Source: MONTANA STATE UNIVERSITY submitted to NRP
A NOVEL BIOFILM-TARGETING VACCINE TO CONTROL MYCOPLASMA OVIPNEUMONIAE INFECTION IN SHEEP.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
Reporting Frequency
Annual
Accession No.
1032179
Grant No.
2024-67016-42407
Cumulative Award Amt.
$300,000.00
Proposal No.
2023-07980
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2026
Grant Year
2024
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Microbiology and Cell Biology
Non Technical Summary
Mycoplasma ovipneumoniae is a bacterium that infects sheep and that contributes to the development of respiratory diseases. There are currently no treatments or vaccines for this pathogen. We have recently shown that Mycoplasma ovipneumoniae can form biofilms. Biofilms are communities of bacteria that stick to each other and to a surface and that therefore are very resistant to treatments. With this project, we seek to develop and test a novel Mycoplasma ovipneumoniae vaccine that targets biofilms in the respiratory tract to clear or prevent Mycoplasma ovipneumoniae infection. We hypothesize that including material from biofilms in the vaccine will lead to a strong immune response that can prevent the formation of biofilms or break up existing biofilms. In our project, we will (1) Design and produce a vaccine to Mycoplasma ovipneumoniae that incorporates biofilm components and that induces an immune response in the respiratory tract and (2) test whether a biofilm-based Mycoplasma ovipneumoniae vaccine can protect sheep from Mycoplasma ovipneumoniae infection. An effective vaccine is expected to prevent infection and improve health and productivity of lambs that are experimentally or naturally exposed to Mycoplasma ovipneumoniae. If this project is successful, farmers will finally have a tool to combat Mycoplasma ovipneumoniae infection. If we can demonstrate that incorporation of biofilm material in a Mycoplasma vaccine is effective, this approach could also be adapted for other pathogenic Mycoplasma in other agricultural species.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
(N/A)
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113610109040%
3113610110040%
3114040109020%
Goals / Objectives
The long-term goal of our research is to develop novel strategies to eliminate Mycoplasma ovipneumoniae (M. ovi) infections from domestic sheep herds. M. ovi is a facultative pathogen that infects the respiratory tract of sheep and that is an important co-factor in ovine atypical pneumonia. With this project, we seek to develop and test a novel M. ovi vaccine that targets biofilms in the respiratory tract to clear or prevent M. ovi infection.We will test the hypothesis that an M. ovi vaccine that incorporates biofilm antigens and triggers anti-biofilm responses may protect sheep from M. ovi infection with two experimental objectives:Objective 1: Design and produce a vaccine to M. ovi that incorporates biofilm components and that induces mucosal antibody responses.Objective 2: Assess the ability of the biofilm-based M. ovi vaccine to protect sheep from M. ovi infection and improve productivity.
Project Methods
Objective 1: Design and produce a vaccine to M. ovi that incorporates biofilm components and that induces mucosal antibody responses1.a. Formulate a biofilm-based M. ovi vaccine: We will use M. ovi biofilms from 3-5 field strains of M. ovi, isolated from naturally infected MAES sheep, to generate a multivalent vaccine formulation. To generate sufficient M. ovi biofilm antigens for inoculation of sheep, we will culture the M. ovi biofilms in drip flow reactors at the CBE Medical Biofilms Laboratory. The biofilms will be homogenized, gently heat-inactivated at 56°C and sterile filtered. The preparation will then be mixed with MetaStim®, a veterinary vaccine adjuvant that acts as a lipid droplet-based delivery system, and carbopol, which activates antigen-presenting cells, promotes T cell responses, and enhances neutralizing antibody formation. A commercial vaccine for porcine M. hyopneumoniae infection that offers significant protection and results in strong mucosal IgG, IgA and IgM responses contains these two adjuvants. For quality control and standardization, we will determine the concentration of protein, carbohydrates, nucleic acids, and lipopolysaccharide (LPS) in the vaccine formulation using standard assays.1.b. Assess the safety of the adjuvanted biofilm-based M. ovi vaccine. Since we have a limited number of SPF sheep available, we first will test the safety of the vaccine in C57BL/6 mice (n=4/group). Mice will be subcutaneously (s.c.) inoculated twice over three weeks with an adjuvanted M. ovi vaccine that contains either (1) planktonic M. ovi or (2) M. ovi