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1.
Pharmaceutics ; 14(4):856, 2022.
Article in English | MDPI | ID: covidwho-1785876

ABSTRACT

The SARS-CoV-2 virus has caused a global crisis, resulting in 0.5 billion infections and over 6 million deaths as of March 2022. Fortunately, infection and hospitalization rates were curbed due to the rollout of DNA and mRNA vaccines. However, the efficacy of these vaccines significantly drops a few months post immunization, from 88% down to 47% in the case of the Pfizer BNT162 vaccine. The emergence of variant strains, especially delta and omicron, have also significantly reduced vaccine efficacy. We propose peptide vaccines as a potential solution to address the inadequacies of the current vaccines. Peptide vaccines can be easily modified to target emerging strains, have greater stability, and do not require cold-chain storage. We screened five peptide fragments (B1–B5) derived from the SARS-CoV-2 spike protein to identify neutralizing B-cell peptide antigens. We then investigated adjuvant systems for efficient stimulation of immune responses against the most promising peptide antigens, including liposomal formulations of polyleucine (L10) and polymethylacrylate (PMA), as well as classical adjuvants (CFA and MF59). Immune efficacy of formulations was evaluated using competitive ELISA, pseudovirion neutralization, and live virus neutralization assays. Unfortunately, peptide conjugation to L10 and PMA dramatically altered the secondary structure, resulting in low antibody neutralization efficacy. Of the peptides tested, only B3 administered with CFA or MF59 was highly immunogenic. Thus, a peptide vaccine relying on B3 may provide an attractive alternative to currently marketed vaccines.

2.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-321184

ABSTRACT

This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin-converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include: 1) serum heat treatment to prevent non-specific interactions, 2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, 3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and 4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 hours for >30 serum samples;this includes the 7.5 hours of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious, time-consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R 2 =0.975, n=25), demonstrating high sensitivity.

3.
Vaccines (Basel) ; 9(12)2021 Dec 16.
Article in English | MEDLINE | ID: covidwho-1580388

ABSTRACT

This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the 'gold standard' assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2 = 0.975, n = 25), demonstrating high sensitivity.

4.
Molecules ; 26(2)2021 Jan 15.
Article in English | MEDLINE | ID: covidwho-1389464

ABSTRACT

Peptides are fragments of proteins that carry out biological functions. They act as signaling entities via all domains of life and interfere with protein-protein interactions, which are indispensable in bio-processes. Short peptides include fundamental molecular information for a prelude to the symphony of life. They have aroused considerable interest due to their unique features and great promise in innovative bio-therapies. This work focusing on the current state-of-the-art short peptide-based therapeutical developments is the first global review written by researchers from all continents, as a celebration of 100 years of peptide therapeutics since the commencement of insulin therapy in the 1920s. Peptide "drugs" initially played only the role of hormone analogs to balance disorders. Nowadays, they achieve numerous biomedical tasks, can cross membranes, or reach intracellular targets. The role of peptides in bio-processes can hardly be mimicked by other chemical substances. The article is divided into independent sections, which are related to either the progress in short peptide-based theranostics or the problems posing challenge to bio-medicine. In particular, the SWOT analysis of short peptides, their relevance in therapies of diverse diseases, improvements in (bio)synthesis platforms, advanced nano-supramolecular technologies, aptamers, altered peptide ligands and in silico methodologies to overcome peptide limitations, modern smart bio-functional materials, vaccines, and drug/gene-targeted delivery systems are discussed.


Subject(s)
Anti-Infective Agents/pharmacology , Antiviral Agents/pharmacology , Peptides/chemistry , Peptides/pharmacology , Peptides/therapeutic use , Amino Acids/chemistry , Anti-Infective Agents/chemistry , Antiviral Agents/chemistry , COVID-19/drug therapy , Computer Simulation , Cosmeceuticals/chemistry , Cosmeceuticals/therapeutic use , Dietary Supplements , Gene Transfer Techniques , Humans , Lactoferrin/chemistry , Lipid Bilayers , Nanostructures/administration & dosage , Nanostructures/chemistry , Peptides/administration & dosage , Stem Cells , Vaccines, Subunit/chemistry , Vaccines, Subunit/pharmacology
5.
Adv Sci (Weinh) ; 8(16): e2100985, 2021 08.
Article in English | MEDLINE | ID: covidwho-1281196

ABSTRACT

COVID-19 is disastrous to global health and the economy. SARS-CoV-2 infection exhibits similar clinical symptoms and immunopathological sequelae to SARS-CoV infection. Therefore, much of the developmental progress on SARS-CoV vaccines can be utilized for the development of SARS-CoV-2 vaccines. Careful antigen selection during development is always of utmost importance for the production of effective vaccines that do not compromise recipient safety. This holds especially true for SARS-CoV vaccines, as several immunopathological disorders are associated with the activity of structural and nonstructural proteins encoded in the virus's genetic material. Whole viral protein and RNA-encoding full-length proteins contain both protective and "dangerous" sequences, unless pathological fragments are deleted. In light of recent advances, peptide vaccines may present a very safe and effective alternative. Peptide vaccines can avoid immunopathological pro-inflammatory sequences, focus immune responses on neutralizing immunogenic epitopes, avoid off-target antigen loss, combine antigens with different protective roles or mechanisms, even from different viral proteins, and avoid mutant escape by employing highly conserved cryptic epitopes. In this review, an attempt is made to exploit the similarities between SARS-CoV and SARS-CoV-2 in vaccine antigen screening, with particular attention to the pathological and immunogenic properties of SARS proteins.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Viral Vaccines/immunology , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , Humans , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology , Viral Vaccines/administration & dosage
6.
Clin Transl Immunology ; 10(3): e1260, 2021.
Article in English | MEDLINE | ID: covidwho-1120050

ABSTRACT

OBJECTIVES: A major COVID-19 vaccine strategy is to induce antibodies that prevent interaction between the Spike protein's receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2). These vaccines will also induce T-cell responses. However, concerns were raised that aberrant vaccine-induced immune responses may exacerbate disease. We aimed to identify minimal epitopes on the RBD that would induce antibody responses that block the interaction of the RBD and ACE2 as a strategy leading to an effective vaccine with reduced risk of inducing immunopathology. METHODS: We procured a series of overlapping 20-amino acid peptides spanning the RBD and asked which were recognised by plasma from COVID-19 convalescent patients. Identified epitopes were conjugated to diphtheria-toxoid and used to vaccinate mice. Immune sera were tested for binding to the RBD and for their ability to block the interaction of the RBD and ACE2. RESULTS: Seven putative vaccine epitopes were identified. Memory B-cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains. CONCLUSION: COVID-19 convalescent patients have SARS-CoV-2-specific antibodies and MBCs, the specificities of which can be defined with short peptides. Epitope-specific antibodies synergistically block RBD-ACE2 interaction.

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