Mortality
and disabilities as outcomes of cardiovascular diseases are primarily
related to blood clotting. Optimization of thrombolytic drugs is aimed
at the prevention of side effects (in particular, bleeding) associated
with a disbalance between coagulation and anticoagulation caused by
systemically administered agents. Minimally invasive and efficient
approaches to deliver the thrombolytic agent to the site of clot formation
are needed. Herein, we report a novel nanocomposite prepared by heparin-mediated
cross-linking of urokinase with magnetite nanoparticles (MNPs@uPA).
We showed that heparin within the composition evoked no inhibitory
effects on urokinase activity. Importantly, the magneto-control further
increased the thrombolytic efficacy of the composition. Using our
nanocomposition, we demonstrated efficient lysis of experimental clots in vitro and in animal vessels followed by complete restoration
of blood flow. No sustained toxicity or hemorrhagic complications
were registered in rats and rabbits after single bolus i.v. injection
of therapeutic doses of MNPs@uPA. We conclude that MNPs@uPA is a prototype
of easy-to-prepare, inexpensive, biocompatible, and noninvasive thrombolytic
nanomedicines potentially useful in the treatment of blood clotting.
The vast majority of SARS-CoV-2 vaccines which are licensed or under development focus on the spike (S) protein and its receptor binding domain (RBD). However, the S protein shows considerable sequence variations among variants of concern. The aim of this study was to develop and characterize a SARS-CoV-2 vaccine targeting the highly conserved nucleocapsid (N) protein. Recombinant N protein was expressed in Escherichia coli, purified to homogeneity by chromatography and characterized by SDS-PAGE, immunoblotting, mass spectrometry, dynamic light scattering and differential scanning calorimetry. The vaccine, formulated as a squalane-based emulsion, was used to immunize Balb/c mice and NOD SCID gamma (NSG) mice engrafted with human PBMCs, rabbits and marmoset monkeys. Safety and immunogenicity of the vaccine was assessed via ELISA, cytokine titer assays and CFSE dilution assays. The protective effect of the vaccine was studied in SARS-CoV-2-infected Syrian hamsters. Immunization induced sustainable N-specific IgG responses and an N-specific mixed Th1/Th2 cytokine response. In marmoset monkeys, an N-specific CD4+/CD8+ T cell response was observed. Vaccinated Syrian hamsters showed reduced lung histopathology, lower virus proliferation, lower lung weight relative to the body, and faster body weight recovery. Convacell® thus is shown to be effective and may augment the existing armamentarium of vaccines against COVID-19.
Background The vast majority of SARS-CoV-2 vaccines which are
licensed or under development focus on the spike (S) protein and its
receptor binding domain (RBD). However, S and RBD from SARS-CoV-2
variants of concerns show considerable sequence variations and repeated
injections for boosting specific immunity are necessary. Aim of this
study was to develop and characterize a SARS-CoV-2 vaccine targeting the
highly conserved nucleocapsid (N) protein. Methods Recombinant
N protein was expressed in Escherichia coli, purified to
homogeneity by chromatography and characterized by SDS-PAGE,
immunoblotting, mass spectrometry, dynamic light scattering and
differential scanning calorimetry. The N protein vaccine was obtained by
formulation of recombinant N as squalane-based emulsion and used to
immunize Balb/c mice, NOD scid gamma (NSG) mice engrafted with human
PBMC, rabbits and marmoset monkeys to study safety as well as antibody
and cellular immunity using ELISA for antibodies, measurement of
N-specific Th1 and Th2 cytokine secretion and carboxyfluorescein
succinimidyl ester (CFSE) dilution assays for CD4
and CD8 T cell responses. The protective effect of
the vaccine was studied in SARS-CoV-2-infected Syrian hamsters.
Results Immunization of mice, rabbits and Syrian hamsters with
the recombinant N protein-based vaccine formulated as squalane-based
emulsion (Convacell®) induced sustainable N-specific IgG responses and a
N-specific mixed Th1/Th2 cytokine response. In marmoset monkeys a
N-specific CD4 as well as CD8 T
cell response was observed. Vaccinated and then infected Syrian hamsters
showed reduced lung histopathology, reduced virus was detected in lung
tissue, lung weight relative to the body was not increased after
challenge and body weight was regained faster than in non-vaccinated
animals. Repeated dose toxicity studies in mice and rabbits showed that
Convacell® was well tolerated and safe. Conclusions Convacell®
induced a SARS-CoV-2-specific protective immune response in Syrian
hamsters. It is a new vaccine targeting the nucleocapsid protein of
SARS-CoV-2 and thus may augment the armamentarium of vaccines for
COVID-19.
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