Peptide-TLR7/8a-Coordinated DNA Vaccines Elicit Enhanced Immune Responses against Infectious Diseases.

DNA vaccines represent an innovative approach for the immunization of diverse diseases. However, their clinical trial outcomes are constrained by suboptimal transfection efficiency and immunogenicity. In this work, we present a universal methodology involving the codelivery of Toll-like receptor 7/8 agonists (TLR7/8a) and antigen gene using TLR7/8a-conjugated peptide-coated poly(ß-amino ester) (PBAE) nanoparticles (NPs) to augment delivery efficiency and immune response. Peptide-TLR7/8a-coated PBAE NPs exhibit advantageous biophysical attributes, encompassing diminutive particle dimensions, nearly neutral ζ potential, and stability in the physiological environment. This synergistic approach not only ameliorates the stability of plasmid DNA (pDNA) and gene delivery efficacy but also facilitates subsequent antigen production. Furthermore, under optimal formulation conditions, the TLR7/8a-conjugated peptide coated PBAE NPs exhibit a potent capacity to induce robust immune responses. Collectively, this nanoparticulate gene delivery system demonstrates heightened transfection efficacy, stability, biodegradability, immunostimulatory effect, and low toxicity, making it a promising platform for the clinical advancement of DNA vaccines.

Construction of Toxoplasma gondii SRS29C nucleic acid vaccine and comparative immunoprotective study of an SRS29C and SAG1 combination.

Toxoplasma gondii is an intracellular protozoan parasite that infects all nucleated cells except the red blood cells. Currently, nucleic acid vaccines are being widely investigated in Toxoplasma gondii control, and several nucleic acid vaccine candidate antigens have shown good protection in various studies. The aim of this study was to construct a nucleic acid vaccine with Toxoplasma gondii SRS29C as the target gene. We explored the nucleic acid vaccine with Toxoplasma surface protein SRS29C and the combined gene of SRS29C and SAG1 and evaluated its immunoprotective effect against Toxoplasma gondii. To amplify the gene fragment and clone it to the expression vector, the recombinant plasmid pEGFP-SRS29C was constructed by PCR. Eukaryotic cells were transfected with the plasmid, and the expression of the target protein was assessed using the Western blot method. The level of serum IgG was determined via ELISA, and the splenic lymphocyte proliferation ability was detected using the CCK-8 method. The percentages of CD4+ and CD8+ T cells were measured by flow cytometry. Mice were immunised three times with single-gene nucleic acid vaccine and combination vaccine. Splenic lymphocytokine expression was determined using ELISA kits. The mice's survival time was monitored and recorded during an in vivo insect assault experiment, and the vaccine's protective power was assessed. The outcomes showed that PCR-amplification of an SRS29C gene fragment was successful. The 4,733-bp vector fragment and the 1,119-bp target segment were both recognised by double digestion. Additionally, after transfection of the recombinant plasmid pEGFP-SRS29C, Western blot examination of the extracted protein revealed the presence of a target protein strip at 66 kDa. The test results demonstrated that the IgG content in the serum of the pEGFP-SRS29C group and the co-immunization group was significantly higher than that of the PBS group and the empty vector group. The IgG potency induced by the co-immunization group was higher than that of the pEGFP-SRS29C group and the pEGFP-SAG1 group, the number of splenic lymphocyte proliferation number was higher than that of the PBS group and the empty vector group. The CD4+/CD8+ T ratio was higher than that of the PBS group and the empty vector group. The expression of IFN-γ and TNF-α in the splenocytes of the pEGFP-SRS29C group and the combined immunisation group was significantly higher following antigen stimulation. In the worm attack experiments, mice in the PBS and empty vector groups perished within 9 days of the worm attack, whereas mice in the pEGFP-SRS29C group survived for 18 days, mice in the pEGFP-SAG1 group survived for 21 days, and mice in the co-immunization group survived for 24 days. This demonstrates that the constructed Toxoplasma gondii nucleic acid vaccine pEGFP-SRS29C and the combined gene vaccine can induce mice to develop certain humoral and cellular immune responses, and enhance their ability to resist Toxoplasma gondii infection.

Plasmid targeting and destruction by the DdmDE bacterial defence system.

Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.

Immunogenic properties of empty pcDNA3 plasmid against zoonotic cutaneous leishmaniasis in mice.

