Preclinical Development of a Novel Epitope-based DNA Vaccine Candidate against SARS-CoV-2 and Evaluation of Immunogenicity in BALB/c Mice.

The protective efficacies of current licensed vaccines against COVID-19 have significantly reduced as a result of SARS-CoV-2 variants of concern (VOCs) which carried multiple mutations in the Spike (S) protein. Considering that these vaccines were developed based on the S protein of the original SARS-CoV-2 Wuhan strain, we designed a recombinant plasmid DNA vaccine based on highly conserved and immunogenic B and T cell epitopes against SARS-CoV-2 Wuhan strain and the Omicron VOC. Literature mining and bioinformatics were used to identify 6 immunogenic peptides from conserved regions of the SARS-CoV-2 S and membrane (M) proteins. Nucleotide sequences encoding these peptides representing highly conserved B and T cell epitopes were cloned into a pVAX1 vector to form the pVAX1/S2-6EHGFP recombinant DNA plasmid vaccine. The DNA vaccine was intranasally or intramuscularly administered to BALB/c mice and evaluations of humoral and cellular immune responses were performed. The intramuscular administration of pVAX1/S2-6EHGFP was associated with a significantly higher percentage of CD8+ T cells expressing IFN-γ when compared with the empty vector and PBS controls. Intramuscular or intranasal administrations of pVAX1/S2-6EHGFP resulted in robust IgG antibody responses. Sera from mice intramuscularly immunized with pVAX1/S2-6EHGFP were found to elicit neutralizing antibodies capable of SARS-CoV-2 Omicron variant with the ACE2 cell surface receptor. This study demonstrated that the DNA vaccine construct encoding highly conserved immunogenic B and T cell epitopes was capable of eliciting potent humoral and cellular immune responses in mice.

The Promising Potential of Reverse Vaccinology-Based Next-Generation Vaccine Development over Conventional Vaccines against Antibiotic-Resistant Bacteria.

The clinical use of antibiotics has led to the emergence of multidrug-resistant (MDR) bacteria, leading to the current antibiotic resistance crisis. To address this issue, next-generation vaccines are being developed to prevent antimicrobial resistance caused by MDR bacteria. Traditional vaccine platforms, such as inactivated vaccines (IVs) and live attenuated vaccines (LAVs), were effective in preventing bacterial infections. However, they have shown reduced efficacy against emerging antibiotic-resistant bacteria, including MDR M. tuberculosis. Additionally, the large-scale production of LAVs and IVs requires the growth of live pathogenic microorganisms. A more promising approach for the accelerated development of vaccines against antibiotic-resistant bacteria involves the use of in silico immunoinformatics techniques and reverse vaccinology. The bioinformatics approach can identify highly conserved antigenic targets capable of providing broader protection against emerging drug-resistant bacteria. Multi-epitope vaccines, such as recombinant protein-, DNA-, or mRNA-based vaccines, which incorporate several antigenic targets, offer the potential for accelerated development timelines. This review evaluates the potential of next-generation vaccine development based on the reverse vaccinology approach and highlights the development of safe and immunogenic vaccines through relevant examples from successful preclinical and clinical studies.

The development of DNA vaccines against SARS-CoV-2.

BACKGROUND: The COVID-19 pandemic exerted significant impacts on public health and global economy. Research efforts to develop vaccines at warp speed against SARS-CoV-2 led to novel mRNA, viral vectored, and inactivated vaccines being administered. The current COVID-19 vaccines incorporate the full S protein of the SARS-CoV-2 Wuhan strain but rapidly emerging variants of concern (VOCs) have led to significant reductions in protective efficacies. There is an urgent need to develop next-generation vaccines which could effectively prevent COVID-19. METHODS: PubMed and Google Scholar were systematically reviewed for peer-reviewed papers up to January 2023. RESULTS: A promising solution to the problem of emerging variants is a DNA vaccine platform since it can be easily modified. Besides expressing whole protein antigens, DNA vaccines can also be constructed to include specific nucleotide genes encoding highly conserved and immunogenic epitopes from the S protein as well as from other structural/non-structural proteins to develop effective vaccines against VOCs. DNA vaccines are associated with low transfection efficiencies which could be enhanced by chemical, genetic, and molecular adjuvants as well as delivery systems. CONCLUSIONS: The DNA vaccine platform offers a promising solution to the design of effective vaccines. The challenge of limited immunogenicity in humans might be solved through the use of genetic modifications such as the addition of nuclear localization signal (NLS) peptide gene, strong promoters, MARs, introns, TLR agonists, CD40L, and the development of appropriate delivery systems utilizing nanoparticles to increase uptake by APCs in enhancing the induction of potent immune responses.

Development of Next Generation Vaccines against SARS-CoV-2 and Variants of Concern.

