Startvac mechanism of action

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startvac new vaccine against mastitis

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  • Good afternoon everybody and thank you very much to come here. I would like to apologize for not speaking in Polish, but I will try to speak in English and Michal will translate the presentation for you. The purpose of my presentation is to introduce our new vaccine STARTVAC for the mastitis. To start with I will make an introduction about the mastitis and vaccination. Then I’ll explain you the characteristics of the STARTVAC vaccine. After that, I’ll talk about the field trials that we performed with STARTVAC, describing the design of the study and showing the efficacy results that we obtained. Finally , I’ll summarize my presentation with some conclusions. Please, do feel free to interrupt me if you have any questions.
  • As we were convinced that a vaccine could be a help to control the mastitis, in the year 2000 we started the STARTVAC project for the development of a mastitis vaccine. The development of a vaccine is a long way that begins with the research on the (1)… Then it’s necessary to perform stability studies (2),…(3), (4) and (5). Finally, we decided to submit the STARTVAC dossier… (6). After the evaluation of the dossier, the European Comission .. (7).
  • Let’s look now at the characteristics of the STARTVAC vaccine: STARTVAC is an inactivated vaccine with two antigenic components : E. coli J5. S. aureus , whose cells contain an extracel·lular component that we refer to as SAAC. I’ll explain the composition in more detail later. Additionally , STARTVAC vaccine have an oil-based adjuvant .
  • STARTVAC should be administered to cows and heifers more than 22 months of age. The vaccination is indicated for herd immunisation of healthy cows and heifers,…
  • The dose of the vaccine is 2 ml per animal and the method of administration is by … The recommended vaccination plan consist of a first vaccination at 45 days before the expected parturition date. A second vaccination 35 days ….And a booster vaccination at 62 days after the second vaccination.
  • The vaccination plan was the indicated in the summary of product characteristics. The first dose of the vaccine was administered at 45 days before the expected parturation dat. Second immunization was 35 days thereafter (corresponding to 10 days before the expected parturition date).Finally, third vaccination was 62 days after the second vaccination (booster vaccination). This slide shows the relation between the vaccination plan and the risc of mastitis, before and after calving. You can see that the immunity conferred by the vaccination, between the Outset and the duration of immunity, includes the maximum risk for S. aureus mastitis and colibacilar mastitis.
  • In this figure we can see a biofilm of S. aureus . Growing cells are surrounded by the extracellular matrix of polysaccharide and keep closer together inside the biofilm.
  • Another characteristic of the S. aureus slime-producing strains is the mucous aspect of the colonies, as you can see in the figure. Next, let’s move on to explain in more detail the STARTVAC composition. First of all, I’ll begin by the definition of slime. In the last years is more frequently used the name of BIOFILM instead of SLIME , but both names are synonymous and refer to the extracellular matrix of S. aureus made up of polysaccharide . The chemical composition of slime is different than capsules. Both are polysaccharides, but chemically different. The production of this extracellular matrix it’s an important virulence factor that defines the ability of bacterial strains to colonize host tissues and biomaterials, participating in the intercellular adhesion among bacterial cells and subsequent development of a biofilm. This leads to chronic infections and bacterial resistance to phagocytosis. In staphylococcical bovine mastitis , bacteria adhere to the mammary gland epithelial cells and grow forming colonies surrounded by a large exopolysaccharide matrix, constituing a biofilm. Because of their aggregate size, biofilms are not susceptible to macrophage o neutrophil phagocytosis and become resistant to some antibiotics, promoting the chronicity of the disease. Poly- N -acetyl  -1,6 glucosamine surface polysaccharide is a major constituent of the staphylococcal biofilm matrix.