BACKGROUND: Leishmania (L) parasite, the causative agent of zoonotic cutaneous leishmaniasis (ZCL), effectively stimulates the mammalian cells to mount strong humoral responses by enhancing T-helper-2 (Th2)-associated cytokines for its survival. The best strategy to decrease the intensity of infection in the host is induction of cellular immunity. METHODS: We evaluated the effects of the empty bacterial pcDNA3 plasmid on mice infected with L. major and quantified the immune mediators including IFN-γ, IL-4, IL-10, IgG2a, IgG1, arginase activity and nitric oxide (NO) in the mice. Moreover, the footpad lesion size and parasite load were assessed. RESULTS: We observed that pcDNA3 could modulate the immune responses in favor of host cells and decrease the disease severity. Th2- associated mediators, including arginase, IL-4, and IL-10 are downregulated, while cellular responses are upregulated in line with an increase in the levels of nitric oxide (NO) and interfero-gamma (IFN-γ). Interestingly, pcDNA3 induced specific Th1-associated antibodies, IgG2a isotype; however, it suppressed the production of humoral IgG1. The stimulation of the immune response by the empty pcDNA3 is able to shift the immune function to predominant cellular responses caused by Th1, and it had a positive effect on the treatment of zoonotic cutaneous leishmaniasis (ZCL). CONCLUSIONS: Altogether, we introduced the pcDNA3 as a potential interfering factor in the modulation of the immune system against ZCL. Since this vector has been widely used as a control group in different studies, we suggest that the potential function of the empty vector should be deeply assessed, as it exerts anti-parasitic effects on mice infected with L. major.

The Invasin and Complement-Resistance Protein Rck of Salmonella is More Widely Distributed than Previously Expected.

The rck open reading frame (ORF) on the pefI-srgC operon encodes an outer membrane protein responsible for invasion of nonphagocytic cell lines and resistance to complement-mediated killing. Until now, the rck ORF was only detected on the virulence plasmids of three serovars of Salmonella subsp. enterica (i.e., Bovismorbificans, Enteritidis, and Typhimurium). The increasing number of Salmonella genome sequences allowed us to use a combination of reference sequences and whole-genome multilocus sequence typing (wgMLST) data analysis to probe the presence of the operon and of rck in a wide array of isolates belonging to all Salmonella species and subspecies. We established the presence of partial or complete operons in 61 subsp. enterica serovars as well as in 4 other subspecies with various syntenies and frequencies. The rck ORF itself was retrieved in 36 subsp. enterica serovars and in two subspecies with either chromosomal or plasmid-borne localization. It displays high conservation of its sequence within the genus, and we demonstrated that most of the allelic variations identified did not alter the virulence properties of the protein. However, we demonstrated the importance of the residue at position 38 (at the level of the first extracellular loop of the protein) in the invasin function of Rck. Altogether, our results highlight that rck is not restricted to the three formerly identified serovars and could therefore have a more important role in virulence than previously expected. Moreover, this work raises questions about the mechanisms involved in rck acquisition and about virulence plasmid distribution and evolution. IMPORTANCE The foodborne pathogen Salmonella is responsible for a wide variety of pathologies depending on the infected host, the infecting serovars, and its set of virulence factors. However, the implication of each of these virulence factors and their role in the specific host-pathogen interplay are not fully understood. The significance of our research is in determining the distribution of one of these factors, the virulence plasmid-encoded invasin and resistance to complement killing protein Rck. In addition to providing elements of reflection concerning the mechanisms of acquisition of specific virulence genes in certain serotypes, this work will help to understand the role of Rck in the pathogenesis of Salmonella.

Nucleic acid vaccines and CpG oligodeoxynucleotides for allergen immunotherapy.

PURPOSE OF REVIEW: Molecular forms of allergen-specific immunotherapy (AIT) are continuously emerging to improve the efficacy of the treatment, to shorten the duration of protocols and to prevent any side effects. The present review covers the recent progress in the development of AIT based on nucleic acid encoding allergens or CpG oligodeoxynucleotides (CpG-ODN). RECENT FINDINGS: Therapeutic vaccinations with plasmid deoxyribonucleic acid (DNA) encoding major shrimp Met e 1 or insect For t 2 allergen were effective for the treatment of food or insect bite allergy in respective animal models. DNA expressing hypoallergenic shrimp tropomyosin activated Foxp3+ T regulatory (Treg) cells whereas DNA encoding For t 2 down-regulated the expression of pruritus-inducing IL-31. Co-administrations of major cat allergen Fel d 1 with high doses of CpG-ODN reduced Th2 airway inflammation through tolerance induction mediated by GATA3+ Foxp3hi Treg cells as well as early anti-inflammatory TNF/TNFR2 signaling cascade. Non-canonical CpG-ODN derived from Cryptococcus neoformans as well as methylated CpG sites present in the genomic DNA from Bifidobacterium infantis mediated Th1 or Treg cell differentiation respectively. SUMMARY: Recent studies on plasmid DNA encoding allergens evidenced their therapeutic potential for the treatment of food allergy and atopic dermatitis. Unmethylated or methylated CpG-ODNs were shown to activate dose-dependent Treg/Th1 responses. Large clinical trials need to be conducted to confirm these promising preclinical data. Moreover, tremendous success of messenger ribonucleic acid (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 must encourage as well the re-exploration of mRNA vaccine platform for innovative AIT.

