Eimeria maxima Rhomboid-like Protein 5 Provided Partial Protection against Homologous Challenge in Forms of Recombinant Protein and DNA Plasmid in Chickens

Eimeria maxima (E. maxima) is one of the most prevalent species that causes chicken coccidiosis on chicken farms. During apicomplexan protozoa invasion, rhomboid-like proteins (ROMs) cleave microneme proteins (MICs), allowing the parasites to fully enter the host cells, which suggests that ROMs have the potential to be candidate antigens for the development of subunit or DNA vaccines against coccidiosis. In this study, a recombinant protein of E. maxima ROM5 (rEmROM5) was expressed and purified and was used as a subunit vaccine. The eukaryotic expression plasmid of pVAX–EmROM5 was constructed and was used as a DNA vaccine. Chickens who were two weeks old were vaccinated with the rEmROM5 and pVAX–EmROM5 vaccines twice, with a one-week interval separating the vaccination periods. The transcription and expression of pVAX–EmROM5 in the injected sites were detected through reverse transcription PCR (RT-PCR) and Western blot (WB) assays. The cellular and humoral immune responses that were induced by EmROM5 were determined by detecting the proportion of CD4+ and CD8+ T lymphocytes, the cytokine levels, and the serum antibody levels. Finally, vaccination-challenge trials were conducted to evaluate the protective efficacy of EmROM5 in forms of the recombinant protein (rEmROM5) and in the DNA plasmid (pVAX–EmROM5) separately. The results showed that rEmROM5 was about 53.64 kDa, which was well purified and recognized by the His-Tag Mouse Monoclonal antibody and the chicken serum against E. maxima separately. After vaccination, pVAX–EmROM5 was successfully transcribed and expressed in the injected sites of the chickens. Vaccination with rEmROM5 or pVAX–EmROM5 significantly promoted the proportion of CD4+/CD3+ and CD8+/CD3+ T lymphocytes, the mRNA levels of the cytokines IFN-γ, IL-2, IL-4, IL-17, TNF SF15, and IL-10, and specific IgG antibody levels compared to the control groups. The immunization also significantly reduced the weight loss, oocyst production, and intestinal lesions that are caused by E. maxima infection. The anticoccidial index (ACI)s of the vaccinated groups were beyond 160, showing moderate protection against E. maxima infection. In summary, EmROM5 was able to induce a robust immune response and effective protection against E. maxima in chickens in the form of both a recombinant protein and DNA plasmid. Hence, EmROM5 could be used as a candidate antigen for DNA vaccines and subunit vaccines against avian coccidiosis.

COVID-eVax, an electroporated plasmid DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain, elicits protective immune responses in animal models of COVID-19

The COVID-19 pandemic caused by the β-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax – a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD – induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.

A Mathematical Model to Expedite Electroporation Based Vaccine Development for COVID-19

Electroporation has an application in the selective delivery of drugs explicitly into cells. However, the challenge is to achieve efficiency in delivering the drugs. The key parameter responsible for successful electroporation-mediated drug delivery is induced transmembrane voltage (ITMV). The Food & Drug Administration (FDA) has recently approved the clinical trials of DNA plasmid delivery of the COVID-19 vaccine through electroporation. The requirement is to develop a COVID-19 vaccine within a limited time. Hence, the extensive amount of laboratory experiments are not feasible. It has increased dependency on simulation-based analysis. The simulations of electroporation depend on ITMV expression for the specified cell and the environment. In this paper, we have derived the closed-form expression of ITMV (∆Vm). The closed-form expression is used in COMSOL Multiphysics simulation to obtain extracellular concentration variation as a function of time. The simulation results match the empirical results from the literature and hence validate the closed-form expression. The closed-form expression will reduce the development time of electroporation-assisted COVID-19 vaccine delivery.

Respiratory FimA-Specific Secretory IgA Antibodies Upregulated by DC-Targeting Nasal Double DNA Adjuvant Are Essential for Elimination of Porphyromonas gingivalis

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.

PANDEMIC AND “INFODEMIC”

Dear Reader, With the passage of about one year since the outbreak of the COVID-19 pandemic, huge amounts of scientific research data on COVID-19 are being generated daily and we have thus substantial information and understanding of the disease. While treatment with known anti-viral drugs, monoclonal antibodies, steroids and APIs continues, focus is now on the development and manufacturing of effective vaccines to protect the population at large. Several Indian companies are already conducting pan-India clinical trials of different vaccines. Bharat Biotech, in cooperation with the Indian Council of Medical Research and the National Institute of Virology, has developed a whole-virion inactivated vaccine on an inactivated platform, which will be administered intramuscularly in two doses (0, 28d). Biological E. is clinically evaluating its adjuvant protein subunit (RBD) vaccine on a protein subunit platform, again administered intramuscularly in two doses (0, 28d). Cadila Healthcare has developed a DNA plasmid vaccine on a DNA platform, administered intradermally in three doses (0, 28, 56d). Dr. Reddy’s Laboratories, together with the Russian Direct Investment Fund, will be conducting clinical trials of the Sputnik V adenovirus-based vaccine on non-replicating viral vector platform (developed by the Gamaleya National Research Institute). The Serum Institute of India, together with ICMR, is conducting clinical trials of the ChAdOx1-S AstraZeneca-Oxford vaccine on a non-replicating viral vector, also administered intramuscularly in two doses (0, 28d).

Immunogenic Potential of DNA Vaccine candidate, ZyCoV-D against SARS-CoV-2 in Animal Models

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We developed a candidate vaccine ZyCoV-D comprising of a DNA plasmid vector carrying the gene encoding the spike protein (S) of the SARS-CoV-2 virus. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500μg dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also provided Th-1 response as evidenced by elevated IFN-γ levels.

CRISPR-Cas Systems: Prospects for Use in Medicine

CRISPR-Cas systems, widespread in bacteria and archaea, are mainly responsible for adaptive cellular immunity against exogenous DNA (plasmid and phage). However, the latest research shows their involvement in other functions, such as gene expression regulation, DNA repair and virulence. In recent years, they have undergone intensive research as convenient tools for genomic editing, with Cas9 being the most commonly used nuclease. Gene editing may be of interest in biotechnology, medicine (treatment of inherited disorders, cancer, etc.), and in the development of model systems for various genetic diseases. The dCas9 system, based on a modified Cas9 devoid of nuclease activity, called CRISPRi, is widely used to control gene expression in bacteria for new drug biotargets validation and is also promising for therapy of genetic diseases. In addition to direct use for genomic editing in medicine, CRISPR-Cas can also be used in diagnostics, for microorganisms’ genotyping, controlling the spread of drug resistance, or even directly as “smart” antibiotics. This review focuses on the main applications of CRISPR-Cas in medicine, and challenges and perspectives of these approaches.

Immunotherapeutic Activities of a DNA Plasmid Carrying the Mycobacterial hsp65 Gene (DNAhsp65)

DNA vaccines have become relevant subject matter, and efforts for their development have been increasing due to their potential as technology platforms applicable for prophylactic and therapeutic approaches for infectious diseases and for cancer treatment, allergies, and autoimmune diseases. This review aimed to summarize current knowledge about the plasmid DNA vaccine carrying the mycobacterial hsp65 gene (DNAhsp65), which demonstrates immunomodulatory and immunoregulatory properties of both the innate and adaptive immune systems. The possible mechanisms associated with the modulation and regulatory role of DNAhsp65 in the control of various conditions is also discussed.