biofilm, each containing 5-10x106 bacteria. A similar dose of biofilm-derived M. ovi in TiterMax adjuvant induced anti-M. ovi IgG antibodies in a preliminary experiment. Mice will be observed for any adverse reactions to the vaccine, and the formulation will be adjusted if needed. If no significant adverse reactions occur and anti-M. ovi-antibody responses are induced (see 1.c. below), we will immunize four groups (n=2) of 3- to 4-month-old SPF lambs with increasing amounts of the adjuvanted, biofilm-based M. ovi vaccine. We will first immunize two lambs with the lowest dose and move on to the next group and dose only if there are no serious adverse effects. Lambs will be inoculated s.c. in the neck region twice over three weeks. Based on previous vaccine studies in sheep, doses between 50 µg and 1 mg/lamb (5x107-1x1010 bacteria) will be tested. Since higher antigen doses led to increased antibody responses in a previous M. ovi vaccine study, we will select the highest dose of the vaccine that is tolerated well by the sheep for subsequent experiments.1.c. Assess the immunogenicity of the M. ovi vaccine: To assess the immunogenicity of the M. ovi vaccine formulation, we will first analyze sera from the immunized mice, collected 3-4 weeks after the second dose, for reactivity with M. ovi. Antigen preparations from M. ovi biofilms or planktonic bacteria will be tested with the sera in an indirect ELISA. Likewise, we will assess antibody responses in the sheep from the experiment in 1.b. Over a period of three months, we will collect weekly serum samples to measure systemic antibodies, and monthly nasal wash fluids and bronchoalveolar lavages (BALs)to measure mucosal antibodies. Samples from SPF sheep inoculated with the adjuvants alone will be used for comparison. Indirect ELISA with planktonic or biofilm antigen preparations and detection antibodies that recognize ovine IgG, IgM and IgA will be used to quantify different antibody classes. To assess biofilm-specific responses, the nasal washes, BAL and sera also will be used in Western blots with antigen preparations from either planktonic M. ovi or biofilms. Cytokine arrays for pro-inflammatory, anti-inflammatory and T cell cytokines will be performed to determine the type of immune response induced, using both sera and BAL samples.To determine whether antibodies from the immunized sheep can actively disrupt existing biofilms, we will test sera, nasal washes, and BALs for anti-biofilm activity in vitro, following published protocols. We will grow M. ovi biofilms on glass bottom plates for 24-48 h in the presence of BAL, nasal washes, or sera from immunized sheep or control animals. Alternatively, we will add the BAL, nasal washes, or sera to established M. ovi biofilms to analyze whether the antibodies can induce active biofilm dispersal. We will measure biofilm mass using confocal microscopy and SYTO9 staining and will compare antibody-treated samples from immunized animals and non-immunized controls to untreated biofilms.Objective 2: Assess the ability of the biofilm-based M. ovi vaccine to protect sheep from M. ovi infection and improve productivity.2.a. Perform an M. ovi challenge experiment in specific-pathogen-free domestic lambs. We have successfully established an M. ovi infection model in our SPF sheepand will use this model to test whether the vaccine protects sheep with no prior exposure from M. ovi infection. To reduce the number of experimental animals, any immunized lambs from the experiments in 1.b/c. that have developed an antibody response will be used for a pilot experiment. Based on the pilot study, a larger group of lambs aged 2-3 months will then be used in a second, sufficiently powered challenge experiment. For experimental design, we will collaborate with the INBRE Statistical Consulting and Research Services. We will nasally challenge vaccinated and control lambs with ~5x108 M.ovi,three weeks after the second vaccine dose. Vaccine efficacy will be assessed based on M. ovi colonization in vaccinated compared to unvaccinated lambs using qPCR analysis of weekly nasal swab samples. We will also monitor clinical signs, weight gain, and feed conversion rate. Four weeks after the challenge, lambs will be euthanized, and tissue samples from the nasal cavity, tonsils, trachea, bronchi, and lungs will be harvested for analysis of M. ovi infection and histopathology. We also will measure anti-M. ovi and anti-biofilm antibody responses in sera, nasal washes, and BAL weekly after challenge (see Obj. 1).2.b. Define the impact of the M. ovi biofilm vaccine on sheep with prior M. ovi infection. Induction of anti-biofilm antibodies in response to systemic application of the biofilm-based, adjuvanted M. ovi vaccine may