Performing Department
(N/A)
Non Technical Summary
Influenza A virus of swine (IAV-S) causes significant economic losses to swine producers and poses a major public health concern due to its zoonotic potential. The constant evolution of the virus in the swine herds necessitates frequent vaccine updates. The goal of this project is to develop a nonreplicating platform for the rapid development and updating of IAV-S vaccines. Our platform will utilize DNA plasmids to express vaccine antigens because the DNA plasmid is non-infectious, highly stable and easy to produce. We will encapsulate the DNA plasmids with lipid nanoparticles (so-called LNP-DNA) to enhance their cellular uptake and protein expression, thereby enhancing the immune responses. This proposal has three objectives: to determine the minimal dose of the LNP-DNA vaccine, to develop a trivalent LNP-DNA vaccine containing antigens of three cocirculating IAV-S subtypes, and to evaluate the influence of maternally-derived antibodies on the vaccine efficacy. If successful, this project will produce a safe and effective vaccine platform that enables rapid vaccine updates to enhance vaccine efficacy and mitigate the economic impact of IAV-S.
Animal Health Component
35%
Research Effort Categories
Basic
30%
Applied
35%
Developmental
35%
Goals / Objectives
The three supporting objectives are:Objective 1: To identify vaccine protective dose 50 of the DNA vaccine.Objective 2: To develop a trivalent DNA vaccine containing hemagglutinin genes of three circulating influenza virusesObjective 3: To assess the potential influence of maternally derived antibodies on DNA vaccine efficacy.
Project Methods
Objective 1: To identify vaccine protective dose 50 of the DNA vaccine.Five groups of seronegative pigs will be housed in the ABSL2 facility at the University of Nebraska-Lincoln (UNL). After 1 week of acclimation, the pigs will be administered i.m. with different vaccine doses, ranging from 0 to 500 microgram of DNA. Whole blood with and without anticoagulant will be collected weekly from all pigs until day 28 post-vaccination. Blood samples with an anticoagulant will be used for peripheral blood mononuclear cell (PBMC) isolation to evaluate the kinetics of cellular immune responses. Blood samples without an anticoagulant will be used for serum isolation to evaluate antibody responses.On day 28 post-vaccination, all pigs will be challenged by a combination of intratracheal and intranasal inoculation of 2 × 105 TCID50 of the selected influenza strains. Our previous studies indicate that this dose and route of challenge consistently results in prominent lung lesions in non-vaccinated control pigs.After challenge infection, nasal swabs will be collected daily to measure viral shedding. On day 5 post-challenge, the pigs will be humanely euthanized, and a necropsy will be performed by a board-certified veterinary pathologist who will be blinded to the experimental design. Gross lung lesions will be visually scored. After that, samples of three lung lobes (apical, middle, and caudal) will be collected and fixed in 10% buffered formalin for histopathologic examination.To assess the immune response and level of protection, we will utilize the following assays.Hemagglutination inhibition (HI) assay to measure antibody responses.IFN-g ELISpot assay to evaluate T-cell responses.Real-time RT-PCR to quantify viral shedding in nasal secretion.Lung lesion scores after challenge infection.Objective 2: To develop a trivalent DNA vaccine containing hemagglutinin genes of three circulating influenza virus subtypes.2.1. Determine the optimal concentration of each hemagglutinin gene in the trivalent vaccine.We will use the knowledge gained from Objective 1 to determine the amount of each DNA plasmid to be included in the trivalent DNA vaccine. The prototype trivalent DNA vaccine will be generated by combining equal amounts of each DNA plasmid, starting with 5x protective dose 50 (PD50). Following this, we will immunize 8 pigs with the prototype trivalent DNA vaccine. Blood samples will be collected weekly after vaccination to measure HI antibody titers against the three homologous influenza strains.We will use HI titers as the parameter to determine if the trivalent vaccine induced balanced immune responses against the three vaccine antigens. This is because HI titers have been demonstrated to be a reliable correlate of vaccine-induced protection.If we observe low HI titers against any of these three viral strains, we will adjust the concentration of the three individual DNA plasmids in the trivalent vaccine formulation. Specifically, we will generate a new trivalent vaccine using a higher concentration of the DNA plasmid that elicits lower HI titers against the homologous virus. We will then repeat the immunization experiment in pigs to evaluate their immune responses against this new trivalent vaccine formulation.2.2. Assess the protective efficacy of a trivalent DNA vaccine.We expect that after 2-3 rounds of optimization, we will identify an optimal formulation of a trivalent vaccine that elicits equal HI titers against all three antigens. The following experiments will be to assess the protective efficacy of the trivalent vaccine.The experiment will involve 24 three-week-old pigs seronegative for influenza virus. The pigs will be randomly assigned into three treatment groups, eight pigs per group. Group 1 will be injected i.m. with PBS as a negative control. Group 2 will be administered i.m. with commercial WIV vaccine while Group 3 will be vaccinated i.m. with the trivalent DNA vaccine.On day 28 p.v., the pigs will be challenged with 2x105 TCID50 of one of the three homologous virus strains. Samples collection, assays to evaluate immune responses and vaccine-mediated protection, and statistical analysis will be the same as in Objective 1.Objective 3: To assess the potential influence of maternally derived antibodies on DNA vaccine efficacy.To prepare piglets with and without maternally derived antibodies (MDA), six pregnant sows confirmed seronegative for influenza virus will be purchased from a high-health farm. Three sows will receive two doses of the WIV vaccine at three-week intervals, starting from midgestation. The remaining three sows will not receive any vaccinations. All sows will be allowed to undergo natural delivery, and the piglets will be allowed to nurse from their own dams. We anticipate that each group of three sows (vaccinated and non-vaccinated) will give birth to at least 30 piglets.Blood samples will be collected from the piglets two weeks after farrowing to measure the MDA titers. We will select 24 piglets born to the sows that receive the WIV vaccine (MAD+ pigs) and 24 piglets born to the sows that do not receive the WIV vaccine (MDA- pigs) and randomly assign them into six groups of eight pigs. Each group of pigs will be housed in a separate room in the ABSL2 facility.At two weeks of age, groups T02 and T05 will be vaccinated i.m. with the WIV vaccine based on the H1N1 strain, while groups T03 and T06 will be vaccinated i.m. with the LNP-H1N1 vaccine. The WIV vaccine will be administered twice at three weeks between doses, whereas the LNP-DNA vaccine will be administered once. Groups T01 and T04 will be injected with PBS as non-vaccinated control groups.Whole blood samples with and without anticoagulant will be collected weekly to assess antibody and T-cell responses, following the methodology outlined in Objective 1.At day 35 after vaccination, all pigs will be challenged by an intratracheal/intranasal inoculation with 2 x 105 TCID50 of the H1N1 virus (homologous to the vaccine) when they are 8 weeks old.Nasal swabs will be collected daily to assess viral shedding. On day 5 post-challenge, the pigs will be humanely euthanized, and necropsies will be performed as described above.