Calcium phosphate nanoparticles conjugated with outer membrane vesicle of Riemerella anatipestifer for vaccine development in ducklings.

Biodegradable calcium phosphate nanoparticles offer a viable substitute for traditional adjuvants such as aluminum in vaccine production. Calcium phosphate nanoparticle adjuvanted with outer membrane vesicle (OMV) of gram negative bacteria may induce efficient immune response in the host. The present study was carried out to evaluate the potential of a mucosal vaccine formulation of calcium phosphate (CAP) nanoparticle using OMV of Riemerella anatipestifer (RA) as antigen against New Duck disease in ducks. The work was initiated with isolation, identification of RA, followed by OMV production and extraction. The CAP-OMV nanoparticle was prepared and characterized. The efficacy of the vaccine formulation and toxicity were studied in ducks. The average OMV yield in terms of protein concentration was found to be 122.33 ± 3.48 mg per liter of BHI broth. In SDS-PAGE, isolated OMVs exhibited presence of 16 distinct protein bands with molecular weight ranging from 142.1 to 12.1 kDa. Seven protein bands of 74.1, 69.3, 55.5, 50.6, 45.6, 25.1 and 13.1 kDa were detected relatively more distinct. The major bands detected in our findings were 42 kDa, 37 kDa and 16 kDa that corresponds to OmpA, OmpH, P6 respectively. The mean zeta size (±SD) and potential of the nanoparticle were 246.20 ± 0.53 nm and -25.60 ± 5.97 respectively. In transmission electron microscopy (TEM), the nanoparticles exhibited an average diameter of 129.80 ± 11.10 nm and displayed spherical morphology. The median protective dose (PD50) of CAP-OMV nanoparticle was 1881.10 µg of protein. Group I ducks received 3762 µg of protein (entrapped protein in CAP-OMV nanoparticle) via intra nasal route and it showed the highest serum IgG and secretory IgA level than other immunized groups. All experimental ducks were challenged with 10 × LD50 on 35 days of post primary immunization. Group I showed 100 % survivability in the challenge study. No gross and biochemical indication of acute or chronic toxicity were recorded. In conclusion, our results suggest that CAP-OMV nanoparticle can be a safe and efficient mucosal vaccine delivery system for RA, eliciting strong immune response in the host.

CpG dinucleotide enrichment in the influenza A virus genome as a live attenuated vaccine development strategy.

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens' eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines.

Ferritin nanocages as efficient nanocarriers and promising platforms for COVID-19 and other vaccines development.

BACKGROUND: The development of safe and effective vaccines against SARS-CoV-2 and other viruses with high antigenic drift is of crucial importance to public health. Ferritin is a well characterized and ubiquitous iron storage protein that has emerged not only as a useful nanoreactor and nanocarrier, but more recently as an efficient platform for vaccine development. SCOPE OF REVIEW: This review discusses ferritin structure-function properties, self-assembly, and novel bioengineering strategies such as interior cavity and exterior surface modifications for cargo encapsulation and delivery. It also discusses the use of ferritin as a scaffold for biomedical applications, especially for vaccine development against influenza, Epstein-Barr, HIV, hepatitis-C, Lyme disease, and respiratory viruses such as SARS-CoV-2. The use of ferritin for the synthesis of mosaic vaccines to deliver a cocktail of antigens that elicit broad immune protection against different viral variants is also explored. MAJOR CONCLUSIONS: The remarkable stability, biocompatibility, surface functionalization, and self-assembly properties of ferritin nanoparticles make them very attractive platforms for a wide range of biomedical applications, including the development of vaccines. Strong immune responses have been observed in pre-clinical studies against a wide range of pathogens and have led to the exploration of ferritin nanoparticles-based vaccines in multiple phase I clinical trials. GENERAL SIGNIFICANCE: The broad protective antibody response of ferritin nanoparticles-based vaccines demonstrates the usefulness of ferritin as a highly promising and effective approaches for vaccine development.

