting temperatures at 132 and 148 °C. This polyester can be chemically recycled through hydrolytic degradation. Under aqueous neutral or acidic conditions, the dominating pathway for polyester hydrolysis is through backbiting from the chain end. Under basic conditions, mid-chain cleavage, accelerated by the ketone carbonyl group in the backbone, promotes the hydrolysis of nearby backbone ester bonds. The final hydrolysis product is 5-hydroxylevulinic acid, the ring opened hydrolysis product of . was also observed to degrade under the action of a Brønsted acid to a bis-spirocyclic dilactone natural product altaicadispirolactone, which is a dimer of . This constitutes a rare example of a one-step synthesis of a secondary metabolite of non-trivial structure in which a polymer was the starting material and the sole source of matter. Analogous ROTEP of the isomeric 4-membered lactone 4-acetyl-β-propiolactone () was also explored, although this chemistry was not as well-behaved as the to polymerization.
PubMed: 33767514
DOI: 10.1021/acs.macromol.0c00787 -
Journal of Microbiological Methods May 2021There are many approaches available to produce inactive bacteria by termination of growth, each with a different efficacy, impact on cell integrity, and potential for...
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.
Topics: Cell Wall; Disinfectants; Disinfection; Ethanol; Formaldehyde; Gram-Negative Bacteria; Gram-Positive Bacteria; Hot Temperature; Microbial Viability; Propiolactone
PubMed: 33766606
DOI: 10.1016/j.mimet.2021.106208 -
Voprosy Virusologii Mar 2021Hemorrhagic fever with renal syndrome (HFRS) holds a leading place among natural focal human diseases in Russian Federation. There is no etiotropic therapy for the...
INTRODUCTION
Hemorrhagic fever with renal syndrome (HFRS) holds a leading place among natural focal human diseases in Russian Federation. There is no etiotropic therapy for the disease now. The vaccine prophylaxis is the most effective method to control this infection. The main criteria for inactivated vaccines evaluation are its immunogenicity and specific activity.The study purposes were to develop a sensitive and specific real-time PCR method for viral RNA quantification in the inactivated vaccine and to study the correlation between the viral RNA amount and vaccine immunogenicity.
MATERIAL AND METHODS
L-segment fragments of the Puumala, Hantaan, and Sochi vaccine strains were selected as diagnostic targets for oligonucleotides and fluorescent probes. The immunogenicity of experimental vaccines was determined by the induction of neutralizing antibodies in BALB/c mice.
RESULTS
A highly specific, sensitive and reproducible real-time PCR method has been developed. The analytical sensitivity was 1.24 ± 1.5 x 102 copies/ml for Puumala virus; 1.16 ± 1.4 * 102 copies/ml for Hantaan; 1.32 ± 1.8 * 102 copies/ ml for Sochi, with a virus content of 1.5 ± 0.5 lg FFU/ml; 1.8 ± 0.5 lg FFU/ml and 2.2 ± 0.5 lg FFU/ml, respectively. The viral RNA amount in experimental vaccine preparations inactivated with β-propiolactone was proportional to the neutralizing antibodies titer observed in mice following the immunization.
DISCUSSION
It was found that different virus inactivators differently affects the detected viral RNA amount, but not the vaccine immunogenicity, which indicates the same degree of the immunogenic proteins damage. The direct relationship between the viral RNA copy number and vaccine immunogenicity makes it possible to use this criterion for vaccine dosage preparation.
CONCLUSION
The developed method for viral RNA quantification is a promising tool for the specific activity control of the HFRS vaccine.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Hemorrhagic Fever with Renal Syndrome; Mice; Mice, Inbred BALB C; RNA, Viral; Real-Time Polymerase Chain Reaction; Vaccines, Inactivated; Viral Vaccines
PubMed: 33683067
DOI: 10.36233/0507-4088-30