Recipient Organization
LONG ISLAND UNIVERSITY
700 NORTHERN BLVD
GREENVALE,NY 115481319
Performing Department
(N/A)
Non Technical Summary
Bovine respiratory disease (BRD) is the most costly disease affecting North American beef cattle, and is responsible for 40-50% of the mortality and 70-80% of the morbidity in these animals, resulting in over $3 billion/year in losses to the cattle industry. Decreased weight gain and performance affects an additional 10% of these animals. BRD in pre-weaned calves alone costs the US beef cow-calf industry ~$165 million annually. BRD is the leading cause of morbidity and mortality in U.S. feedlot and stocker cattle and the second leading cause of loss in dairy calves. The primary bacterial agents responsible for BRD are H. somni, Mannheimia haemolytica, Pasteurella multocida, and Mycoplasma bovis. However, H. somni can also cause a wide range of infections other than BRD, and is therefore the target bacterium for this investigation. Current commerical vaccines consisting of killed bacteria that have been grown in vitro are not adequately effective at preventing BRD and some other systemic infections. We and others have shown that opportunistic bacteria express novel proteins in thehost in order to sequester iron that is bound to host proteins, such as transferrin and hemin. These proteins are not produced in vitro where adequte iron is available. Furthermore, during chronic infections bacteria persist in the host in theform of a biofilm. We have shown that half of the bacterial genome is differentially expressed when the bacteria are in a biofilm, compared to when grown shaking or in a fermentor, and that many novel proteins are present when the bacteria are in a biofilm. In additiion, H. somniand other bacteria may be able to presist in phagocytic or other cells and avoid recognition by antibodies. Therefore, many potentially protective antibodies are not producedor able to reach the bacteria, and an important cellular immune response is lacking.H. somniand other gram-negative pathogens also produce endotoxin, which results in serious side-effects in some immunized animals. As a result of the deficiencies in current vaccines, we will produce a vaccine that includes the components of the bacteria that are expressed in the host (iron binding proteins and biofilm matrix components) in the form of outer membrane vessicles (OMV), which are relatively simple to prepare, combined withthe biofilm matrix anda novel vaccine that has been shown to greatly stimulate both the humoral and cellular immune responses of the host to desired antigenic components. To produce a safe vaccine we will generate a mutant that lacks only endotoxic activity through the mutation of one gene by an established allelic exchange method. OMV from the mutant bacteria will be grown to produce iron-binding proteins and other ourter membrane proteins, which are combined with biofilm matrix and the adjuvant. Thorough genetic and biochemical characterization of the mutant will be carried out. The optimal concentrationof all components will first be determined in Holstein calves for immune efficacy and safety, and compared to commercial bacterins. Once optimized, additional safety data will be collected on a large number of commercial beef calves in comparison to commercial vaccines. Approved statistical analyses will be used for all data analysis.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Goals / Objectives
There is strong evidence that Histophilus somni bacterin vaccinesare not adequate to prevent respiratory, cardiac, or septicemic diseases caused by H. somni infection of cattle. Our hypothesis is that current bacterins, which are most commonly administered to prevent bovine respiratory disease (BRD), the leading cause of death in U.S. cattle, are inadequate because the physiology and antigens expressed by the bacterium in the host is very different from those expressed in culture typically used for vaccine manufacture. Furthermore, typical whole cell bacterins contain large amounts of the endotoxin, to which cattle are particularly sensitive. Thus, bacterins may cause adverse reactions, making veterinarians and farmers hesitant to use them. The virulence properties of H. somni have been thoroughly studied by our group and others, with research demonstrating that the natural growth state of H. somni is a biofilm, which can be prevalent in cattle during BRD or myocarditis. We have established that antigens are expressed in the hostthat are not present during planktonic growth (i.e., in vitro broth culture, as used for vaccine manufacture). We have also shown that half of the bacterial genome is differentially expressed when the bacteria form a biofilm, compared to planktonic growth. In addition, other key antigens are expressed in the host that are not expressed during in vitro culture, such as iron binding proteins, which are required by the bacterium to sequester host iron required for growth. It is also likely that current H. somni vaccines fail to induce cell-mediated immunity that can eliminate the agent surviving within host phagocytic cellsdue to inadequate activation of T helper cell type 1 (Th1) responses. Our Goal is to apply our knowledge of H. somni biology, outer membrane vesicles (OMV), and biofilms to develop an "Intelligently-Designed" vaccine that will induce an effective host immune response recognizing the pathogen's antigenic profile as it occurs in the host, and will lack inflammatory properties due to endotoxin. To accomplish our goal our aims are: 1. Generate an lpxL1 mutant of H. somni pneumonia isolate strain 2336 to provide a non-endotoxic source of in vivo-like outer membrane vesicles (ivOMV) and biofilm matrix (BM) material; 2. Prepare the ivOMV-BM vaccine; 3. Test the ivOMV-BM vaccine and compare it to current bacterin vaccines in an experimental model of bovine BRD; 4. Test the ivOMV-BM vaccine in a commercial cattle herd for safety. Successful completion of these aims will form the foundation for larger future trials to confirm efficacy of the ivOMV-BM vaccine, positioning us to engage involvement of pharmaceutical companies who could broadly market the vaccine. This vaccine has the potential to substantially improve calf protection against H. somni disease due to its inclusion of H. somni antigens that are not present under conditions of traditional vaccine manufacture.
