Corrigendum to "PRAK-03202: A triple antigen virus-like particle vaccine candidate against SARS CoV-2"[Heliyon 7 (10) (October 2021) e08124].

[This corrects the article DOI: 10.1016/j.heliyon.2021.e08124.].

PRAK-03202: A triple antigen virus-like particle vaccine candidate against SARS CoV-2.

The rapid development of safe and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is a necessary response to coronavirus outbreak. Here, we developed PRAK-03202, the world's first triple antigen virus-like particle vaccine candidate, by cloning and transforming SARS-CoV-2 gene segments into a highly characterized S. cerevisiae-based D-Crypt™ platform, which induced SARS CoV-2 specific neutralizing antibodies in BALB/c mice. Immunization using three different doses of PRAK-03202 induced an antigen-specific (spike, envelope, and membrane proteins) humoral response and neutralizing potential. Peripheral blood mononuclear cells from convalescent patients showed lymphocyte proliferation and elevated interferon levels suggestive of epitope conservation and induction of T helper 1-biased cellular immune response when exposed to PRAK-03202. These data support further clinical development and testing of PRAK-03202 for use in humans.

Small-molecule quinolinol inhibitor identified provides protection against BoNT/A in mice.

Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC(50) values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).

Deletion mutant comprising 198 residues of BoNT/A toxin receptor binding domain retained gt1b binding property but failed to induce protective antibody response in a mouse model.

The most effective protection against toxin is inducing protective immune response through vaccination that will produce neutralizing antibodies. Antibodies will bind to and clear toxin from the circulation before it can enter nerve cells and block neurotransmission and can also be used for development of detection system. In the present study we constructed a deletion mutant of the binding domain (1098-1296) to produce smallest toxin fragment as vaccine candidate against BoNT/A. The BoNT/A-HCC protein was highly expressed in Escherichia coli SG13009 and found to form inclusion bodies. The purified inclusion bodies were solubilized in 6 M guanidine-HCl containing 10 mM ß-mercaptoethanol and 20 mM imidazole and the rBoNT/A-HCC was purified and refolded in a single step on Ni2+ affinity column. The purified protein was ∼98 % pure as assessed by SDS-polyacrylamide gel with the yield of 8 mg/L and showed binding to polysialoganglioside (GT1b). The rBoNT/A-HCC at dose of 40 µg/mouse generated high IgG antibody titre with predominance of IgG1 subtype, but failed to protect animals against BoNT/A challenge. Antibody titre in serum was determined by enzyme linked immunosorbent assay and specific binding to rBoNT/A-HCC was demonstrated by surface plasmon resonance (SPR), with a dissociation constant of 0.8 pM.

Recombinant Shiga toxin B subunit elicits protection against Shiga toxin via mixed Th type immune response in mice.

Shigella dysenteriae is the causative agent of the third commonest bacterial disease for childhood diarrhoea and responsible for millions of deaths per year. It produces potent toxin termed Shiga toxin which is listed in category B biological warfare agent of CDC, USA. Earlier we have reported production of recombinant Shiga toxin B subunit that produced antibodies which neutralized Shiga toxin toxicity in HeLa cells. In the present study, we have evaluated the immunomodulatory potential of rStxB protein in Balb/c mice using Freunds adjuvants. Animal protection with recombinant StxB was conferred through both humoral and cellular immune responses as indicated by an increased antibody titre with predominance of IgG2a and IgG2b isotypes along with elevated levels of IgG1 subtype. Cytokine profile of the mice antiserum and splenocyte also indicates Th2 and Th1 type of immune responses where former dominates in early stage of immunization. Histopathology study of kidneys of vaccinated mice showed remarkable differences when compared to the animals infected with Shigella dysenteriae type1. The present study indicates that recombinant StxB is a promising vaccine candidate and can be used for production of therapeutic antibodies to counter Shiga intoxication.

Prevention of aggregation and autocatalysis for sustaining biological activity of recombinant BoNT/A-LC upon long-term storage.