A subtractive proteomics and immunoinformatics approach towards designing a potential multi-epitope vaccine against pathogenic Listeriamonocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, which is dangerous for pregnant women, the elderly or individuals with a weakened immune system. Individuals with leukaemia, cancer, HIV/AIDS, kidney transplant and steroid therapy suffer from immunological damage are menaced. World Health Organization (WHO) reports that human listeriosis has a high mortality rate of 20-30% every year. To date, no vaccine is available to treat listeriosis. Thereby, it is high time to design novel vaccines against L. monocytogenes. Here, we present computational approaches to design an antigenic, stable and safe vaccine against the L. monocytogenes that could help to control the infections associated with the pathogen. Three vital pathogenic proteins of L. monocytogenes, such as Listeriolysin O (LLO), Phosphatidylinositol-specific phospholipase C (PI-PLC), and Actin polymerization protein (ActA), were selected using a subtractive proteomics approach to design the multi-epitope vaccine (MEV). A total of 5 Cytotoxic T-lymphocyte (CTL) and 9 Helper T-lymphocyte (HTL) epitopes were predicted from these selected proteins. To design the multi-epitope vaccine (MEV) from the selected proteins, CTL epitopes were joined with the AAY linker, and HTL epitopes were joined with the GPGPG linker. Additionally, a human ß-defensin-3 (hBD-3) adjuvant was added to the N-terminal side of the final MEV construct to increase the immune response to the vaccine. The final MEV was predicted to be antigenic, non-allergen and non-toxic in nature. Physicochemical property analysis suggested that the MEV construct is stable and could be easily purified through the E. coli expression system. This in-silico study showed that MEV has a robust binding interaction with Toll-like receptor 2 (TLR2), a key player in the innate immune system. Current subtractive proteomics and immunoinformatics study provides a background for designing a suitable, safe and effective vaccine against pathogenic L. monocytogenes.

Calcium phosphate adjuvanted nanoparticles of outer membrane proteins of Salmonella Typhi as a candidate for vaccine development against Typhoid fever.

Introduction. The conventional adjuvants used in vaccines have limitations like induction of an imbalanced Th1 and Th2 immune response. To overcome this limitation, novel adjuvants and newer forms of existing adjuvants like calcium phosphate nanoparticles are being tested.Hypothesis/Gap Statement. Calcium phosphate adjuvanted outer membrane proteins vaccine may work as an efficient, safe and cost effective vaccine against Salmonella Typhi.Aim. Our goals were to evaluate the potential of calcium phosphate nanoparticles as an adjuvant using outer membrane proteins (Omps) of Salmonella Typhi as antigens for immune response, with montanide (commercially available adjuvant) as control, and its toxicity in rats.Methodology. Calcium phosphate adjuvanted outer membrane proteins nanoparticles were synthesized and characterized. The efficacy of vaccine formulation in mice and toxicity assay were carried out in rats.Results. The calcium phosphate nanoparticles varying in size between 20-50 nm had entrapment efficiency of 41.5% and loading capacity of 54%. The calcium phosphate nanoparticle-Omps vaccine formulation (nanoparticle-Omps) induced a strong humoral immune response, which was significantly higher than the control group for the entire period of study. In the montanide-Omps group the initial very high immune response declined steeply and then remained steady. The immune response induced by nanoparticle-Omps did not change appreciably. The cell mediated immune response as measured by lymphocyte proliferation assay and delayed type hypersensitivity test showed a higher response (P<0.01) for the nanoparticles-Omps group as compared to montanide-Omps group. The bacterial clearance assay also showed higher clearance in the nanoparticles-Omps group as compared to montanide-Omps group (approx 1.4%). The toxicity analysis in rats showed no difference in the values of toxicity biomarkers and blood chemistry parameters, revealing vaccine formulation was non-toxic in rats.Conclusion. Calcium phosphate nanoparticles as adjuvant in vaccines is safe, have good encapsulation and loading capacity and induce a strong cell mediated, humoral and protective immune response.

Withanone and Withaferin-A are predicted to interact with transmembrane protease serine 2 (TMPRSS2) and block entry of SARS-CoV-2 into cells.

Coronavirus disease 2019 (COVID-19) initiated in December 2019 in Wuhan, China and became pandemic causing high fatality and disrupted normal life calling world almost to a halt. Causative agent is a novel coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/2019-nCoV). While new line of drug/vaccine development has been initiated world-wide, in the current scenario of high infected numbers, severity of the disease and high morbidity, repurposing of the existing drugs is heavily explored. Here, we used a homology-based structural model of transmembrane protease serine 2 (TMPRSS2), a cell surface receptor, required for entry of virus to the target host cell. Using the strengths of molecular docking and molecular dynamics simulations, we examined the binding potential of Withaferin-A (Wi-A), Withanone (Wi-N) and caffeic acid phenethyl ester to TPMRSS2 in comparison to its known inhibitor, Camostat mesylate. We found that both Wi-A and Wi-N could bind and stably interact at the catalytic site of TMPRSS2. Wi-N showed stronger interactions with TMPRSS2 catalytic residues than Wi-A and was also able to induce changes in its allosteric site. Furthermore, we investigated the effect of Wi-N on TMPRSS2 expression in MCF7 cells and found remarkable downregulation of TMPRSS2 mRNA in treated cells predicting dual action of Wi-N to block SARS-CoV-2 entry into the host cells. Since the natural compounds are easily available/affordable, they may even offer a timely therapeutic/preventive value for the management of SARS-CoV-2 pandemic. We also report that Wi-A/Wi-N content varies in different parts of Ashwagandha and warrants careful attention for their use.Communicated by Ramaswamy H. Sarma.