enable sheep with ongoing M. ovi infection to clear the bacteria. We will test this hypothesis in the MAES flock, which has a high level of natural M. ovi infection. Experimental group sizes will be determined based on the results from the challenge experiment in 2.a. For safety reasons, we will first immunize non-pregnant, 7-8 months-old ewe lambs, using the protocol described in 2.a. M. ovi infection status will be determined prior to immunization using qPCR analysis of nasal swab samples. To determine whether the M. ovi vaccine can induce sterilizing immunity, we will measure M. ovi colonization by qPCR of nasal swab samples monthly for at least three months. Impact on productivity will be assessed based on weight gains and carcass weights, where available.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:The target audiences for our study are scientists interested in sheep infectious disease and immunology, veterinarians, sheep farmers and individuals involved in wildlife conservation. In addition, our team is organizing a monthly seminar series at MSU, termed the Sheep Health Research Updates, that is focused on domestic and wild sheep health (https://nios.montana.edu/mbi/sheephealthresearchuntitled.html). This seminar series is attended by MSU faculty and research trainees from the Departments of Microbiology and Cell Biology, the Animal and Range Sciences Department, the Chemistry and Biochemistry Department, MSU Extension and FWP and serves as a local forum to exchange new data and ideas with researchers interested in sheep health and production. Through a virtual option, our seminars also have gained an increasing audience of university and government researchers from other institutions in the Northwest. Presenters include MSU graduate students and faculty as well as collaborators from other institutions. We have discussed our ongoing work on M. ovi vaccine development at these meetings. Initial data on the development of the M. ovipneumoniae vaccine was presented in a public seminar at Montana State University this April by a graduate student in the Bimczok lab, Dr. B. Tegner Jacobson, as part of his dissertation defense. In addition, PI Dr. Bimczok gave an interview discussing our ongoing research with a representative from the Wild SheepFoundation in November 2024, which will be distributed to an audience of wild sheep conversationists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided research training opportunities for two Ph.D. students in the Bimczok laboratory, Mr. B.T. Jacobson and Ms. Sobha Sonar, and for a postdoctoral fellow, Dr. Hannah Marahrens, a small ruminant veterinarian. Ms. Sonar gained experience in the isolation of M. ovipneumoniae from lung samples in the Bimczok lab and isolated multiple vaccine candidate strains. She also received training in ELISA development from co-investigator Dr. Rynda-Apple and was able to validate an in-house ELISA protocol to measure antibody responses to the vaccine and to natural M. ovipneumoniae infection. Mr. Jacobson has been leading the studies on Mycoplasma biofilms in the Bimczok laboratory. He analyzed the genotype of the candidate vaccine strains, performed strain selection for the vaccine, and confirmed biofilm growth for the selected genotyping. He also received internship training in an industry lab, working with our collaborators at SAN group to manufacture the biofilm-based vaccine at their production facility in Germany. Postdoctoral fellow Dr. Marahrens joined our team in February. She has received training in M. ovipneumoniae culture, has established culture conditions for monocyte-derived dendritic cells, and has gained experience with managing large animal experiments. Dr. Marahrens also has gained experience in performing respiratory tract endoscopies in sheep, so that we can administer challenge infections and collect bronchoalveolar lavage samples. The project also provided research training opportunities for three undergraduate students in the Bimczok lab. Two Montana State University students who contributed to the project obtained research credit and gained co-authorships on publications. One of the students enrolled in veterinary school after graduation and is continuing to work on the project through a Summer Veterinary Research Fellowship sponsored by Washington State University. A third undergraduate student is working on the project over the summer with NSF funding through the Extreme Biofilms Research Experience for Undergraduates. Approximately fifteen additional undergraduate students, mostly pre-vet students from Montana State University, were able to gain experience helping with lambing and newborn care forour specific pathogen free sheep flock, but were not supported by this award. How have the results been disseminated to communities of interest?