Efficacy of a plant-produced virus-like particle vaccine in chickens challenged with Influenza A H6N2 virus.

The efficacy, safety, speed, scalability and cost-effectiveness of producing hemagglutinin-based virus-like particle (VLP) vaccines in plants are well-established for human influenza, but untested for the massive poultry influenza vaccine market that remains dominated by traditional egg-grown oil-emulsion whole inactivated virus vaccines. For optimal efficacy, a vaccine should be closely antigenically matched to the field strain, requiring that influenza A vaccines be updated regularly. In this study, an H6 subtype VLP transiently expressed in Nicotiana benthamiana was formulated into a vaccine and evaluated for efficacy in chickens against challenge with a heterologous H6N2 virus. A single dose of the plant-produced H6 VLP vaccine elicited an immune response comparable to two doses of a commercial inactivated H6N2 vaccine, with mean hemagglutination inhibition titres of 9.3 log2 and 8.8 log2 , respectively. Compared to the non-vaccinated control, the H6 VLP vaccine significantly reduced the proportion of shedders and the magnitude of viral shedding by >100-fold in the oropharynx and >6-fold in the cloaca, and shortened oropharyngeal viral shedding by at least a week. Despite its potency, the cost of the antigenic mismatch between the inactivated H6N2 vaccine and challenge strain was evident not only in this vaccine's failure to reduce viral shedding compared to the non-vaccinated group, but its apparent exacerbation of oropharyngeal viral shedding until 21 days post-challenge. We estimate that a kilogram of plant leaf material can produce H6 VLP vaccines sufficient for between 5000 and 30 000 chickens, depending on the effective dose and whether one or two immunizations are administered.

Development of a sandwich ELISA to detect virus-like-particles in enterovirus A71 vaccines.

The goal of this paper was to develop a sandwich ELISA that can detect intact human enterovirus A71 (EV-A71) virus-like particles (VLPs) in vaccines. This assay specifically detected EV-A71 viruses from different sub-genogroups as well as EV-A71 VLPs, and treatment of VLPs with high heat and low pH reduced or completely abolished detection of the VLPs suggesting that the ELISA detected assembled particles. Using a purified VLP as a reference standard, a quantitative sandwich ELISA (Q-ELISA) was established which was used to monitor the yield and purity of the VLPs during manufacturing. Coupled with immunogenicity studies, the Q-ELISA was used to evaluate the performance of the VLPs and formalin-inactivated EV-A71 vaccine. This assay has the potential to play an important role in the development of an efficient process to produce and purify the VLPs and in examining the quality of EV-A71 vaccines.

Platform technologies for modern vaccine manufacturing.

Improved understanding of antigenic components and their interaction with the immune system, as supported by computational tools, permits a sophisticated approach to modern vaccine design. Vaccine platforms provide an effective tool by which strategically designed peptide and protein antigens are modularized to enhance their immunogenicity. These modular vaccine platforms can overcome issues faced by traditional vaccine manufacturing and have the potential to generate safe vaccines, rapidly and at a low cost. This review introduces two promising platforms based on virus-like particle and liposome, and discusses the methodologies and challenges.

Evaluation of protective efficacy induced by virus-like particles containing a Trichinella spiralis excretory-secretory (ES) protein in mice.

BACKGROUND: The frequent outbreaks of human trichinellosis globally underscore the need to develop effective vaccine. We hypothesized that a novel vaccine could improve vaccine efficacy against Trichinella spiralis. METHODS: In this study, we developed virus-like particles (VLPs) containing the 53 KDa excretory/secretory (ES) protein of T. spiralis and the influenza matrix protein 1 (M1) as a core protein, and investigated the protective efficacy of the VLPs alone or with cholera toxin (CT) in a mouse model. RESULTS: Intramuscular immunization induced T. spiralis-specific IgG, IgG1 and IgG2a antibody responses before and after challenge infections in the sera. These antibody responses were significantly enhanced in mice immunized with adjuvanted VLPs. Upon challenge infection, vaccinated mice showed significantly reduced worm burden in the diaphragm. Protective immune responses and efficacy of protection were significantly improved by immunization with VLPs together with CT adjuvant. CONCLUSIONS: Our results are informative for a better understanding of the protective immunity induced by T. spiralis VLPs, and will provide insight into designing safe and effective vaccines.