Tumor perfusion evaluation using dynamic contrast-enhanced ultrasound after electrochemotherapy and IL-12 plasmid electrotransfer in murine melanoma.

Electrochemotherapy with bleomycin (ECT BLM) is an effective antitumor treatment already used in clinical oncology. However, ECT alone is still considered a local antitumor therapy because it cannot induce systemic immunity. When combined with adjuvant gene electrotransfer of plasmid DNA encoding IL-12 (GET pIL-12), the combined therapy leads to a systemic effect on untreated tumors and distant metastases. Although the antitumor efficacy of both therapies alone or in combination has been demonstrated at both preclinical and clinical levels, data on the predictors of efficacy of the treatments are still lacking. Herein, we evaluated the results of dynamic contrast-enhanced ultrasound (DCE-US) as a predictive factor for ECT BLM and GET pIL-12 in murine melanoma. Melanoma B16F10 tumors grown in female C57Bl/6NCrl mice were treated with GET pIL-12 and ECT BLM. Immediately after therapy, 6 h and 1, 3, 7 and 10 days later, tumors were examined by DCE-US. Statistical analysis was performed to inspect the correlation between tumor doubling time (DT) and DCE-US measurements using semilinear regression models and Bland-Altman plots. Therapeutic groups in which DCE-US showed reduced tumor perfusion had longer tumor DTs. It was confirmed that the DCE-US parameter peak enhancement (PE), reflecting relative blood volume, had predictive value for the outcome of therapy: larger PE correlated with shorter DT. In addition, perfusion heterogeneity was also associated with outcome: tumors that had more heterogeneous perfusion had faster growth, i.e., shorter DTs. This study demonstrates that DCE-US can be used as a method to predict the efficacy of electroporation-based treatment.

Impact of a Plasmid DNA-Based Alphavirus Vaccine on Immunization Efficiency.

Alphavirus vectors have been engineered for high-level gene expression relying originally on replication-deficient recombinant particles, more recently designed for plasmid DNA-based administration. As alphavirus-based DNA vectors encode the alphavirus RNA replicon genes, enhanced transgene expression in comparison to conventional DNA plasmids is achieved. Immunization studies with alphavirus-based DNA plasmids have elicited specific antibody production, have generated tumor regression and protection against challenges with infectious agents and tumor cells in various animal models. A limited number of clinical trials have been conducted with alphavirus DNA vectors. Compared to conventional plasmid DNA-based immunization, alphavirus DNA vectors required 1000-fold less DNA to elicit similar immune responses in rodents.

Molecular Adjuvants for DNA Vaccines: Application, Design, Preparation, and Formulation.

Compared with conventional vaccines, the main advantage of DNA vaccine-based methods is its continued expression of the plasmid-encoded antigens followed by the induction of subsequent humoral and cellular immunities. DNA vaccines are currently used in animal models, but limited success has been obtained for use in clinical applications due to their poor immunogenicity. Various strategies are attempted to improve the induced immune response of DNA vaccines. It has been demonstrated that co-administration of molecular adjuvants with DNA vaccines is a promising approach to effectively elicit protective immunity by increasing the transfection efficiency of DNA vaccines. Genetic adjuvants are incorporated to promote activation of the transfected local antigen-presenting cells (APCs) and immune cells in the draining lymph node and polarization of T-cell subsets to decrease T-cell tolerance to the specific antigen. Here we provide an overview of different types of genetic adjuvants. The aim of the current chapter is to present a framework for the construction of a gene-based vaccine and adjuvant. Moreover, we describe the application of DNA vaccines co-administered with different types of genetic adjuvants and the methods to evaluate their potency in the mouse models.

Enhanced Biosynthesis of Plasmid DNA from Escherichia coli Applying Experimental Design.

