Virosome: An engineered virus for vaccine delivery.

The purpose of immunization is the effective cellular and humoral immune response against antigens. Several studies on novel vaccine delivery approaches such as micro-particles, liposomes & nanoparticles, etc. against infectious diseases have been investigated so far. In contrast to the conventional approaches in vaccine development, a virosomes-based vaccine represents the next generation in the field of immunization because of its balance between efficacy and tolerability by virtue of its mechanism of immune instigation. The versatility of virosomes as a vaccine adjuvant, and delivery vehicle of molecules of different nature, such as peptides, nucleic acids, and proteins, as well as provide an insight into the prospect of drug targeting using virosomes. This article focuses on the basics of virosomes, structure, composition formulation and development, advantages, interplay with the immune system, current clinical status, different patents highlighting the applications of virosomes and their status, recent advances, and research associated with virosomes, the efficacy, safety, and tolerability of virosomes based vaccines and the future prospective.

Cell-free protein synthesis of influenza virus hemagglutinin HA2-integrated virosomes for siRNA delivery.

It is well known that the difficulty of siRNA therapeutic application is the lack of safe and effective delivery vector. Virosome is a nano vesicle composed of lipid membrane and membrane protein. It retains fusion protein without virus genetic material, and therefore has the reduced immunogenicity compared with viral vector. Virosomes have the potential to deliver protein and nucleic acid drugs, but the traditional preparation method of virosomes is quite limited. In this study, we firstly proposed to synthesize influenza virus hemagglutinin HA2 virosomes by cell-free protein synthesis. In this study, liposomes provided the hydrophobic lipid bilayer environment for the formation of HA2 protein multimer, which inhibited the aggregation of hydrophobic HA2 and improved HA2 protein expression. Chitosan as a rigid core adsorbed siRNA and improved the encapsulation efficiency of siRNA. In conclusion, the cell-free protein synthesis was used to prepare HA2 virosomes, which paves the way for constructing a novel nano vector with high delivery efficiency and biosafety for the delivery of siRNA.

Comparative efficacy and safety of vaccines to prevent seasonal influenza: A systematic review and network meta-analysis.

Background: Influenza is one of the most common respiratory viral infections worldwide. Numerous vaccines are used to prevent influenza. Their selection should be informed by the best available evidence. We aimed to estimate the comparative efficacy and safety of seasonal influenza vaccines in children, adults and the elderly. Methods: We conducted a systematic review and network meta-analysis (NMA). We searched the Cochrane Library Central Register of Controlled Trials, MEDLINE and EMBASE databases, and websites of regulatory agencies, through December 15th, 2020. We included placebo- or no vaccination-controlled, and head-to-head randomized clinical trials (RCTs). Pairs of reviewers independently screened the studies, abstracted the data, and appraised the risk of bias in accordance to the Cochrane Handbook for Systematic Reviews of Interventions. The primary outcome was laboratory-confirmed influenza. We also synthesized data for hospitalization, mortality, influenza-like illness (ILI), pneumonia or lower respiratory-tract disease, systemic and local adverse events (AEs). We estimated summary risk ratios (RR) using pairwise and NMA with random effects. This study is registered with PROSPERO, number CRD42018091895. Findings: We identified 13,439 citations. A total of 231 RCTs were included after screening: 11 studies did not provide useful data for the analysis; 220 RCTs [100,677 children (< 18 years) and 329,127 adults (18-60 years) and elderly (≥ 61 years)] were included in the NMA. In adults and the elderly, all vaccines, except the trivalent inactivated intradermal vaccine (3-IIV ID), were more effective than placebo in reducing the risk of laboratory-confirmed influenza, with a RR between 0.33 (95% credible interval [CrI] 0.21-0.55) for trivalent inactivated high-dose (3-IIV HD) and 0.56 (95% CrI 0.41-0.74) for trivalent live-attenuated vaccine (3-LAIV). In adults and the elderly, compared with trivalent inactivated vaccine (3-IIV), no significant differences were found for any, except 3-LAIV, which was less efficacious [RR 1.41 (95% CrI 1.04-1.88)]. In children, compared with placebo, RR ranged between 0.13 (95% CrI 0.03-0.51) for trivalent inactivated vaccine adjuvanted with MF59/AS03 and 0.55 (95% CrI 0.36-0.83) for trivalent inactivated vaccine. Compared with 3-IIV, 3-LAIV and trivalent inactivated adjuvanted with MF59/AS03 were more efficacious [RR 0.52 (95% CrI 0.32-0.82) and RR 0.23 (95% CrI 0.06-0.87)] in reducing laboratory-confirmed influenza. With regard to safety, higher systemic AEs rates after vaccination with 3-IIV, 3-IIV HD, 3-IIV ID, 3-IIV MF59/AS03-adj, quadrivalent inactivated (4-IIV), quadrivalent adjuvanted (4-IIV MF59/AS03-adj), quadrivalent recombinant (4-RIV), 3-LAIV or quadrivalent live attenuated (4-LAIV) vaccines were noted in adults and the elderly [RR 1.5 (95% CrI 1.18-1.89) to 1.15 (95% CrI 1.06-1.23)] compared with placebo. In children, the systemic AEs rate after vaccination was not significantly higher than placebo. Interpretation: All vaccines cumulatively achieved major reductions in the incidence of laboratory-confirmed influenza in children, adults, and the elderly. While the live-attenuated was more efficacious than the inactivated vaccine in children, many vaccine types can be used in adults and the elderly. Funding: The directorate general of welfare, Lombardy region.

