Performing Department
(N/A)
Non Technical Summary
Our team'sproposal tackles a big problem affecting cattle: the impact of bovine respiratory syncytial virus (BRSV). BRSV is a primary cause of respiratory disease in cattle and is also a major pathogen involved in the Bovine Respiratory Disease Complex (BRDC) in calves, the most costly cause of infection-related economic losses across the beef and dairy sectors. Current vaccines offer variable efficacy and have raised safety concerns, underscoring the need for innovative solutions. Our main goal is to pioneer the development of a novel single cycle vaccine strategy that promises not only efficacy but also heightened safety.Single-cycle vaccines consist of a live-attenuated version of a pathogen. Although categorized as live vaccines, single-cycle vaccines are unable to fully replicate in animals. This type of vaccine operates ingeniously: the virus has the ability to infect animal cells and, like any other virus, uses the host cell to generate viral proteins. The infection of the host and the production of these many viral proteins triggersan immune response against the virus.However, our vaccine is designed to prevent the virus from ever completing its replication cycle. Although the virus produces most of the proteins necessary for replication, one essential piece is missing, preventing the formation of infectious virus particles. This means that the animal's immune system can learn to combat the virus using a real virus model, but without the risk of the virus in the vaccine spreading. Additionally,weare focusing on making a vaccine that can be given through the nose, which is not only easier to administer but also leads to antibodies and immune cells being located at mucosa sites, where infections usually take place. Nasal vaccines also avoid issues with the calf's immune system eliminating the vaccine before it can trigger the animal's immune system. This issue arises in young animals from interference by maternal antibodies, which are passed on through colostrum ingestion (maternal immunity).Single-cycle vaccines function through the rational design of deletions or modifications in the BRSV genes necessary for viral particle formation and infectivity. This strategy ensures the vaccine's safety by eliminating the possibility of virus shedding and reversion to a virulent form.Our proposal outlines three principal objectives: (1) the creation of a single-cycle BRSV vaccine in which the essential viral Matrix protein gene is absent, preventing the generation of progeny since the M protein is a necessary component of the viral particle (termed Mnull BRSV single-cycle vaccine); (2) the development of a second single-cycle vaccine in which all essential viral proteins are present, but in which the viral Fusion protein is modified to lose its function (preF BRSV single-cycle vaccine). This change renders the virus unable to spread after a first cycle in the host, as the Fusion protein is necessary for the virus to bind to and enter host cells. It is only possible to grow these viruses under laboratory settings with the help of unique cell lines especially developed to provide the missing viral protein (in the case of the matrix protein) or to complement the function of the modified protein (in the case of the Fusion protein). We will then (3) carry out the evaluation of these vaccines' immunogenicity and efficacy in calves. This initiative builds upon our prior research and applies proven single-cycle replication technologies to BRSV, aiming to produce vaccine prototypes that are both broadly effective and exceptionally safe.The anticipated outcome of our project is the generation of a mucosal single-cycle BRSV vaccine prototype that is both highly effective and very safe, promising a significant advancement in the fight against BRDC in calves. This could have a big impact on the cattle industry by keeping calves healthier, reducing the need for antibiotics, and saving farmers money. Ultimately, our project aims to solve a major problem in animal health, benefiting both animals and the people who rely on them.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
We have previously developed a pioneer live-attenuated mucosal vaccines platform for human RSV (hRSV) and propose translating our technology to create a prototype for the BRSV counterpart. We aim to develop this prototype vaccine and assess immunogenicity vaccine efficacy and protection against virulent BRSV in calves. Multiple vaccines are currently marketed for BRSV, including for parenteral and mucosal delivery, but variable efficacy and safety concerns have been reported. Parenteral vaccine delivery faces interference by MDA that hampers vaccine uptake and diminishes responses to vaccination. Moreover, current live attenuated vaccines (including nasal delivered) are replication competent, retaining the risk of reversal to- and residual -virulence.The success of this vaccine would significantly improve cattle health, reduce antibiotic use associated with secondary infection in cattle, and increase production efficiency, contributing to food safety and nutrition security. The objectives of this study are related to the program area of Animal Health and Disease (Priority code A1221). Successful completion of this work will significantly impact the priority areas of "disease prevention" by developing improved "vaccination delivery systems and/or vaccines" for use in livestock species.To achive this goal, our objectives are:Generation of Mnull BRSV single-cycle vaccine.Generation of preF BRSV single-cycle vaccine.Immunogenicity and efficacy of single-cycle BRSV vaccines in calves.