Therapeutic applications of plasmid DNA (pDNA) have significantly advanced during the last years. Currently, several pDNA-based drugs are already in the market, whereas several others have entered phases 2 and 3 of clinical trials. The present and future demand for pDNA requires the development of efficient bioprocesses to produce it. Commonly, pDNA is produced by cultures of Escherichia coli. It has been previously demonstrated that specific strains of E. coli with a modified substrate transport system can be able to attain high cell densities in batch mode, due to the very low overflow metabolism displayed. However, the large amounts of oxygen demanded can lead to microaerobic conditions after some hours of cultivation, even at small scale. Typically, the inherent problems for these cultures are the high oxygen demand and the accumulation of acetate, a metabolic byproduct that is synthesized aerobically when the glucose rate exceeds the limits.In recent years, several researches have been focused on the study of induction of plasmid DNA as well as strategies for fermentation using semi-defined mediums. These studies conceived relevant results that allow us to design a production platform for enhanced plasmid DNA. So, the main goal of this chapter is to show how the development of an experimental design directed to aromatic amino acids pathway can improve the yield of a therapeutic plasmid DNA by culture of a new strain of Escherichia coli VH33.

Main Features of DNA-Based Vectors for Use in Lactic Acid Bacteria and Update Protocols.

DNA vaccines have been used as a promising strategy for delivery of immunogenic and immunomodulatory molecules into the host cells. Although, there are some obstacles involving the capability of the plasmid vector to reach the cell nucleus in great number to promote the expected benefits. In order to improve the delivery and, consequently, increase the expression levels of the target proteins carried by DNA vaccines, alternative methodologies have been explored, including the use of non-pathogenic bacteria as delivery vectors to carry, deliver, and protect the DNA from degradation, enhancing plasmid expression.

Cross-Protection against MERS-CoV by Prime-Boost Vaccination Using Viral Spike DNA and Protein.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness and has a high mortality of ∼34%. However, since its discovery in 2012, an effective vaccine has not been developed for it. To develop a vaccine against multiple strains of MERS-CoV, we targeted spike glycoprotein (S) using prime-boost vaccination with DNA and insect cell-expressed recombinant proteins for the receptor-binding domain (RBD), S1, S2, SΔTM, or SΔER. Our S subunits were generated using an S sequence derived from the MERS-CoV EMC/2012 strain. We examined humoral and cellular immune responses of various combinations with DNA plasmids and recombinant proteins in mice. Mouse sera immunized with SΔER DNA priming/SΔTM protein boosting showed cross-neutralization against 15 variants of S-pseudovirions and the wild-type KOR/KNIH/002 strain. In addition, these immunizations provided full protection against the KOR/KNIH/002 strain challenge in human DPP4 knock-in mice. These findings suggest that vaccination with the S subunits derived from one viral strain can provide cross-protection against variant MERS-CoV strains with mutations in S. DNA priming/protein boosting increased gamma interferon production, while protein-alone immunization did not. The RBD subunit alone was insufficient to induce neutralizing antibodies, suggesting the importance of structural conformation. In conclusion, heterologous DNA priming with protein boosting is an effective way to induce both neutralizing antibodies and cell-mediated immune responses for MERS-CoV vaccine development. This study suggests a strategy for selecting a suitable platform for developing vaccines against MERS-CoV or other emerging coronaviruses.IMPORTANCE Coronavirus is an RNA virus with a higher mutation rate than DNA viruses. Therefore, a mutation in S-protein, which mediates viral infection by binding to a human cellular receptor, is expected to cause difficulties in vaccine development. Given that DNA-protein vaccines promote stronger cell-mediated immune responses than protein-only vaccination, we immunized mice with various combinations of DNA priming and protein boosting using the S-subunit sequences of the MERS-CoV EMC/2012 strain. We demonstrated a cross-protective effect against wild-type KOR/KNIH/002, a strain with two mutations in the S amino acids, including one in its RBD. The vaccine also provided cross-neutralization against 15 different S-pseudotyped viruses. These suggested that a vaccine targeting one variant of S can provide cross-protection against multiple viral strains with mutations in S. The regimen of DNA priming/Protein boosting can be applied to the development of other coronavirus vaccines.

Construction and identification of influenza plasmid pool imparting high yields to candidate vaccine viruses in Vero cell at low temperature.