Screening HLA-A-restricted T cell epitopes of SARS-CoV-2 and the induction of CD8+ T cell responses in HLA-A transgenic mice.

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific T cells have been found to play essential roles in host immune protection and pathology in patients with coronavirus disease 2019 (COVID-19), this study focused on the functional validation of T cell epitopes and the development of vaccines that induce specific T cell responses. A total of 120 CD8+ T cell epitopes from the E, M, N, S, and RdRp proteins were functionally validated. Among these, 110, 15, 6, 14, and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; in addition, four epitopes from the S protein displayed one amino acid that was distinct from the current SARS-CoV-2 variants. Then, 31 epitopes restricted by the HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or poly (lactic-co-glycolic acid) nanoparticles, and these vaccines elicited robust and specific CD8+ T cell responses in HLA-A2/DR1 transgenic mice as well as wild-type mice. In contrast to previous research, this study established a modified DC-peptide-PBL cell coculture system using healthy donor PBMCs to validate the in silico predicted epitopes, provided an epitope library restricted by nine of the most prevalent HLA-A allotypes covering broad Asian populations, and identified the HLA-A restrictions of these validated epitopes using competitive peptide binding experiments with HMy2.CIR cell lines expressing the indicated HLA-A allotype, which initially confirmed the in vivo feasibility of 9- or 10-mer peptide cocktail vaccines against SARS-CoV-2. These data will facilitate the design and development of vaccines that induce antiviral CD8+ T cell responses in COVID-19 patients.

Colloidal Manganese Salt Improves the Efficacy of Rabies Vaccines in Mice, Cats, and Dogs.

Rabies, caused by rabies virus (RABV), remains a serious threat to public health in most countries worldwide. At present, the administration of rabies vaccines has been the most effective strategy to control rabies. Herein, we evaluate the effect of colloidal manganese salt (Mn jelly [MnJ]) as an adjuvant of rabies vaccine in mice, cats, and dogs. The results showed that MnJ promoted type I interferon (IFN-I) and cytokine production in vitro and the maturation of dendritic cells (DCs) in vitro and in vivo. Besides, MnJ serving as an adjuvant for rabies vaccines could significantly facilitate the generation of T follicular helper (Tfh) cells, germinal center (GC) B cells, plasma cells (PCs), and RABV-specific antibody-secreting cells (ASCs), consequently improve the immunogenicity of rabies vaccines, and provide better protection against virulent RABV challenge. Similarly, MnJ enhanced the humoral immune response in cats and dogs as well. Collectively, our results suggest that MnJ can facilitate the maturation of DCs during rabies vaccination, which can be a promising adjuvant candidate for rabies vaccines. IMPORTANCE Extending the humoral immune response by using adjuvants is an important strategy for vaccine development. In this study, a novel adjuvant, MnJ, supplemented in rabies vaccines was evaluated in mice, cats, and dogs. Our results in the mouse model revealed that MnJ increased the numbers of mature DCs, Tfh cells, GC B cells, PCs, and RABV-specific ASCs, resulting in enhanced immunogenicity and protection rate of rabies vaccines. We further found that MnJ had the same stimulative effect in cats and dogs. Our study provides the first evidence that MnJ serving as a novel adjuvant of rabies vaccines can boost the immune response in both a mouse and pet model.

Chitosan and alginate salt as biomaterials are potential natural adjuvants for killed cholera vaccine.

Chitosan and alginate salts are natural biopolymers that have gained recent attention in the biomedical sectors. Their properties allow them to become potential candidates as safe, cheap, and effective vaccine adjuvants. The present study aimed to enhance the immunogenic response of a current injectable killed cholera vaccine (KCV) using chitosan and alginate salt as natural adjuvants against alum. We tested KCV adjuvanted with alum, chitosan, and sodium alginate in mice. Mice were immunized intraperitoneally with KCV adjuvanted with alum, chitosan, or alginate salt and compared with a control unadjuvanted immunized group. Humoral, cellular, and functional immune responses were evaluated in all groups. The addition of adjuvants, particularly natural adjuvants, to KCV significantly improved the immune response as demonstrated by specific antibody increase, strong proliferation effects, and high protection rate against different challenge doses of cholera strains. Our findings demonstrate that chitosan and alginate salt are superior adjuvants for boosting the KCV immune response and highlights the requirement for further vaccine development.