Project Methods
Aim 1.Generate an lpxL1 mutant of H. somni pneumonia isolate strain 2336 to provide a non-endotoxic source of in vivo-like outer membrane vesicles (ivOMV) and biofilm matrix (BM) material. To accomplish this aim we will use allelic exchange mutagenesis by procedures established in our laboratory to isolate an H. somni mutant (2336::LpxL1) in which lpxL1 has been replaced with a chloramphenicol resistance (CmR) cassette. We will alsocomplement the mutation to confirm the mutant is isogenic. We will thoroughly characterize 2336::lpxL1 to confirm it is isogenic and lacks substantial endotoxic activity, and thatthe ivOMV from 2336::lpxL1 are identical to ivOMV previously derived from strain 2336. Mutations wil be confirmed by PCR and reverse transcriptase-PCR. The lipooligosaccharide will be charcterized by gel electrophoresis, and endotoxic activity will be determined byLimulusamoebocyte lysate assayand TL4 agonistactivity. Proteins in the ivOMB and BM will be characterized by Western blotting and proteomic analysis (liquid chrmatography-mass spectrometry). ivOMV will be prepared by gowing the bacteria in medium with no available iron due to chelation byethylenediamine-N,N′-bis-2-hydroxyphenylacetic acid (EDDHA). The OMV will be isolated by differential ultracentrifugation.and density gradient centrifugation. BM will be grown in 1L cultures and rotated at only 25 RPMfor 5 days. The top 900 mls is removed and the BM is recovered from the bottom 100 mls after removal of medium components and cells by differential centrifutation, sonication, and filtration. The ivOMV-BM vaccine is combined with adjuvant provided by our collaboratorDr. Robert Ernst. We will optimize the concentration of 2336::lpx1 ivOMV-BM vaccine and adjuvant by vaccinating calves and determiningthe maximum Th1 and Th2 response with minimal reactivity. Calves are healthy 4- to 5-month-old Holsteins, and seronegative to H. somni.The calves are immunized twice 3 weeks apart with the 2336::lpxL1 ivOMV-BM vaccine, or a commercial bacterin. Theefficacy of each vaccine is determined by measuring the cellular and humoral immune responses from all immunized animals, and gross and cellular pathology of the animals five days after challenge.Sera will be analyzed by ELISA for IgM, IgG1, IgG2, and IgE directed against H. somni killed whole cells, and by Western blotting to detect antibodies to specific antigens. Plasma will be collected at the same time points for measurement of Th1, Th2 and pro-inflammatory cytokine analyses. Cytokines indicative of a Th1 response (interferon gamma([IFN-g), tumor necrosis factor alpha (TNF-a), interleukin-12 (IL-12), or Th2 response (IL-4, IL-6, IL-10), will be determined by Luminex assay. The effects of vaccine formulation on cytokine and antibody expression are determined by comparing outcomes for calves vaccinated with different formulations using repeated measures ANOVA or Kruskall-Wallis test for parametric or nonparametric outcomes, respectively, with a P value set at 0.05 for significance. The ideal formulation determined above will be used for safety in a commercial herd in comparison to commercial H. somni vaccines.Five- to 7-month-old Angus and Charolais crossbred beef calves born at Mississippi State Universitywill be used, with 52 calves assigned to receive either 1) optimized 2336::lpxL1 ivOMV-BM vaccine, 2) commercial bacterin SomnuShield (Elanco), or 3) commercial bacterin SomnuBac (Zoetis). Inclusion of 52 cattle per group provides 80% power at alpha = 0.05 to detect a difference of 2% versus 20% cattle experiencing any adverse reactions within 24 hours of vaccination in the experimental versus bacterin groups, respectively. Cattle will be vaccinated twice subcutaneously 3-weeks apart. Blood for evaluation of antibodies (IgG1 and IgE directed against H. somni) and cytokines (IFN-g and IL-4) will be collected before the first vaccine dose, before the second dose, and 4 weeks after the second dose. Cattle will be observed for 30 minutes following each vaccination, and at 6, 12, and 24 hours after vaccination by a veterinarian blinded to the treatment assignment and scored for signs of rapid or difficult breathing, nasal or ocular discharge, unsteady gait, diarrhea, or inability to stand.