startvac mechanism of action

STARTSTARTVACVAC®®

INTRODUCTION

SPC

VACCINAL PLAN

SLIME

COLIFORMS

2000 – 2009: from a project to an authorizated vaccine

1. Research on the antigenic and adjuvant components of the vaccine (formulation) and development of the analytical techniques to control the product.

2. Stability studies.

3. Safety studies.

4. Efficacy studies in laboratory trials.

5. Safety and efficacy studies in field trials.

6. Submission of the STARTVAC dossier to the European Medicines Agency (EMEA) to obtain the Community marketing authorisation via the centralised procedure.

7. The European Commission granted a marketing authorisation valid throughout the European Union for STARTVAC on 11 February 2009.

STARTVAC: Introduction

Product name: STARTVACInactivated vaccine against Bovine mastitis.

Composition:Active substances:- Escherichia coli (J5) inactivated - Staphylococcus aureus (SP140) inactivated, expressing Slime

Associated Antigenic Complex (SAAC)Adjuvant:- Oil-based

STARTVAC: Summary of product characteristics

Target species:

Cattle: cows and heifers.

Age: from 22 months of age onwards.

Therapeutic Indications:

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.


Amounts to be administered and administration route

Bovine: 1 dose of 2 ml / animal.

The method of administration is by deep intramuscular injection in the neck muscles.

Recommended vaccination scheme:

-First vaccination at 45 days before the expected parturition date.

-Second vaccination 35 days thereafter (corresponding to 10 days before the expected parturition date).

-Third vaccination 62 days after the second vaccination (booster vaccination).


Immunity of STARTVAC

Risk of S.aureus mastitis

Risk of colibacilar mastitis

Drying off Lactation

calv

ing

0 13 35 45 97

-60 -45 -32 -10 0 52 130 dp calving

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OOI DOI

STARTVAC

STARTVAC

STARTVAC

Low risk Medium risk Very High riskHigh risk

STARTVAC: Vaccination plan

• SLIME (= BIOFILM) is an extracellular layer, excreted by many bacteria, composed of polysaccharide, that covers the cell wall and is physically connected with it.

Scanning Electron micrograph of Staphylococcus aureus

biofilm grown in-vitro using the colony biofilm model. The biofilm of

coccoid cells is blanked by extracellular polymer matrix

(www.erc.montana.edu).

STARTVAC: What is the Slime ?

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-

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STARTVAC: The Biofilm as survival mechanism

Scanning electron micrograph of a biofilm on a metal surface from an industrial water system.

• Biofilms are survival mechanisms of clinically relevant microorganisms.

• 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.

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) 20Pseudomonas aeruginosa ATCC 27853

Imipenem 1 (MIC) 1,024

E. coli ATCC 25922 Ampicillin 2 (MIC) 512P. pseudomallei Ceftazidime 8 (MBC) 800Streptococcus sanguis Doxycycline 0.063 (MIC) 3.15

• 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.

• 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.

DONLAN AND COSTERTON CLIN. MICROBIOL. REV. 2002

STARTVAC: The Biofilm as survival mechanism

STARTVAC: Biofilm in staphylococcal bovine mastitis

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• 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.

•Biofilm structure cartoon. Copyright Center for Biofilm Engineering

Montana State University

Slime Associated Antigenic Complex (SAAC) from the vaccinal S. aureus, strain SP 140:

• 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.

• SAAC production is specific to S. aureus strains phenotypically characterised as SP (slime producing) and is involved in biofilm formation on microtiter plates.

• Poly-N-acetyl -1,6 glucosamine (PNAG) surface polysaccharide is a major constituent of the staphylococcal biofilm matrix.

STARTVAC: Some general features of the vaccinal strain

• The strain SP 140 has the ica operon, that is associated to the biofilm production.

• The ica operon and the biofilm antigens are expressed by 95% of S. aureus strains (1) and a high precentage of coagulase negative staphylococci.

(1) CUCARELLA C, et al. Role of biofilm-associated protein bapin (the pathogeny of of bovine Staphylococcus aureus. Infect. Immun.2004, 72:2177-2185.


• Phenotypical characteristics of slime-producing

S. aureus (I)

•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.

•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.


• Phenotypical characteristics of slime-producing

S. aureus (II)

•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

•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

•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

•Negative control (non-inoculated culture media) in rows G and H: OD at 492nm: 0.060

•Stain:safranin at 0.1% w/v

A

BC

D

E

FG

H


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.

STARTVAC: : Biofilm research and efficacy results

•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.

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.

STARTVAC: : EFFICACY results: Humoral immunityEFFICACY results: Humoral immunity

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).

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.

FIELD TRIALS. FIELD TRIALS. Results of EFFICACY: Humoral immunityResults of EFFICACY: Humoral immunity

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).

FIELD TRIALS. FIELD TRIALS. Results of EFFICACY: Local immunityResults of EFFICACY: Local immunity

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.

Anti-slime response

Anti- E. coli J5 response

Significance of the

regression

(p value)

0.0028 0.0009

FIELD TRIALS. FIELD TRIALS. Results of EFFICACY: Correlation between Results of EFFICACY: Correlation between Humoral immunity and Local immunityHumoral immunity and Local immunity

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

ANY QUESTIONS ?

STARTVAC R&D TEAM

startvac mechanism of action

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