Virosome-based nanovaccines; a promising bioinspiration and biomimetic approach for preventing viral diseases: A review.

Vaccination is the most effective means of controlling infectious disease-related morbidity and mortality. However, due to low immunogenicity of viral antigens, nanomedicine as a new opportunity in new generation of vaccine advancement attracted researcher encouragement. Virosome is a lipidic nanomaterial emerging as FDA approved nanocarriers with promising bioinspiration and biomimetic potency against viral infections. Virosome surface modification with critical viral fusion proteins is the cornerstone of vaccine development. Surface antigens at virosomes innovatively interact with targeted receptors on host cells that evoke humoral or cellular immune responses through antibody-producing B cell and internalization by endocytosis-mediated pathways. To date, several nanovaccine based on virosome formulations have been commercialized against widespread and life-threatening infections. Recently, Great efforts were made to fabricate a virosome-based vaccine platform against a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Thus, this review provides a novel overview of the virosome based nanovaccine production, properties, and application on the viral disease, especially its importance in SARS-CoV-2 vaccine discovery.

Comparison of Virosome vs. Liposome as drug delivery vehicle using HepG2 and CaCo2 cell lines.

AIM: The present work involves encapsulation of herbal drug nanocurcumin into the virosomes and compared with a liposome in terms of their in vitro anti-proliferative, anti-inflammatory, and anti-migratory efficacy. METHODS: The anti-proliferative, anti-inflammatory, and anti-migratory efficacy of virosome and liposome were compared in HepG2 and CaCo2 cells by using MTT, Nitric oxide scavenging, and Wound healing assay, respectively. RESULTS: Size of the optimised NC-Virosome and NC-Liposome was 70.06 ± 1.63 and 265.80 ± 1.64 nm, respectively. The prepared NC-Virosome can be stored at -4 °C up to six months. The drug encapsulation efficiency of NC-Virosome and NC-Liposome was found to be 84.66 ± 1.67 and 62.15 ± 1.75% (w/w). The evaluated minimum inhibitory concentration (IC50 value) for NC-Virosome was 102.7 µg/ml and 108.1 µg/ml, while NC-Liposome showed 129.2 µg/ml and 160.1 µg/ml for HepG2 and CaCo2 cells, respectively. Morphological examination depicts detachment of the cells from substratum after exposure to NC-Virosome for 48 h. CONCLUSION: The prepared NC-Virosome provides remarkable in vitro efficacy in both the cell lines with site-specific drug-targeting potential as compared to the liposome, results proved its potential as a drug delivery vehicle for future therapy with reduced toxicity.

Niosomal virosome derived by vesicular stomatitis virus glycoprotein as a new gene carrier.

Virosomes as membranous vesicles with viral fusion protein in their membrane are versatile vehicles for cargo delivery. The vesicular stomatitis virus glycoprotein (VSV-G) is a common fusogenic protein used in virosome preparation. This glycoprotein has been used in liposomal systems so far, but in this study, we have tried to use the niosomal form instead of liposome for. Niosomes are vesicular systems composed of non-ionic surfactants. Niosomes were constructed by the thin-film hydration method. VSV-G gene in pMD2.G plasmid was expressed in the HEK293T cell line and then was reconstituted in the niosome bilayer. The formation of niosomal virosomes was confirmed with different methods such as SDS-PAGE gel, western blotting, and transmission electron microscopy (TEM). The efficiency of niosomal virosome was investigated with the pmCherry reporter gene. SDS-PAGE and western blotting proved the expression and successful insertion of protein into the bilayer. The TEM images showed the spike projection of VSV-G on the surface of niosomes. The transfection results showed high efficiency of niosomal virosomes as a novel carrier. This report has verified that niosome could be used as an efficient bilayer instead of liposome to construct virosomes.

