Evaluation Of Immunotherapies Targeting Capsules And Exotoxins In The Control Of Infectious Diseases Caused By Staphylococcus Aureus

Staphylococcus aureus(S. aureus) is one of the most common pathogens that are responsible for the infections in both human and livestocks. And, the capsule and exotoxins of S. aureus are two major virulent factors in the pathogenisis of these infections. To develop an alternative to antibiotic therapy, this study was conducted to evaluate antibody-based immunotherapies which targeting capsule and exotoxins in bovine mastitis and human infections models, including bacteremia, pneumonia and endocarditis. Our preliminary research indicated that egg yolk antibody (IgY) has great potential to treat bovine mastitis caused by S. aureus. However, IgY targeting capsule and exotoxins were not investigated up to now. And, glycoengineering is a novel technology through which S. aureus capsular polysacharrides (CP) could be conjugated to exotoxin to creat a multivalent vaccine. In this study, the bioconjugate vaccines were evaluated in three infection models induced by multiple strains of S. aureus.(1) Capsule-specific IgY (IgY-T5, IgY-T8and IgY-T336) was obtained by immunizing laying hens with vaccines containing inactivated whole cells of serotypes5,8and336S. aueus and partially purified to80%purity. ELISA indicated that IgY binded the S. aureus cell wall specifically and showed no binding to the cell wall of Lactobacillus bulgarius, a commercially used probiotic for yorgert production. In a growth inhibition assay conducted in Columbia broth,15mg/ml of IgY-T5or IgY-T8were not able to inhibit the growth of Lowenstein or Wright strain during an8h of incubation. However, IgY-T336inhibated the growth of types5,8and336strains by about2log units in CFU. In an internalization assay,5mg/ml of specific IgYs were able to block the invasion of their homogolous strains to MAC-T cells. These results suggest that capsule-specific IgY has great potential in the treatment of bovine mastitis.(2) Exotoxins-specific IgY (IgY-toxins) was obtained by immunizing laying hens with the total toxins isolated from culture supernatant of S. aueus V8and RN6390strains. IgY-toxins was shown to protect MAC-T cells and bovine pneutrophils from lysis induced by S. aureus exotoxins, with a dose-dependent mode. And,5mg/ml of IgY-T336markly blocked the invasion of GFP-expressing RN6390to MAC-T cells. Furthermore, IgY-T336provided greater protection when combined with0.5mg/ml of IgY-toxins, at same time, improved the cell growth morphology. Treatment with20mg of IgY-T5, IgY-T8, IgY-T336each or20mg of IgY-toxins for6days significantly reduced the somatic cell counting (SCC) of milk sampled from the cow with clinical mastitis caused by S. aureus. Intramammary infusion with a combination of capsule and exotoxin specific IgYs showed an even lower level of SCC (1～2×104cells/ml), with no difference with the positive control (100mg of penicillin treatment). And, milk samples from the all treatment groups contained significantly lower level of clots compared to the PBS control. However, no difference was observed between all four treatment groups. None of the treatments, with exception of penicillin, significantly reduced the bacterial level in the milk samples. Together, these results suggest that application of IgY in the treatment of mastitis should focus on the toxin neutralizing and cell-invasion blocking activities, but not bacterial growth inhibition activity of IgY.(3) Through glycoengineering, CPs were successfully conjugated to Pseudomonas aeruginosa exotoxin A (Epa) or S. aureus a-toxin (Hla). In vitro function studies showed that a CP5-Epa, a CP5-Hla and a CP8-Epa specifically opsonized the types5or8strains for killing by human neutrophils. Besides, a CP5-Hla was able to to protect red blood cells against lysis induced by native α-toxin. These results indicated that glycoengineering effectively produced antigenic capsular polysacharrides and toxin with right epitops of S. aureus.(4) S. aureus is one of the most common pathogens responsible for bacterimia in human clinical practice. Mouse model study demonstrated that active immunization with0.2μg CP5-2μg Epa protected mice against bacteremia, body weight loss and renal abscess formation. Passive transfer with1mg of a CP5-Epa significantly reduced the bactereia level induced by S. aureus Reynolds, USA100or USA200. Active immunization with2μg CP5-20μg Hla or passive immunization with1mg of a CP5-Hla protected mice against bacteremia induced by S. aureus Reynolds (CP5+). Active immunization with0.2μg CP8-2μg Epa or passive transfer with1mg of a CP8-Epa protected mice against bacteremia induced by Reynolds (CP8+). These results indicated bioconjugate vaccine targeting CP5and CP8effectively fended off the systemic infection-bacteremia caused by S. aureus.(5) Cases of pneumonia caused by S. aureus is reported frequently and characterized with high lethallty rate. Mice immunized with2μg CP5-20μg Hla were protected against lethal pneumonia induced by Newman and MRSA strains LAC (CP-) and ST80(CP8+) and bacteremia induced by Reynolds. Depletion of CD4+or CD8+T cells in mice immunized with2μg CP5-20μg Hla did not affect the survival rate of mice intranally challenged with Newman. Passive transfer of1mg of a CP5-Hla, but not a CP5-Epa, significantly extended the survival time of mice, however, did not improve the survival rate in lethal pneumonia model. Passive immunization with two doses of1mg of a CP5-Hla at24h and2h before challenge significantly protected mice against lethal pneumonia. This phenomenon indicated that antibody, but not T cell, plays an important role in the protection against lethal pneumonia. And, Hla, but not CP, is the major virulent factor in S. aureus pneumonia model.(6) S. aureus could colonize on some medical implants, catheter for example, and form biofilm. In catheter-associated endocarditis model, passive transfer of1or15mg of a CP5-Hla showed a trend of, but not significant reduction in bactereial level in aortic valve vegetation, bacteremia, body weight loss, renal bactereial burden and survival rate.In conclusion, a combination of IgY targeting capsule and exotins of S. aureus showed great efficacy in the treatment of bovine mastitis. And, CP-toxin bioconjugate vaccines produced by glycoengineering block these two virulent factors and demonstrated protection in bacteremia (systemic infection), pneumonia (local infection) and showed a trend of protection in catheter-associated endocarditis (catheter associated infection). Together, immunotherapies targeting capsules and exotoxins of S. aureus have great potential in the treatment and prevetion of the infectious diseases in human and livestocks.