Development of a Novel Vaccine against Potentially Deadly MRSA in MiceMary Rose Doherty

Department of Biological Sciences, York College of Pennsylvania

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 researchhas 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. These proteins 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 thesimilarity in function between HarA and IsdB, it hasnot determined the effectiveness of HarA as a potentialvaccine. Additionally, researchers have not yet tested IsdC, which possesses a similar genetic sequence andactivity to the previous proteins.

REVIEW OF LITERATURE

Increase in MRSA due to antibiotic overuse (Fig 1.)

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.

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)

RESEARCH DESIGN

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’

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.

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)

LITERATURE CITED

I want to extend my thanks to Dr. Mathur, my mentor, for her guidance in this research project.

ACKNOWLEDGEMENTS

IsdA IsdB IsdC HarA

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IsdA has broad binding affinity and attaches a variety of extra-cellular matrix proteins: fibrinogen, fibronectinand hemoglobin, which serve as camouflage (1,3) IsdB directly binds host hemoglobin (most abundantiron source in humans), induces humoral immunity in mice and removal of IsdB decreases virulence (2,6) IsdC binds heme iron and helps deliver heme ironinto bacterial cells; removing IsdC reduces expression of other Isd surface proteins(1,6) HarA (IsdH) binds haptoglobin, hemoglobinand haptoglobin-hemoglobin complexes; the removal of IsdH decreases virulence (1)

Vaccine design focuses on subunit S. aureus surface proteins (Fig 2.)

Focus = Isd’s, iron acquisition system in MRSA

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).

Figure 3. Geometric mean titer (GMT); IsdB immunized (black dots), mock Immunized(white dots) in monkeys at weeks 0+4.

Fig 6. Gel electrophoresis: expected basePairs of cloned Isd proteins.

EXPECTED RESULTS

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 aureus IsdB Is a hemoglobin receptor required for heme Iron utilization. Journal of Bacteriology. 188:8421-8429.

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 mice2each vaccine administered in 3 each 50uL doses on days 0, 7, 213combined vaccine (IsdA, IsdB, IsdC, HarA)4amorphous aluminum hydroxyphosphate sulfate

5number of mice introduced to each strain (n=10) after immunization 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 32each vaccine administered in 3 each 50uL doses on days 0, 7, 213single vaccine IsdA4single vaccine IsdB5single vaccine IsdC6single vaccine HarA7combined vaccine (IsdA, IsdB, IsdC, HarA)8amorphous aluminum hydroxyphosphate sulfate9other non-virulent strains

Figure 2. Potential vaccine candidates against MRSA derived from virulence factors in S. aureus (3).

Fig 7. Expected percent survival (%) of mice exposed to vaccines, PBS data taken from previous study (5).

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 antibodytiters after vaccination (Fig 3.) (2)

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)

Figure 4. Combined vaccine generates protective immunity (p<0.03) (Fisher’s exact test) compared tosingle proteins or PBS (little-no effect).

Perfect DNA™ 1 kbp Ladder (Novagen)

Figure 1. *CDC recognizes increase MRSA since 1999.

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REVIEW OF LITERATURE CONTINUED