- Public presentation by graduate student at the Montana State University Microbiology and Cell Biology Seminar Series (dissertation defense) - Interview of Dr. Bimczok by representative of the Wild Sheep Foundation (not yet published) - What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will continue our ongoing immunization experiment in the SPF lambs. If antibodies to M. ovipneumoniae are successfully induced by the vaccines, we will move on to the challenge phase of our study. All immunized lambs and placebo-treated control animals will be inoculated intratracheally with the bacterial strains that were included in the two experimental vaccines. During the course of this experiment, we will continue to collect health scoring data including adverse vaccine reactions, will monitor weight gains, and will collect blood, serum, nasal swabs. At the experimental endpoint, lambs will be euthanized and necropsied, and tissue samples and bronchoalveolar lavages will be collected. We will use PCR to assess M. ovipneumoniae infection levels post-challenge, so that we can measure vaccine-mediated protection from infection. We also will measure adaptive immune responses to M. ovipneumoniae by ELISA and T cell stimulation assay. Upon completion of the vaccine and challenge experiments, we will analyze all data from this project and will prepare at least one manuscript describing our study. We also will present our findings at scientific meetings and through local seminars. If the study with the SPF lambs shows that either the regular or the biofilm-based vaccine confer significant protection from M. ovipneumoniae infection, we will test the vaccine in a field setting in a flock of domestic sheep at MSU's Red Bluff research ranch that has a high level of natural infection withM. ovipneumoniae.

Impacts
What was accomplished under these goals? Over the past year, we have designed two multi-strain vaccines that are based on planktonic or biofilm-grown Mycoplasma ovipneumoniaeand that we are currently testing in an immunization and challenge experiment in specific-pathogen-free domestic lambs. Vaccine development: For the vaccine, we isolated field strains of M. ovipneumoniaefrom lungs of lambs that were naturally or experimentally infected withM. ovipneumoniae.Strains were identified asM. ovipneumoniae by microbiology techniques and PCR, and the ability of the strains for form biofilms was confirmed. Illumina and Nanopore sequencing then were used to obtain high resolution genomes of the strains. Three strains that were genetically distinct based on their virulence gene profiles were selected for the vaccines. To produce safe and effective vaccines for administration to lambs, we collaborated with SAN Vet, an autogenic vaccine manufacturer that has experience in the production ofM. ovipneumoniaevaccines for the European market. Two different vaccine formulations were prepared, each containing 1 x 10^8 formalin-killed bacteria mixed with 10% Montanide gel 02 adjuvant: a standard vaccine that was based on planktonicM. ovipneumoniae, and a biofilm vaccine composed of bacteria grown as an adherent biofilm on glass plates, which includes additional antigens found in the biofilm extracellular polymeric matrix. Identity, sterility, and successful inactivation of the bacteria was confirmed. Analysis of immunogenicity: In an ongoing study, we are currently testing the safety and immunogenicity of the two M. ovipneumoniae vaccine formulations. Twenty-one 2-3-month-old male and female specific-pathogen-free lambs were randomly assigned to three groups (n=7 each) and received a subcutaneous injection of (1) saline placebo; (2) the M. ovipneumoniaevaccine generated from bacteria grown under standard culture conditions; or (3) theM. ovipneumoniaevaccine that was generated from bacteria grown as a biofilm.No serious adverse effects beyond occasional fevers and local responses were detected following vaccine administration. A booster injection is scheduled for three weeks after the first dose. Our team is currently collecting health and weight data, data on local and systemic vaccine reactions, nasal swabs, and blood and serum samples to measure vaccine safety and efficacy.To analyze adaptive immune responses to the two vaccines, we developed and validated an enzyme-linked immunosorbent assay (ELISA) to quantify serum antibody responses to M. ovipneumoniae. We also have optimized culture conditions for ovine blood monocyte-derived dendritic cells, so that we can perform T cell stimulation assays with M. ovipneumoniae antigens to measure antigen-specific T cell responses in vaccinated and infected sheep.

Publications