We generated plasmid pools for the rapid preparation of candidate vaccine strains, which could grow in the Vero cells at low temperature. Firstly, we cloned in the pHW2000 plasmid each of the eight gene segments (PB2, PB1, PA, hemagglutinin [HA], neuraminidase [NA], NS, NP, M) of two master donor strains (MDS), respectively, A/Yunnan/1/2005Vca(H3N2) and B/Yunnan/2/2005Vca(By), which had Vca phenotype (cold-adapted phenotype in Vero cells). Secondly, the similar operation was implemented with each of the HA, NA and NP segments of circulating strains with epidemic potential (parental strains). The virus rescue techniques were employed in this study, according to the homology rate of HA segments between MDS and parental strains. Then, we harvested amount of new Vca virus strains. By transmission electron microscope, it could observe new viruses' diameter and length were from 100 to 120 nm. Importantly, these reassortant viruses could get high-yield production in Vero cells at 25℃ from the beginning to the fourth generation, which was significantly differ from their original parental viruses. Additional, these production 16 new Vca strains could maintain enough antibody binding capacity and attenuation phenotype, which consisted with their MDS. So these plasmid pools constructed by mount of different influenza A and B virus gene fragments could present desired working performance and provide convenience and realization for more Vca reassortant virus as candidate vaccine strain if needing.

Enhancement of a biotechnological platform for the purification and delivery of a human papillomavirus supercoiled plasmid DNA vaccine.

New biotechnological strategies are being explored, aimed at rapid and economic manufacture of large quantities of DNA vaccines with the required purity for therapeutic applications, as well as their correct delivery as biopharmaceuticals to target cells. This report describes the purification of supercoiled (sc) HPV-16 E6/E7 plasmid DNA (pDNA) vaccine from a bacterial lysate, using an arginine-based monolith, presenting a spacer arm in its configuration. To enhance the performance of the purification process, monolith modification with the spacer arm can improve accessibility of the arginine ligand. By using a low NaCl concentration at pH 7.0, a condition to eliminate the RNA impurity directly in the flow through was established. The pH increase to 7.5 allowed the elimination of non-functional pDNA isoforms, the sc pDNA being recovered by increasing the ionic strength. As well as a binding capacity of 2.53 mg/mL obtained with a pre-purified sc pDNA sample, the column also purified sc pDNA from high lysate loading, with capacities above 1 mg/mL. Due to the sample displacement phenomena, non-functional pDNA isoforms were eliminated throughout column loading, favoring the degree of purity of final sc pDNA of 93.3%-98.5%. Thereafter, purified sc pDNA was successfully encapsulated into CaCO3-gelatin nano-complexes. Delivery of the pDNA-carriers to THP-1 cells was assessed through pDNA cellular uptake evaluation and correct E6 expression was verified by mRNA and protein detection. A biotechnological platform was established for sc pDNA purification and delivery to dendritic cells, stimulating further in vivo studies.

Astrovirus reverse genetics systems, a story of success.

Astroviruses are one of the main causes of gastroenteritis of medical and veterinary relevance worldwide. Recently, these viruses were associated with neurological disease in mammals, including humans. Reverse genetics systems are the most powerful tool to improve our understanding of the virus replication, and eventually to develop safe vaccine candidates. In the present review, it is summarized the current knowledge on the different strategies used to develop reverse genetics systems for mamastroviruses and avastroviruses, and some of the biological answers that have provided are discussed.

Reverse genetics approaches of Borna disease virus: applications in development of viral vectors and preventive vaccines.

The plasmid-based reverse genetics system, which involves generation of recombinant viruses from cloned cDNA, has accelerated the understanding of clinical and virological aspects of different viruses. Borna disease virus (BoDV) is a nonsegmented, negative-strand RNA virus that causes persistent intranuclear infection in various vertebrate species. Since its first report, reverse genetics approaches with modified strategies have greatly improved rescue efficiency of recombinant BoDV and enhanced the understanding of function of each viral protein and mechanism of intranuclear persistency. Here, we summarize different reverse genetics approaches of BoDV and recent developments in the use of reverse genetics for generation of viral vectors for gene therapy and virus-like particles for potential preventive vaccines.

Co-occurrence of a novel VIM-1 and FosA3-encoding multidrug-resistant plasmid and a KPC-2-encoding pKP048-like plasmid in a clinical isolate of Klebsiella pneumoniae sequence type 11.