Robust manufacturing and comprehensive characterization of recombinant hepatitis E virus-like particles in Hecolin(®).

The hepatitis E virus (HEV) vaccine, Hecolin(®), was licensed in China for the prevention of HEV infection and HEV-related diseases with demonstrated safety and efficacy [1,2]. The vaccine is composed of a truncated HEV capsid protein, p239, as the sole antigen encoded by open reading frame 2 and produced using Escherichia coli platform. The production of this virus-like particle (VLP) form of the antigen was successfully scaled up 50-fold from a bench scale to a manufacturing scale. Product consistency was demonstrated using a combination of biophysical, biochemical and immunochemical methods, which revealed comparable antigen characteristics among different batches. Particle size of the nanometer scale particulate antigen and presence of key epitopes on the particle surface are two prerequisites for an efficacious VLP-based vaccine. The particle size was monitored by several different methods, which showed diameters between 20 and 30nm for the p239 particles. The thermal stability and aggregation propensity of the antigen were assessed using differential scanning calorimetry and cloud point assay under heat stress conditions. Key epitopes on the particulate antigen were analyzed using a panel of murine anti-HEV monoclonal antibodies (mAbs). The immuno reactivity to the mAbs among the different antigen lots was highly consistent when analyzed quantitatively using a surface plasmon resonance technique. Using a sandwich ELISA to probe the integrity of two different epitopes in the antigen, the specific antigenicity of multiple batches was assessed to demonstrate consistency in these critical product attributes. Overall, our findings showed that the antigen production process is robust and scalable during the manufacturing of Hecolin(®).

Analytical technologies for influenza virus-like particle candidate vaccines: challenges and emerging approaches.

Influenza virus-like particle vaccines are one of the most promising ways to respond to the threat of future influenza pandemics. VLPs are composed of viral antigens but lack nucleic acids making them non-infectious which limit the risk of recombination with wild-type strains. By taking advantage of the advancements in cell culture technologies, the process from strain identification to manufacturing has the potential to be completed rapidly and easily at large scales. After closely reviewing the current research done on influenza VLPs, it is evident that the development of quantification methods has been consistently overlooked. VLP quantification at all stages of the production process has been left to rely on current influenza quantification methods (i.e. Hemagglutination assay (HA), Single Radial Immunodiffusion assay (SRID), NA enzymatic activity assays, Western blot, Electron Microscopy). These are analytical methods developed decades ago for influenza virions and final bulk influenza vaccines. Although these methods are time-consuming and cumbersome they have been sufficient for the characterization of final purified material. Nevertheless, these analytical methods are impractical for in-line process monitoring because VLP concentration in crude samples generally falls out of the range of detection for these methods. This consequently impedes the development of robust influenza-VLP production and purification processes. Thus, development of functional process analytical techniques, applicable at every stage during production, that are compatible with different production platforms is in great need to assess, optimize and exploit the full potential of novel manufacturing platforms.

Advances in virus-like particle vaccines for filoviruses.

Ebola virus (EBOV) and Marburg virus (MARV) are among the deadliest human pathogens, with no vaccines or therapeutics available. Multiple vaccine platforms have been tested for efficacy as prophylactic pretreatments or therapeutics for prevention of filovirus hemorrhagic fever. Most successful vaccines are based on a virus-vectored approach expressing the protective glycoprotein (GP); protein-based subunit and DNA vaccines have been tested with moderate success. Virus-like particle (VLP) vaccines have realized promising results when tested in both rodents and nonhuman primates. VLPs rely on the natural properties of the viral matrix protein (VP) 40 to drive budding of filamentous particles that can also incorporate ≥ 1 other filovirus protein, including GP, VP24, and nucleoprotein (NP). Filovirus VLP vaccines have used particles containing 2 or 3 (GP and VP40, with or without NP) viral proteins generated in either mammalian or insect cells. Early studies successfully demonstrated efficacy of bivalent VLP vaccines in rodents; more recent studies have shown the ability of the VLP vaccines containing GP, NP, and VP40 to confer complete homologous protection against Ebola virus and Marburg virus in a prophylactic setting against in macaques. This review will discuss published work to date regarding development of the VLP vaccines for prevention of lethal filovirus hemorrhagic fever.