  • Next, let’s move on to explain in more detail the STARTVAC composition. First of all, I’ll begin by the definition of slime. In the last years is more frequently used the name of BIOFILM instead of SLIME , but both names are synonymous and refer to the extracellular matrix of S. aureus made up of polysaccharide . The chemical composition of slime is different than capsules. Both are polysaccharides, but chemically different. The production of this extracellular matrix it’s an important virulence factor that defines the ability of bacterial strains to colonize host tissues and biomaterials, participating in the intercellular adhesion among bacterial cells and subsequent development of a biofilm. This leads to chronic infections and bacterial resistance to phagocytosis. In staphylococcical bovine mastitis , bacteria adhere to the mammary gland epithelial cells and grow forming colonies surrounded by a large exopolysaccharide matrix, constituing a biofilm. Because of their aggregate size, biofilms are not susceptible to macrophage o neutrophil phagocytosis and become resistant to some antibiotics, promoting the chronicity of the disease. Poly- N -acetyl  -1,6 glucosamine surface polysaccharide is a major constituent of the staphylococcal biofilm matrix.
  • I said before that S. aureus inactivated cells in the SARTVAC vaccine contain SAAC. So, what is the relation between SAAC and slime or biofilm? SAAC is an extracellular component that we isolated from biofilm-producing S. aureus isolates . SAAC was chemically analysed and it showed to be constituted by 55 % polysaccharide and 45 % peptide. We refer to this component as “Slime Associated Antigenic Complex” because we saw that SAAC is only produced by S. aureus strains phenotypically characterised as slime-producing. On the other hand, we demonstrated that SAAC is involved in biofilm formation, because we found a correlation between SAAC production and the biofilm formation ability in vitro . Further analyses demonstrated that the SAAC-specific antigen is a polysaccharide. The polysaccharidic component of SAAC contains 6 % glucosamine and galactosamine. So, probably, the glucosamine we detect in SAAC could be deacetylated sugars from the PNAG (the main polysaccharide of the biofilm matrix).
  • This figure shows the method to determine the slime-producing phenotype of S. aureus isolates.
  • Another characteristic of the S. aureus slime-producing strains is the mucous aspect of the colonies, as you can see in the figure on the left. On the right side, you can see the result of a biofilm assay (o late adherence test). Basically, it consists in growing the strain in a microtiter plate. Then, wells are emptied and washed with PBS. Adhered cells are fixed and stained with safranin. Finally, the OD of the biofilm is measured with an ELISA microplate reader. In A, B and C rows, grew a biofilm producing strain. In D, E and F rows grew a non-producing strain. Rows G and H were non-inoculated negative controls.
  • The levels of specific anti-SAAC and anti- E . coli J5 antibodies in serum of the STARTVAC group was…
  • Well, I’ve told you about the S. aureus component of the STARTVAC. Now let’s look at the Escherichia coli J5 component. E. coli J5 is a natural mutant strain that laks the enzyme responsible for binding the Outer polysaccharide to the Core region of the LPS. So, the core antigen is exposed in the external membrane of this strain. The interesting thing of this strain is that the core region of LPS is highly conserved among a broad spectrum of Gram-negative bacteria.
  • The levels of specific anti-SAAC and anti- E . coli J5 antibodies in serum of the STARTVAC group was…
  • The results indicated that the serological response in milk…
  • Furthermore, we observed a significant correlation between the…
  • I’ll finish the characteristics of STARTVAC with the mechanisms of protection. The STARTVAC vaccination confers protection through the induction of antibodies against SAAC components present…. The vaccination also induce the production of antibodies against core epitopes of LPS …. When an intramammary infection occurs, antibodies anti-SAAC bind to the initial exopolysaccharide matrix production….; In the case of a coliform infection, antibodies anti- E. coli J5 bind to the core… Then, PMN phagocytose the opsonized bacterial cells, resulting in a clearance of the intramammary infection due to S. aureus an CNS, or E. coli and coliforms.
