Development of a Novel Vaccine against Potentially Deadly MRSA in Mice

1. Development of a Novel Vaccine against Potentially Deadly MRSA in Mice Mary Rose DohertyDepartment of Biological Sciences, York College of Pennsylvania RESEARCH DESIGN REVIEW OF LITERATURE CONTINUED EXPECTED RESULTS • INTRODUCTION • Methicillin Resistant Staphylococcus aureus (MRSA) • is a potentially fatal bacterial pathogen implicated in a • wide variety of clinically significant infectious diseases • (endocarditis, pneumonia, necrotizing fasciitis, toxic • shock syndrome, etc). Due to antibiotic overuse, MRSA • has rapidly developed into a difficult-to-treat antibiotic • resistant disease. Once confined to healthcare settings, • this threatening strain is now pervading communities, • clearly demonstrating the need for an effective • MRSA vaccine. • At present, no MRSA vaccine exists. Recent research • has focused on developing subunit vaccines derived • from S. aureus surface proteins. Such proteins include a • group of nine iron-regulated surface determinants (Isd); • IsdA, IsdB, IsdC, IsdD, IsdE, IsdF, IsdG, IsdH (HarA) • and IsdI. Theseproteins are essential for bacterial • pathogenicity due to their role in obtaining and • utilizing host iron. • In studying Isd proteins, researchers have • established IsdA and IsdB as potential vaccine • candidates. Although research has demonstrated the • similarity in function between HarA and IsdB, it has • not determined the effectiveness of HarA as a potential • vaccine. Additionally, researchers have not yet tested • IsdC, which possesses a similar genetic sequence and • activity to the previous proteins. PCR- Isd proteins (MRSA252) gel electrophoresis transform E.coli via Pet-15b plasmids purify protein (affinity chromatography) sequence proteins’ amino acids (mass spectrometry) Pre-screen: antibody production ELISA prepare single and combination vaccines immunize/mock immunize female BALB/c mice monitor survival terminate mice, extract livers, count cfu’s statistics: Kaplan-Meier/SAS Proc Logistics (survival), parametric, unpaired t-tests (cfu) • Iron, an essential nutrient sequestered within host hemoproteins, is needed by bacteria in order to: evade host defenses, carry out cellular replication, metabolism and virulence (3,4,5) • Bacteria obtain iron: siderophores or direct binding to hemoproteins (3) • Isd proteins are highly conserved and are apart of direct binding, MRSA’s pathogenic strategy to obtain iron (1,5) • Research study tested IsdB as a vaccine in monkeys • (Rhesus Macaques); results showed increase in antibody • titers after vaccination (Fig 3.) (2) Perfect DNA™ 1 kbp Ladder (Novagen) IsdA IsdB IsdC HarA 2670 1959 1065 684 Fig 6. Gel electrophoresis: expected base Pairs of cloned Isd proteins. Figure 2. Potential vaccine candidates against MRSA derived from virulence factors in S. aureus (3). Figure 3. Geometric mean titer (GMT); IsdB immunized (black dots), mock Immunized (white dots) in monkeys at weeks 0+4. • Another experimental study tested IsdA, IsdB, SdrE, SdrD as single or combination vaccines in BALB/c mice; comparison demonstrated greater immunogenicity resulting from a combination vaccine (Fig 4.) and lack of abscesses in mice receiving this immunization (Fig 5.) (5) Fig 7. Expected percent survival (%) of mice exposed to vaccines, PBS data taken from previous study (5). MRSA252 PCR PRIMERS IsdAF 5’-TTATTTAGATTCTTTTCTTTT-3’ IsdAR 5’-ATGACAAAACATTATTTAAAC-3’ IsdBF 5’-TTAGTTTTTACGTTTTCTAGG-3’ IsdBR 5’-ATGAACAAACAGCAAAAAGAA-’3 IsdCF 5’-TTGAAAAATATTTTAAAAGTT-3’ IsdCR 5’-TTATTCCACATTGCCTTTAGA-3’ HarAF 5’-ATGAACAAACATCACCCAAAA-3’ HarAR 5’-TTACTTAGATTCTTTTCTGAA-3’ • STATISTICAL ANALYSIS • Survivorship: Kaplan-Meier (graphpad prism), SAS Proc Logistics • Liver cfu’s: Student t-tests parametric, unpaired, two-tail t-tests with significance (*) p <_ 0.05 or non-significance (ns) • p>0.05 (graphpad prism) RESEARCH OBJECTIVE The goal of this research proposal is to further extend the Isd vaccine study by determining whether or not a combination of IsdA,IsdB, IsdC, and HarA surface proteins are more effective in combating MRSA than single vaccines in murine models. Table 1. Experimental MRSA vaccine design BALB/c female mice 4-5 weeks