Targeted Delivery of CRISPR/Cas13 as a Promising Therapeutic Approach to Treat SARS-CoV-2.

On a worldwide scale, the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to extensive damage to the health system as well as the global economy. Hitherto, there has been no approved drug or vaccine for this disease. Therefore, the use of general antiviral drugs is at the first line of treatment, though complicated with limited effectiveness and systemic side effects. Given the pathophysiology of the disease, researchers have proposed various strategies not only to find a more specific therapeutic way but also to reduce the side effects. One strategy to accomplish these goals is to use CRISPR/Cas13 system. Recently, a group of scientists has used the CRISPR/Cas13 system, which is highly effective in eliminating the genome of RNA viruses. Due to the RNA nature of the coronavirus genome, it seems that this system can be effective against the disease. The main challenge regarding the application of this system is to deliver it to the target cells efficiently. To solve this challenge, it seems that using virosomes with protein S on their membrane surface can be helpful. Studies have shown that protein S interacts with its specific receptor in target cells named Angiotensin-Converting Enzyme 2 (ACE2). Here, we propose if CRISPR/Cas13 gene constructs reach the infected cells efficiently using a virosomal delivery system, the virus genome will be cleaved and inactivated. Considering the pathophysiology of the disease, an important step to implement this hypothesis is to embed protein S on the membrane surface of virosomes to facilitate the delivery of gene constructs to the target cells.

Site-specific phosphorylation of villin remodels the actin cytoskeleton to regulate Sendai viral glycoprotein-mediated membrane fusion.

Connivance of cellular factors during virus-host cell membrane fusion is poorly understood. We have recently shown that cellular villin plays an important role during membrane fusion of reconstituted Sendai virosomes with hepatocytes. Here, we employed villin-null Chinese Hamster Ovary (CHO) cells, where villin expression led to an increased fusion with virosomes, which was further enhanced due to tyrosine phosphorylation in the presence of c-src. However, the villin RRI mutant, lacking actin-severing function, failed to augment membrane fusion. Furthermore, quantitative mass spectrometry and detailed analysis revealed Tyr499 to be the key phosphorylation site of villin responsible for the enhancement of virosome-CHO cell fusion. Overall, our results demonstrate a critical role for villin and its cell-type dependent phosphorylation in regulating membrane fusion.

Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner.

The Ebola virus glycoprotein (EBOV-GP) forms GP-containing microvesicles, so-called virosomes, which are secreted from GP-expressing cells. However, determinants of GP-virosome release and their functionality are poorly understood. We characterized GP-mediated virosome formation and delineated the role of the antiviral factor tetherin (BST2, CD317) in this process. Residues in the EBOV-GP receptor-binding domain (RBD) promote GP-virosome secretion, while tetherin suppresses GP-virosomes by interactions involving the GP-transmembrane domain. Tetherin from multiple species interfered with GP-virosome release, and tetherin from the natural fruit bat reservoir showed the highest inhibitory activity. Moreover, analyses of GP from various ebolavirus strains, including the EBOV responsible for the West African epidemic, revealed the most efficient GP-virosome formation by highly pathogenic ebolaviruses. Finally, EBOV-GP-virosomes were immunomodulatory and acted as decoys for EBOV-neutralizing antibodies. Our results indicate that GP-virosome formation might be a determinant of EBOV immune evasion and pathogenicity, which is suppressed by tetherin.

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells.

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24 h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.

A novel chimeric influenza virosome containing Vesicular stomatitis G protein as a more efficient gene delivery system.

OBJECTIVES: To enhance the efficiency of influenza virosome-mediated gene delivery by engineering this virosome. RESULTS: A novel chimeric influenza virosome was constructed containing the glycoprotein of Vesicular stomatitis virus (VSV-G), along with its own hemagglutinin protein. To optimize the transfection efficiency of both chimeric and influenza cationic virosomes, HEK cells were transfected with plasmid DNA and virosomes and the transfection efficiency was assessed by FACS analysis. The chimeric virosome was significantly more efficient in mediating transfection for all amounts of DNA and virosomes compared to the influenza virosome. CONCLUSIONS: Chimeric influenza virosome, including VSV-G, is superior to the conventional influenza virosome for gene delivery.