  • Startvac mechanism of action

    1. 1. START VAC ® INTRODUCTION SPC VACCINAL PLAN SLIME COLIFORMS
    2. 2. <ul><li>2000 – 2009: from a project to an authorizated vaccine </li></ul><ul><li>Research on the antigenic and adjuvant components of the vaccine (formulation) and development of the analytical techniques to control the product. </li></ul><ul><li>Stability studies. </li></ul><ul><li>Safety studies. </li></ul><ul><li>Efficacy studies in laboratory trials. </li></ul><ul><li>Safety and efficacy studies in field trials. </li></ul><ul><li>Submission of the STARTVAC dossier to the European Medicines Agency (EMEA) to obtain the Community marketing authorisation via the centralised procedure. </li></ul><ul><li>The European Commission granted a marketing authorisation valid throughout the European Union for STARTVAC on 11 February 2009. </li></ul>STARTVAC: Introduction
    3. 3. <ul><li>Product name: STARTVAC </li></ul><ul><li>Inactivated vaccine against Bovine mastitis. </li></ul><ul><li>Composition: </li></ul><ul><li>Active substances: </li></ul><ul><li>Escherichia coli (J5) inactivated </li></ul><ul><li>Staphylococcus aureus (SP140) inactivated, expressing Slime Associated Antigenic Complex (SAAC) </li></ul><ul><li>Adjuvant: </li></ul><ul><li>Oil-based </li></ul>STARTVAC: Summary of product characteristics
    4. 4. <ul><li>Target species:   </li></ul><ul><li>Cattle: cows and heifers. </li></ul><ul><li>Age: from 22 months of age onwards. </li></ul><ul><li>Therapeutic Indications: </li></ul><ul><li>For herd immunisation of healthy cows and heifers, in dairy cattle herds with recurring mastitis problems, to reduce the incidence of sub-clinical mastitis and the incidence and the severity of the clinical signs of clinical mastitis caused by Staphylococcus aureus, coliforms and coagulase-negative staphylococci. </li></ul>STARTVAC: Summary of product characteristics
    5. 5. <ul><li>Amounts to be administered and administration route </li></ul><ul><li>Bovine: 1 dose of 2 ml / animal. </li></ul><ul><li>The method of administration is by deep intramuscular injection in the neck muscles. </li></ul><ul><li>Recommended vaccination scheme: </li></ul><ul><li>-First vaccination at 45 days before the expected parturition date. </li></ul><ul><li>-Second vaccination 35 days thereafter (corresponding to 10 days before the expected parturition date). </li></ul><ul><li>-Third vaccination 62 days after the second vaccination (booster vaccination). </li></ul>STARTVAC: Summary of product characteristics
    6. 6. calving 0 13 35 45 97 -60 -45 -32 -10 0 52 130 dp calving ▼ ▼ ▼ ▼ ▼ ▼ ▼ OOI DOI S T A R T V A C S T A R T V A C S T A R T V A C Low risk Medium risk Very High risk High risk STARTVAC: Vaccination plan Immunity of STARTVAC Risk of S.aureus mastitis Risk of colibacilar mastitis Drying off Lactation
    7. 7. <ul><li>SLIME (= BIOFILM) is an extracellular layer, excreted by many bacteria, composed of polysaccharide, that covers the cell wall and is physically connected with it. </li></ul><ul><li>Scanning Electron micrograph of Staphylococcus aureus </li></ul><ul><li>biofilm grown in-vitro using the colony biofilm model. The biofilm of </li></ul><ul><li>coccoid cells is blanked by extracellular polymer matrix </li></ul><ul><li>(www.erc.montana.edu). </li></ul>STARTVAC: What is the Slime ?