old). _________________________________________________________________________________________ Number of Mice given Specific S. aureus strain5 _________________________________________________________________________________________ Test1# Vaccine2 Adjuvant4 500ug USA100 USA400 MRSA252 COL N315 Total _________________________________________________________________________________________ 1 IsdA with 10 10 10 10 10 50 2 IsdB with | | | | | | 3 IsdC with | | | | | | 4 HarA with | | | | | | 5 Combination3 with | | | | | | 6 Combination3 with/out 10 10 10 10 10 50 _________________________________________________________________________________________ 1each test contained 50 mice 2each vaccine administered in 3 each 50uL doses on days 0, 7, 21 3combined vaccine (IsdA, IsdB, IsdC, HarA) 4amorphous aluminum hydroxyphosphate sulfate 5number of mice introduced to each strain (n=10) after immunization EXPECTED CONCLUSION It is anticipated that a mixture of all four proteins will increase the immune response. Thus a combination vaccine will afford greater immunity than a single vaccine in combating MRSA infection. • REVIEW OF LITERATURE • Increase in MRSA due to antibiotic overuse (Fig 1.) Figure 4. Combined vaccine generates protective immunity (p<0.03) (Fisher’s exact test) compared to single proteins or PBS (little-no effect). Figure 5. Mock immunized (A+B) = abscesses (black arrow) S. aureus concentration (white arrow) combined immunization (C+D) = no abscesses. Whole kidneys (A+ C) magnification (B+D). • IsdA has broad binding affinity and attaches a variety of • extra-cellular matrix proteins: fibrinogen, fibronectin • and hemoglobin, which serve as camouflage (1,3) • IsdB directly binds host hemoglobin (most abundant • iron source in humans), induces humoral immunity in mice • and removal of IsdB decreases virulence (2,6) • IsdC binds heme iron and helps deliver heme iron • into bacterial cells; removing IsdC reduces expression of other Isd surface proteins(1,6) • HarA (IsdH) binds haptoglobin, hemoglobin • and haptoglobin-hemoglobin complexes; the removal of • IsdH decreases virulence (1) LITERATURE CITED Table 2. Control MRSA vaccine design BALB/c mice (4-5 weeks old). _________________________________________________________________________ Number of Mice __________________________________________________ Vaccine2 S. aureus strain ________________ _________________________________ CTRL1# Adjuvant8 Other9 Total A3 B4 C5 D6 E7 USA100 USA400 MRSA252 COL N315 500ug ____________________________________________________________________________________ 1 NO VACCINE 10 10 10 10 10 W - 50 2 10 10 10 10 10 NO BACTERIAL STRAIN W - 50 3 NO VACCINE NO BACTERIAL STRAIN W S.epidermidis 10 4 10 10 10 10 10 NO BACTERIAL STRAIN W S.epidermidis 50 ____________________________________________________________________________________ 1each control contained 50 mice, except CTRL 3 2each vaccine administered in 3 each 50uL doses on days 0, 7, 21 3single vaccine IsdA 4single vaccine IsdB 5single vaccine IsdC 6single vaccine HarA 7combined vaccine (IsdA, IsdB, IsdC, HarA) 8amorphous aluminum hydroxyphosphate sulfate 9other non-virulent strains 1. Dryla et al. 2007 High-affinity binding of the Staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded B-Barrel fold. Journal of Bacteriology. 2. Kuklin et al. 2006. A novel S. aureus vaccine: Induces rapid antibody responses in Rhesus Macaques and specific increased survival in a murine S. aureus sepsis model. Infection and Immunity. 74:2215-2223. 3. Lowy, F.D. 1998. Staphylococcus aureus infections. NEJM. 339:520-532. 4. Mazmanian et al. 2003. Passage of heme-iron across the envelope of Staphylococcus aureus. Science. 299:906-909. 5. Stranger-Jones et al. 2006. Vaccine assembly from surface proteins of Staphylococcus aureus. PNAS. 103:16942-16947. 6. Torres et al. 2006. Staphylococcus aureusIsdB Is a hemoglobin receptor required for heme Iron utilization. Journal of Bacteriology. 188:8421-8429. Figure 1. *CDC recognizes increase MRSA since 1999. • Vaccine design focuses on subunit S. aureus surface • proteins (Fig 2.) • Focus = Isd’s, iron acquisition system in MRSA ACKNOWLEDGEMENTS I want to extend my thanks to Dr. Mathur, my mentor, for her guidance in this research project.