SARS-CoV-2 has caused the COVID-19 pandemic, with over 673 million infections and 6.85 million deaths globally. Novel mRNA and viral-vectored vaccines were developed and licensed for global immunizations under emergency approval. They have demonstrated good safety and high protective efficacy against the SARS-CoV-2 Wuhan strain. However, the emergence of highly infectious and transmissible variants of concern (VOCs) such as Omicron was associated with considerable reductions in the protective efficacy of the current vaccines. The development of next-generation vaccines that could confer broad protection against both the SARS-CoV-2 Wuhan strain and VOCs is urgently needed. A bivalent mRNA vaccine encoding the Spike proteins of both the SARS-CoV-2 Wuhan strain and the Omicron variant has been constructed and approved by the US FDA. However, mRNA vaccines are associated with instability and require an extremely low temperature (-80 °C) for storage and transportation. They also require complex synthesis and multiple chromatographic purifications. Peptide-based next-generation vaccines could be developed by relying on in silico predictions to identify peptides specifying highly conserved B, CD4+ and CD8+ T cell epitopes to elicit broad and long-lasting immune protection. These epitopes were validated in animal models and in early phase clinical trials to demonstrate immunogenicity and safety. Next-generation peptide vaccine formulations could be developed to incorporate only naked peptides, but they are costly to synthesize and production would generate extensive chemical waste. Continual production of recombinant peptides specifying immunogenic B and T cell epitopes could be achieved in hosts such as E. coli or yeast. However, recombinant protein/peptide vaccines require purification before administration. The DNA vaccine might serve as the most effective next-generation vaccine for low-income countries, since it does not require an extremely low temperature for storage or need extensive chromatographic purification. The construction of recombinant plasmids carrying genes specifying highly conserved B and T cell epitopes meant that vaccine candidates representing highly conserved antigenic regions could be rapidly developed. Poor immunogenicity of DNA vaccines could be overcome by the incorporation of chemical or molecular adjuvants and the development of nanoparticles for effective delivery.

miRNA: A Promising Therapeutic Target in Cancer.

microRNAs are small non-coding RNAs that regulate several genes post-transcriptionally by complementarity pairing. Since discovery, they have been reported to be involved in a variety of biological functions and pathologies including cancer. In cancer, they can act as a tumor suppressor or oncomiR depending on the cell type. Studies have shown that miRNA-based therapy, either by inhibiting an oncomiR or by inducing a tumor suppressor, is effective in cancer treatment. This review focusses on the role of miRNA in cancer, therapeutic approaches with miRNAs and how they can be effectively delivered into a system. We have also summarized the patents and clinical trials in progress for miRNA therapy.

Magnitude and pattern of anxiety levels with gender wise predilection of coping strategies amid resident doctors of emergency department.

Objectives: To screen and assess the severity level of anxiety, its influencing factors along with the gender-wise predilection of coping strategies among resident doctors working in accident and emergency departments. Methods: A transverse study was conducted amongst 260 resident doctors of accident and emergency department of different teaching hospitals of Karachi from October 2020 until March 2021. A demographic sheet containing questions related to factors, GAD-7 (Generalized Anxiety Disorder) and Brief COPE were used to measure the severity level of anxiety and coping strategies. Data was scored according to the standard scoring procedure for each subscale and for further statistical analysis SPSS Version 21 was used. Results: Out of all participants, 68.1% were <30 years of age, 63.1% were females while 54.2% were single. The findings of the study showed the prevalence of anxiety within the range of normal (38.1%), mild (35.0%), moderate (16.9%) and severe (10.0%). Gender (p= 0.001), marital status (p= 0.040) and job satisfaction (p= 0.007) among resident doctors were significantly associated with level of anxiety. Deemed to coping strategies, the most frequently were planning (n=145, 90.0%), acceptance (n=141, 87.6%), and religion (n=137, 85.1 %). All coping strategies were mostly opted by females except substance abuse. Conclusion: More than a half of the resident doctors manifested with mild to severe anxiety disorder, which highlights the need for psychological support programs to minimize anxiety levels and to ensure a healthy environment at workplace for the health practitioners.

Identification of B-Cell Epitopes for Eliciting Neutralizing Antibodies against the SARS-CoV-2 Spike Protein through Bioinformatics and Monoclonal Antibody Targeting.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global public health crisis. Effective COVID-19 vaccines developed by Pfizer-BioNTech, Moderna, and Astra Zeneca have made significant impacts in controlling the COVID-19 burden, especially in reducing the transmission of SARS-CoV-2 and hospitalization incidences. In view of the emergence of new SARS-CoV-2 variants, vaccines developed against the Wuhan strain were less effective against the variants. Neutralizing antibodies produced by B cells are a critical component of adaptive immunity, particularly in neutralizing viruses by blocking virus attachment and entry into cells. Therefore, the identification of protective linear B-cell epitopes can guide epitope-based peptide designs. This study reviews the identification of SARS-CoV-2 B-cell epitopes within the spike, membrane and nucleocapsid proteins that can be incorporated as potent B-cell epitopes into peptide vaccine constructs. The bioinformatic approach offers a new in silico strategy for the mapping and identification of potential B-cell epitopes and, upon in vivo validation, would be useful for the rapid development of effective multi-epitope-based vaccines. Potent B-cell epitopes were identified from the analysis of three-dimensional structures of monoclonal antibodies in a complex with SARS-CoV-2 from literature mining. This review provides significant insights into the elicitation of potential neutralizing antibodies by potent B-cell epitopes, which could advance the development of multi-epitope peptide vaccines against SARS-CoV-2.