Project Methods
Our objectives are:Generation of Mnull BRSV single-cycle vaccine.Generation of preF BRSV single-cycle vaccine.Immunogenicity and efficacy of single-cycle BRSV vaccines in calves. To achive these objectives the following methods will be emplyed: Objectives 1 and 2:a) Construction of cDNAs. The full genome sequence of the publicly available BRSV ATCC51908 was cloned into a modified pUC57-Brick plasmid using standard cloning techniques. To generate BRSV-Mnull cDNA, the plasmid containing the above sequenced BRSV genome will be modified. First, the SH gene will be replaced by the coding sequence for green fluorescent protein (GFP). GFP can be removed or replaced by the original SH sequence at a later stage; Then, the G protein will be modified to lack secreted G production by mutating the second AUG (methionine 48) at BRSV G, abolishing G cleavage into its secreted form, (secreted G protein is a viral virulence factor).We will then generate the single cycle cDNA from the BRSV cDNA above (which has GFP and lacks secreted G protein). For Mnull BRSV, the M ORF will be replaced by a Tet transactivator (Tet) protein. The Tet protein is required to drive M expression in the producer line, in which the M gene is preceded by a Tet-responsive promoter. For the BRSV-preF cDNA, the bovine F gene will be subcloned in a plasmid and known DS-Cav-1-based preF-stabilizing mutations (S155C, S290C, S190F, and V207L) will be edited by standard site-directed mutagenesis.b) Single cycle virus rescue from cDNA: Initial transfection of the cDNA and support plasmids containing an internal ribosome entry site (IRES) preceding the BRSV N, P, M2-1, and L ORFs (as well as M - for Mnull BRSV rescue or the functional F protein- for preF BRSV rescue) in BHK-21 cells expressing T7 polymerase from a nonpathogenic alphavirus replicon. Following, rescued viruses will be amplified in Hep2-M cells (stably expressing RSV M protein for Mnull BRSV) or Vbac cells (expressing GP64 for PreF BRSV).c) In vitro characterization of the novel vaccine viruses. Sequences will be confirmed by PCR to verify the stability of clones after amplification in cell lines. To confirm single-cycle replication, we will grow the virus in regular and production cell lines. Additionally, virus titrations will be conducted to confirm further the vaccine's inability to generate infectious progeny. The absence of M protein and the presence of preF protein (relative to wild-type BRSV) will also be confirmed by western blot and immunofluorescence assays.Objective 3. Immunogenicity and efficacy of single-cycle BRSV vaccines in calves. a) Study design: The animal study will be conducted in BSL-2 rooms of the Animal Resources department. Animals will be kept following the Ag Guide recommendations and after IACUC approval. BRSV antibody and virus negative calves will be randomly allocated into 3 experimental groups (n=6 per group, 18 calves total), as follows: BRSV Mnull (G1); BRSV preF Prime (G2), and non-vaccinated control group (G3). Calves will be allowed to acclimate for 5 days. Five milliliters, 2.5ml/nostril, of vaccine preparation (titer ~ 104 - 105 TCID50/ml) will be applied intranasally using a mucosal atomization device. Non-vaccinated control animals will receive supernatant of non-infected cells (2.5ml/nostril). Animals will be challenged 28 days later 28, with virulent BRSV-375 (~104.5 TCID50 in 5 ml by aerosol). Animals will be monitored daily and scored based on respiratory clinical parameters.b)Sample collection, processingand data analysis:Collected samples will include whole blood, serum, and triplicate nasal swabs (two nasal swab replicates will be placed in RNALater®, and the third replicate swab will be placed in cell culture media for virus isolation and titration). Serum will be used to assess humoral responses after vaccination and challege. Whole blood will be harvested, and PBMCs isolated using Ficoll-Paque Plus (GE Healthcare) according to the manufacturer's instructions to assess cellular immune responses to vaccination and post-challenge. Whole blood and clot tubes will be collected on experimental days -2, 0, 7, 14, 21, 28, 30, 35, and 38. Nasal swabs will be collected daily from day 0 to 7, then weekly until the challenge. Post-challenge swabs will be collected daily until day 38. Viral neutralization will be performed by standard VN assays. Cell-mediated immune responses elicited by vaccination in all calves will be evaluated by flow cytometry.BRSV-specific cell-mediated responses will be assessed by carboxyfluorescein succinimidyl ester (CFSE) dilution and intracellular cytokine staining (ICS) assays (interferon-gamma) in response to stimulation with UV-inactivated virus (MOI 1:5), or live virus (MOI 1:1). Pokeweed mitogen(PW) (10 µg/mL; Sigma Aldrich) and unstimulated RPMI 1640 media will be used as positive and negative controls, respectively. Antibodies that will be used for subset identification are CD3 (MIA11), CD4 (ILA11), CD8 (BAQ111A), and TCR1 δ chain (GB21A). We will use mouse anti-bovine IFN-γ (clone CC302) to detect cytokine secreting cells. All antibodies are commercially available, and the PI has previously optimized. BD Aria II cell analyzer (BD Biosciences) will be used to acquire cells, and FlowJo (Tree Star Inc.) to analyze data. Statistical analysis between groups will be performed using ANOVA to determine differences in kinetics and magnitude of BRSV-specific immune responses.Euthanasia will be conducted on day 38, 10 days post-challenge. Euthanasia will be conducted using barbiturate overdose, and pathology will be assessed by a pathologist. We will harvest nasal turbinate, trachea, bronchoalveolar lavage (BAL), and lung samples. Tissue samples will be placed in formalin for subsequential processing and histological assessment or snap-frozen for viral load assessment (RNA detection and viral isolation). Fresh lung samples (5-10 g, cranial lobe) will also be collected to evaluate lung T-cell populations, following standard digestion protocol. Viral isolation and virus titration will be conducted in all positive RT-qPCR samples (virulent BRSV detection will be based on NS2). We will attempt to assess genome replication in vivo swabs collected after immunization and prior to challenge by RT-qPCR for detection of viral genes (M, F, NS1, NS2 detection). The number of animals was estimated as the numbers needed to assess statistical differences between treatment groups, with a power of 0.8 and a significance level of 0.05. We hypothesize that single-cycle vaccines will lead to measurable immune responses and protect animals from virulent BRSV infection.