    8. 8. <ul><li>- </li></ul>STARTVAC: The Biofilm as survival mechanism <ul><li>- </li></ul><ul><li>- </li></ul>Scanning electron micrograph of a biofilm on a metal surface from an industrial water system. <ul><li>Biofilms are survival mechanisms of clinically relevant microorganisms. </li></ul><ul><li>The production of this extracellular matrix defines the ability of bacterial strains to colonize host tissues and biomaterials, participating in the intercellular adhesion among bacterial cells and subsequent development of a BIOFILM, leading to chronic infections and bacterial resistance to phagocytosis and antibiotic treatments. </li></ul>
    9. 9. <ul><li>The nature of biofilm structure and the physiological attributes of biofilm organisms confer an inherent resistance to antimicrobial agents, whether these antimicrobial agents are antibiotics, disinfectants, or germicides. </li></ul><ul><li>Mechanisms responsible for resistance may be one or more of the following: (i) delayed penetration of the antimicrobial agent through the biofilm matrix, (ii) altered growth rate of biofilm organisms, and (iii) other physiological changes due to the biofilm mode of growth. </li></ul><ul><li>D ONLAN AND COSTERTON CLIN. MICROBIOL. REV. 2002 </li></ul>STARTVAC: The Biofilm as survival mechanism Microorganism Antibiotic Reference Organism Antibiotic MIC or MBC of planktonic phenotype (µg/ml) Concentration effective against biofilm phenotype (µg/ml) S. aureus NCTC 8325-4 Vancomycin 2 (MBC) 20 Pseudomonas aeruginosa ATCC 27853 Imipenem 1 (MIC) 1,024 E. coli ATCC 25922 Ampicillin 2 (MIC) 512 P. pseudomallei Ceftazidime 8 (MBC) 800 Streptococcus sanguis Doxycycline 0.063 (MIC) 3.15
    10. 10. STARTVAC: Biofilm in staphylococcal bovine mastitis <ul><li>- </li></ul><ul><li>The bacteria adhere to the mammary gland epithelial cells and grow forming colonies surrounded by a large exopolysaccharide matrix, constituing a BIOFILM. Because of their aggregate size, biofilms are not susceptible to macrophage or neutrophil phagocytosis and, moreover, become resistant to some antibiotics, promoting chronicity of the disease. </li></ul><ul><li>Biofilm structure cartoon. Copyright Center for Biofilm Engineering </li></ul><ul><li>Montana State University </li></ul>
    11. 11. <ul><li>Slime Associated Antigenic Complex (SAAC) from the vaccinal S. aureus , strain SP 140: </li></ul><ul><li>Component of S. aureus chemically characterised to be constituted by 55% polysaccharide (formed mostly by glucose and galactose) and 45% peptide, with a molecular weigh closed to 1,000 kDa. </li></ul><ul><li>SAAC production is specific to S. aureus strains phenotypically characterised as SP (slime producing) and is involved in biofilm formation on microtiter plates. </li></ul><ul><li>Poly- N -acetyl  -1,6 glucosamine (PNAG) surface polysaccharide is a major constituent of the staphylococcal biofilm matrix. </li></ul>STARTVAC: Some general features of the vaccinal strain
    12. 12. <ul><li>The strain SP 140 has the ica operon, that is associated to the biofilm production. </li></ul><ul><li>The ica operon and the biofilm antigens are expressed by 95% of S. aureus strains (1) and a high precentage of coagulase negative staphylococci. </li></ul><ul><li>(1) CUCARELLA C, et al. Role of biofilm-associated protein bap in (the pathogeny of of bovine Staphylococcus aureus. Infect. Immun.2004, 72 :2177-2185. </li></ul>STARTVAC: Some general features of the vaccinal strain
    13. 13. <ul><li>Phenotypical characteristics of slime-producing </li></ul><ul><li>S. aureus (I) </li></ul>STARTVAC: Some general features of the vaccinal strain <ul><li>Fig. 2. Staphylococcus aureus , strain SP140, slime producing phenotype : black and rough colonies, showing an irregular outline. Incubation at 37 ºC/24 hours in TSA medium supplemented with 2% dextrose and 0.8% Congo red. </li></ul><ul><li>Fig. 3. Staphylococcus aureus , strain 240, slime non-producing phenotype : black and smooth colonies, showing a regular and well defined outline. Incubation at 37 ºC/24 hours in TSA medium supplemented with 2% dextrose and 0.8% Congo red. </li></ul>
    14. 14. <ul><li>Phenotypical characteristics of slime-producing </li></ul><ul><li>S. aureus (II) </li></ul>STARTVAC: Some general features of the vaccinal strain <ul><li>Fig.4. Staphylococcus aureus , strain SP140, slime producing phenotype : a mucous, slimy substance formed between the colony and the loop can be observed. Incubation at 37 ºC/24 hours in TSA medium supplemented with 2% dextrose </li></ul><ul><li>Fig.5. Staphylococcus aureus , strain SP140, slime producing phenotype in rows A, B and C: formation of a biofilm adhered to the microplate; OD at 492nm: 1.597 </li></ul><ul><li>Staphylococcus aureus , strain 240, slime non-producing phenotype in rows D, E and F: a very weak biofilm formation can be observed; OD at 492nm: 0.130 </li></ul><ul><li>Negative control (non-inoculated culture media) in rows G and H: OD at 492nm: 0.060 </li></ul><ul><li>Stain:safranin at 0.1% w/v </li></ul>A B C D E F G H
    15. 15. STARTVAC : Biofilm research and efficacy results Bacterins from strong biofilm-producing bacteria triggered the highest production of antibodies to PNAG and conferred the highest protection against infection and mastitis, compared with weak biofilm-producing bacteria and non-cellular inocula.