The spread of carbapenem-resistant Enterobacteriaceae (CRE) worldwide remains a major threat to public health. Notably, carbapenemase-encoding genes are usually located in plasmids harboring other resistance determinants, and isolates having multiple plasmids are often highly resistant to carbapenems. In this study, we characterized the genetic context of coproduction of KPC-2, VIM-1, and FosA3 via two plasmids in the multidrug-resistant Klebsiella pneumoniae sequence type 11 (ST11) isolate JS187, recovered during an outbreak of KPC-2-producing K. pneumoniae in a Chinese teaching hospital in 2008. Plasmid p187-1, coharboring blaVIM-1 and fosA3, consisted of a pKOX-R1-like backbone and two multidrug resistant (MDR) regions separated by pKHS1-like backbone sequences involving plasmid replication and stability. The MDR region 1 was a chimera composed of the blaVIM-1-bearing In916-like integron and Tn1721-like transposon, and was disrupted by sequential insertion of an IS26-based transposition unit carrying blaCTX-M-3 and a ΔTn3-like transposon bearing blaTEM-1. MDR region 2 was an IS26-array structure with fosA3 and blaSHV-12. Plasmid p187-2 harboring blaKPC-2 was closely related to pKP048. blaKPC-2 in p187-2 was carried by a Tn1721 variant, which differed from the prototype Tn1721-blaKPC-2 transposon observed in pKP048 by disruption of an IS26 at Tn3 and deletion of a 31-kb MDR fragment. Co-existence of the novel VIM-1- and FosA3-encoding MDR plasmid p187-1 and the KPC-2-encoding pKP048-like plasmid p187-2 made K. pneumoniae JS187 highly resistant to carbapenems. Moreover, p187-1 still carried genes conferring resistance to cephalosporins, fosfomycin, chloramphenicol, and quaternary ammonium, posing substantial challenges for the treatment of Enterobacteriaceae infections. Thus, monitoring the prevalence and evolution of these plasmids and/or strains is critical.

Potential of plasmid only based reverse genetics of rotavirus for the development of next-generation vaccines.

Plasmid only based reverse genetics (RG) systems for species A rotaviruses (RVA) are available since 2017 and are beginning to be explored in structure-function and viral replication studies as well as for testing of antivirals. The rescue of human rotavirus genomes into viable particles, the expression of heterologous genes from fusion constructs with rotavirus genes, and the possibility to obtain safely attenuated recombinant rotaviruses are of great promise for the production of next-generation rotavirus vaccine candidates. In the context attention is drawn to issues, which arose during the development of reverse genetics-created recombinant influenza virus vaccine candidates.

Immunoinformatics-guided designing of epitope-based subunit vaccines against the SARS Coronavirus-2 (SARS-CoV-2).

SARS Coronavirus-2 (SARS-CoV-2) pandemic has become a global issue which has raised the concern of scientific community to design and discover a counter-measure against this deadly virus. So far, the pandemic has caused the death of hundreds of thousands of people upon infection and spreading. To date, no effective vaccine is available which can combat the infection caused by this virus. Therefore, this study was conducted to design possible epitope-based subunit vaccines against the SARS-CoV-2 virus using the approaches of reverse vaccinology and immunoinformatics. Upon continual computational experimentation, three possible vaccine constructs were designed and one vaccine construct was selected as the best vaccine based on molecular docking study which is supposed to effectively act against the SARS-CoV-2. Thereafter, the molecular dynamics simulation and in silico codon adaptation experiments were carried out in order to check biological stability and find effective mass production strategy of the selected vaccine. This study should contribute to uphold the present efforts of the researches to secure a definitive preventative measure against this lethal disease.

The development of ghost vaccines trials.

INTRODUCTION: Bacterial ghosts are intact bacterial cell envelopes that are emptied of their content by gentle biological or chemical poring methods. Ghost techniques increase the safety of the killed vaccines, while maintaining their antigenicity due to mild preparation procedures. Moreover, ghost-platforms may express and/or carry several antigens or plasmid-DNA encoding for protein epitopes. AREAS COVERED: In this review, the development in ghost-vaccine production over the last 30 years is classified and discussed. The different applications of ghost-vaccines, how they trigger the immune system, their advantages and limitations are displayed. The phage-mediated lysis, molecular manipulation of the lysis-genes, and the biotechnological production of ghosts are described. The trials are classified according to the pattern of lysis and to the type of bacteria. Further subdivision includes chronological ordered application of the ghost as alternative-killed vaccine, recombinant antigen platform, plasmid DNA carrier, adjuvants, and dendritic cell inducer. Particular trials for specific pathogens or from distinct research schools are gathered. EXPERT OPINION: Ghosts are highly qualified to act as immune-presenting platforms that express and/or carry several recombinant and DNA vaccines, as well as, being efficient alternative-killed vaccines. The coming years will show more molecular advances to develop ghost-production and to express more antigens.