Protective efficacy of a plant-produced beta variant rSARS-CoV-2 VLP vaccine in golden Syrian hamsters.

In the quest for heightened protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, we engineered a prototype vaccine utilizing the plant expression system of Nicotiana benthamiana, to produce a recombinant SARS-CoV-2 virus-like particle (VLP) vaccine presenting the S-protein from the Beta (B.1.351) variant of concern (VOC). This innovative vaccine, formulated with either a squalene oil-in-water emulsion or a synthetic CpG oligodeoxynucleotide adjuvant, demonstrated efficacy in a golden Syrian Hamster challenge model. The Beta VLP vaccine induced a robust humoral immune response, with serum exhibiting neutralization not only against SARS-CoV-2 Beta but also cross-neutralizing Delta and Omicron pseudoviruses. Protective efficacy was demonstrated, evidenced by reduced viral RNA copies and mitigated weight loss and lung damage compared to controls. This compelling data instills confidence in the creation of a versatile platform for the local manufacturing of potential pan-sarbecovirus vaccines, against evolving viral threats.

The production of Newcastle disease virus-like particles in Nicotiana benthamiana as potential vaccines.

Newcastle disease (ND) is a highly contagious viral respiratory and neurological disease that has a severe impact on poultry production worldwide. In the present study, an expression platform was established for the transient production in N.bethamiana of ND virus-like particles (VLPs) for use as vaccines against ND. The expression of the ND Fusion (F) and/or Hemagglutinin-neuraminidase (HN) proteins of a genotype VII.2 strain formed ND VLPs in planta as visualized under the transmission electron microscope, and HN-containing VLPs agglutinated chicken erythrocytes with hemagglutination (HA) titres of up to 13 log2.The immunogenicity of the partially-purified ND VLPs was confirmed in specific-pathogen-free White leghorn chickens. Birds receiving a single intramuscular immunization with 1024 HA units (10 log2) of the F/HN ND VLPs administered with 20% [v/v] Emulsigen®-P adjuvant, seroconverted after 14 days with F- and HN-specific antibodies at ELISA titres of 5705.17 and HI geometric mean titres (GMTs) of 6.2 log2, respectively. Furthermore, these ND-specific antibodies successfully inhibited viral replication in vitro of two antigenically closely-related ND virus isolates, with virus-neutralization test GMTs of 3.47 and 3.4, respectively. Plant-produced ND VLPs have great potential as antigen-matched vaccines for poultry and other avian species that are highly immunogenic, cost-effective, and facilitate prompt updating to ensure improved protection against emerging ND field viruses.

Immunogenicity of adjuvanted plant-produced SARS-CoV-2 Beta spike VLP vaccine in New Zealand white rabbits.

The outbreak of the SARS-CoV-2 global pandemic heightened the pace of vaccine development with various vaccines being approved for human use in a span of 24 months. The SARS-CoV-2 trimeric spike (S) surface glycoprotein, which mediates viral entry by binding to ACE2, is a key target for vaccines and therapeutic antibodies. Plant biopharming is recognized for its scalability, speed, versatility, and low production costs and is an increasingly promising molecular pharming vaccine platform for human health. We developed Nicotiana benthamiana-produced SARS-CoV-2 virus-like particle (VLP) vaccine candidates displaying the S-protein of the Beta (B.1.351) variant of concern (VOC), which triggered cross-reactive neutralising antibodies against Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs. In this study, immunogenicity of the VLPs (5 µg per dose) adjuvanted with three independent adjuvants i.e. oil-in-water based adjuvants SEPIVAC SWETM (Seppic, France) and "AS IS" (Afrigen, South Africa) as well as a slow-release synthetic oligodeoxynucleotide (ODN) adjuvant designated NADA (Disease Control Africa, South Africa) were evaluated in New Zealand white rabbits and resulted in robust neutralising antibody responses after booster vaccination, ranging from 1:5341 to as high as 1:18204. Serum neutralising antibodies elicited by the Beta variant VLP vaccine also showed cross-neutralisation against the Delta and Omicron variants with neutralising titres ranging from 1:1702 and 1:971, respectively. Collectively, these data provide support for the development of a plant-produced VLP based candidate vaccine against SARS-CoV-2 based on circulating variants of concern.

Dose immunogenicity study of a plant-produced influenza virus-like particle vaccine in layer hens.

Avian influenza poses one of the largest known threats to global poultry production and human health, but effective poultry vaccines can reduce infections rates, production losses and prevent mortalities, and reduce viral shed to limit further disease spread. The antigenic match between a vaccine and the circulating field influenza A viruses (IAV) is a critical determinant of vaccine efficacy. Here, an Agrobacterium tumefaciens-mediated transient tobacco plant (Nicotiana benthamiana) system was used to rapidly update an H6 influenza subtype virus-like particle (VLP) vaccine expressing the hemagglutininn (HA) protein of South African H6N2 IAVs circulating in 2020. Specific pathogen free White Leghorn layer hens vaccinated twice with ≥125 hemagglutinating unit (HAU) doses elicited protective antibody responses associated with prevention of viral shedding, i.e. hemaglutination inhibition (HI) mean geometric titres (GMTs) of ≥7 log2, for at least four months before dropping to approximately 5-6 log2 for at least another two months. A single vaccination with a 250 HAU dose induced significantly higher HI GMTs compared lower or higher doses, and was thus the optimal dose for chickens. Use of an adjuvant was essential, as the plant-produced H6 HA VLP alone did not induce protective antibody responses. Plant-produced IAV VLPs enable differentiation between vaccinated and infected animals (DIVA principle), and with sucrose density gradient-purified yields of 20,000 doses per kg of plant material, this highly efficacious, safe and economical technology holds enormous potential for improving poultry health in lower and middle-income countries.

Multimodal analysis for human ex vivo studies shows extensive molecular changes from delays in blood processing.

Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMC up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states.

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.