    16. 16. STARTVAC : Biofilm research and efficacy results <ul><li>The study reports the immunogenicity of SAAC in dairy cows when this component is embedded in a S. aureus bacterin of a strong biofilm-producing strain. Results indicated that immunization with a S. aureus bacterin with high SAAC content was able to reduce S. aureus multiplication in the mammary gland after challenge, and suggests that the SAAC-specific antibody response could be involved in the protection against S. aureus intramammary infection. </li></ul>
    17. 17. <ul><li>The production of specific anti-slime (anti-SAAC) in serum of the STARTVAC group was significantly higher (p<0.05) with respect to the Placebo group until 167 days post-vaccination. </li></ul>STARTVAC : EFFICACY results: Humoral immunity
    18. 18. STARTVAC: Some general features of Escherichia coli E.coli J5 is a strain that lacks the enzyme Uridin Diphosphate Galactose 4-Epimerase, which is responsible for binding the somatic antigen (O-Antigen of polysaccharide) to the LPS molecule of the cell wall . The vaccine was developed with the concept of the exposure of the core antigen common to Gram-negative organisms in the mutant J5 strain (rough strain).
    19. 19. <ul><li>The production of specific anti- E.coli J5 antibodies in serum of the STARTVAC group was significantly higher (p<0.05) with respect to the Placebo group until day 132 days post-vaccination. </li></ul>FIELD TRIALS. Results of EFFICACY: Humoral immunity
    20. 20. <ul><li>The serological response in milk remained higher in the STARTVAC group than in the placebo group until day 167 post-vaccination for anti-slime and until day 132 post-vaccination for anti- E.coli J5). </li></ul>FIELD TRIALS. Results of EFFICACY: Local immunity
    21. 21. <ul><li>A significant correlation between the serological response in serum (humoral immunity) and in milk (local immunity), was confirmed against both antigens in field conditions, which confirmed the previously observed in the challenge laboratory trials. </li></ul>FIELD TRIALS. Results of EFFICACY: Correlation between Humoral immunity and Local immunity Anti-slime response Anti- E. coli J5 response Significance of the regression (p value) 0.0028 0.0009
    22. 22. STARTVAC: Mechanism of protection Induction of antibodies against core epitopes of LPS common to gram-negative bacteria Induction of antibodies against SAAC components present in the biofilm matrix that produce staphylococcal bacteria during the initial phase of infection. Startvac Vaccination Antibodies anti-SAAC bind to the initial exopolysaccharide matrix production before the biofilm is established. Antibodies anti- E. coli J5 bind to the core antigens during the multiplication of invading bacterial cells, exposed just before the synthesis of LPS is completed. Intramammary infection (IMI) Phagocytosis of the opsonized bacterial cells by neutrophils Clearance of IMI due to S. aureus and CNS Clearance of IMI due to E. coli and coliforms
    23. 23. ANY QUESTIONS ? STARTVAC R&D TEAM

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