Protein aggregation during expression, purification, storage, or transfer into requisite assay buffers hampers the use of proteins for in vitro studies. The formation of these aggregates represents a major obstacle in the study of biological activity and also restricts the spectrum of protein products being available for the biomedical applications. The catalytic light chain of botulinum neurotoxin type A undergoes autocatalysis and aggregation after purification upon long-term storage and freeze-thawing. In present study the conditions for the high level expression and purification of biologically active light chain protein of botulinum neurotoxin were optimized from a synthetic gene. Several co-solvents were screened in order to prevent autocatalysis and aggregation of rBoNT/A-LC. The effect of the co-solvents is studied on endopeptidase activity during long term storage of the recombinant protein. The purified rBoNT/A-LC was also evaluated for its immunogenicity.

Characterization of LC-HCC fusion protein of botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The gene for encoding the full length light chain with H(CC) (binding) domain of Clostridium botulinum neurotoxin A was synthesized and cloned into a bacterial expression vector pQE30-UA and produced as an N-terminally six-histidine-tagged fusion protein (rBoNT/A LC-H(CC)). This protein was expressed in two different strains of Escherichia coli namely BL21(DE3) and SG13009. Expression at 37 °C revealed localization of rBoNT/A LC- H(CC) in inclusion body whereas it was expressed in soluble form at 21°C. The recombinant fusion protein was purified by nickel affinity gel column chromatography and identified by monoclonal antibody and peptide mass fingerprinting. The recombinant protein was shown to bind with synaptic vesicles and gangliosides (GT1b) using enzyme-linked immunosorbent assay. The rBoNT/A LC-H(CC) was also found to be highly active on its substrate (SNAP-25) from rat brain, indicating that the expressed and purified rBoNT/A LC-H(CC) protein retains a functionally active conformation. Biologically active recombinant fusion protein was also evaluated for its immunological potential.

Botulinum toxin: bioweapon & magic drug.

Botulinum neurotoxins, causative agents of botulism in humans, are produced by Clostridium botulinum, an anaerobic spore-former Gram positive bacillus. Botulinum neurotoxin poses a major bioweapon threat because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need for prolonged intensive care among affected persons. A single gram of crystalline toxin, evenly dispersed and inhaled, can kill more than one million people. The basis of the phenomenal potency of botulinum toxin is enzymatic; the toxin is a zinc proteinase that cleaves neuronal vesicle associated proteins responsible for acetylcholine release into the neuromuscular junction. As a military or terrorist weapon, botulinum toxin could be disseminated via aerosol or by contamination of water or food supplies, causing widespread casualties. A fascinating aspect of botulinum toxin research in recent years has been development of the most potent toxin into a molecule of significant therapeutic utility . It is the first biological toxin which is licensed for treatment of human diseases. In the late 1980s, Canada approved use of the toxin to treat strabismus, in 2001 in the removal of facial wrinkles and in 2002, the FDA in the United States followed suit. The present review focuses on both warfare potential and medical uses of botulinum neurotoxin.

Antibodies against recombinant shiga toxin subunit B neutralize shiga toxin toxicity in HeLa cells.

Shigella dysenteriae type 1 and Escherichia coli O157:H7 produce Shiga toxin 1 (Stx) and Shiga toxin1 (Stx1), respectively and these two toxins are almost identical. E. coli O157:H7 is the major cause of diarrhea-associated hemolytic uremic syndrome. Stx and Stx1 are AB5 type of toxin with a molecular weight of 70 kDa, comprising an enzymaticaly-active A subunit (32 kDa) and five receptor-binding B subunits (7.7 kDa). In this study DNA fragment (289 bp, Gene Bank Accn No. EF685161) coding for B chain of Stx was amplified from S. dysenteriae type1 and cloned. Shiga toxin-binding subunit was expressed and purified in native conditions by affinity and gel permeation chromatography with the yield of 5.1 mg/L in shake flask culture. For the purpose of immunization, the polypeptide was polymerized with glutaraldehyde. Hyper immune serum produced in mice reacted with the purified polypeptide and intact Shiga toxin. The anti-StxB antiserum effectively neutralized the cytotoxicity of Shiga toxin towards HeLa cells.