An H1-H3 chimeric influenza virosome confers complete protection against lethal challenge with PR8 (H1N1) and X47 (H3N2) viruses in mice.

Annual health threats and economic damages caused by influenza virus are still a main concern of the World Health Organization and other health departments all over the world. An influenza virosome is a highly efficient immunomodulating carrier mimicking the natural antigen presentation pathway and has shown an excellent tolerability profile due to its biocompatibility and purity. The major purpose of this study was to construct a new chimeric virosome influenza vaccine containing hemagglutinin (HA) and neuraminidase (NA) proteins derived from the A/PR/8/1934 (H1N1) (PR8) and A/X/47 (H3N2) (X47) viruses, and to evaluate its efficacy as a vaccine candidate in mice. A single intramuscular vaccination with the chimeric virosomes provided complete protection against lethal challenge with the PR8 and X47 viruses. The chimeric virosomes induced high IgG antibody responses as well as hemagglutination inhibition (HAI) titers. HAI titers following the chimeric virosome vaccination were at the same level as the whole inactivated influenza vaccine. Mice immunized with the chimeric virosomes displayed considerably less weight loss and exhibited significantly reduced viral load in their lungs compared with the controls. The chimeric virosomes can be used as an innovative vaccine formulation to confer protection against a broad range of influenza viruses.

Status and progress of research on mucosal vaccines / 中国药学杂志

OBJECTIVE: Mucosal surfaces are a major portal of entry for many human pathogens that are the cause of infectious diseases. Vaccines that immunize by mucosal routes may induce protective immunity against mucosal pathogens at their sites of entry thus to be more effective and economical. We try to overview the status and progress of research on mucosal vaccines. METHODS: The databases of CNKI and Pubmed were used to search the related articles about mucosal vaccines with key words "mucosal vaccine, mucosal adjuvant, mucosal particulate delivery vectors" in Chinese and English. Articles closely related to mucosal vaccines were selected. RESULTS: Thirty-six articles were included at last. Live-attenuated or inactivated mucosal vaccines and vaccines based on new concepts such as subunit vaccines, virus-like particles and virosomes have been marketed, and related research work are undergoing. Safe and effective mucosal adjuvants and delivery vectors are being sought to enhance the magnitude and quality of the protective immune response. The composition, size, surface chemistry and ligands of particulate carrier systems may influence the efficacy. Great progress has been made in several particulate delivery systems. CONCLUSION: Although the research and development of mucosal vaccines are facing many difficulties and challenges, the progress of research work will bring new opportunities to mucosal vaccines development.

Head-to-head comparison of an intradermal and a virosome influenza vaccine in patients over the age of 60: evaluation of immunogenicity, cross-protection, safety and tolerability.

In the present study we first compare immunogenicity against vaccine and heterologous circulating A(H1N1)pdm09 strains, tolerability and safety of intradermal Intanza 15 µg and of virosomal adjuvanted, intramuscularly delivered influenza vaccine, Inflexal V, in healthy elderly volunteers. Five-hundred participants were enrolled in the study and randomly assigned to the two vaccine groups to receive either one dose of Intanza 15 µg or Inflexal V vaccine. All subjects reported solicited local and systemic reactions occurred within 7 d after vaccination and unsolicited adverse events up to 21 d post-immunization and any serious adverse event appeared during the study. A subset of 55 participants was randomly selected for immunogenicity and cross-protection evaluations. Serum samples were collected before and 1 and 3 mo after immunization. Antibody responses were measured using hemagglutination inhibition (HI) against all viruses used in the study and neutralization (NT) assays against A(H1N1)pdm09 strains. At least one of the CHMP criteria for influenza vaccine approval in the elderly was met by virosomal vaccine against all the tested viruses; intradermal vaccine met all criteria against all strains. Several parameters of immune response against strains with a different antigenic pattern from that of vaccine A/California/04/09(H1N1)pdm09 were significantly higher in the intradermal vaccine group compared with the virosomal group. Safety and systemic tolerability of both vaccines were excellent, but injection site reactions occurred significantly more frequently in the intradermal vaccination group. Immunogenicity of Intanza 15 µg intradermal vaccine tended to be higher than that of Inflexal V against heterologous strains in healthy elderly.