Evaluation Of The Protective Immunity Of A Novel Subunit Fusion Protein Vaccine HI In A Murine Model Of Systemic MRSA Infection

Objective:Staphylococcus aureus is a common commensal organism in humans and a majorcause of bacteremia and hospital acquired infection. Because of the spread of strainsresistant to antibiotics, of particular concern is the emergence of methicillin-resistant S.aureus (MRSA), these infections are becoming more difficult to treat. Therefore,exploration of anti-staphylococcal vaccines is currently a high priority.The concept of developing a vaccine based on multivalent antigens has beenpopularized in recent years. The purported benefit of multivalent antigens has previouslybeen described as targeting multiple virulence factors of pathogens that often utilizenumerous virulence factors to cause disease, and the inclusion of multiple staphylococcalantigens would likely result in a more effective vaccine.Both humoral and cellular immunity play important roles in host defense against S.aureus infection. Ideally, anti-staphylococcal vaccines should contain secreted as well ascell wall-associated antigens. The evoked immune responses should lead to the productionof antibodies and T cells producing IFN-γ and/or IL-17, the latter being important for themobilization and activation of neutrophils.This study is to construct bivalent vaccines based on iron-responsive surfacedeterminant B and alpha-toxin and to compare the protective efficacy of the bivalentvaccine to that of the individual proteins in a murine model of systemic MRSA infection.This study is also to learn wheather this protection correlate with neutralizing antibodiesagainst alpha-toxin, opsonic antibodies specific for S. aureus IsdB, and both IL-17A-andIFN-γ-producing memory T cells.Methods1. Genomic DNA extracted from S. aureus strain MRSA252was used as the PCR template. The HI gene was amplified by PCR and BamHI and NotI sites were introduced atthe beginning and end of the PCR products by primers. Double digested PCR products wereligated into pGEX-6P-2. The resulting constructs were transformed into Escherichia colistrain BL21for IPTG-induced expression and were expressed as GST fusion proteins.GST-tagged proteins were affinity-purified from cleared lysates with glutathione–Sepharose.The recombinant protein was purified by CaptoTMMMC and the protein eluate wassubjected to endotoxin removal by Q HP separation. The resulting protein was analyzed bySDS-PAGE.2. BALB/c mice were immunized with recombinant protein or with PBS plusadjuvant alum as a control. The mice were bled and the sera were screened by ELISA.BALB/c mice were infected with MRSA252(1×10~9CFU per mouse) after last boost. Thesurvival rates were monitored for14days after infection. BALB/c mice were infected with2.5×10~8of S. aureus strain MRSA252after last boost, and the target tissues were assessedfor bacterial colonization, cytokine production and histopathology.3. To evaluate the efficacy of the recombinant protein vaccine in actively immunizedmice, the titers of specific antibodies against the different recombinant proteins weredetermined by ELISA. To investigate the cytokine profiles in the spleen cells of miceimmunized with recombinant proteins and control mice immunized with alum adjuvant,spleen cells from all of the groups were stimulated with the recombinant proteins, andcytokine production was analyzed in the supernatants. To determine the nature of protection,cytokines were monitored in the supernatants from cells stimulated with recombinantproteins and analyzed the supernatants for their ability to induce staphylococcal killing inthe presence of PMNs. To determine the levels of cytokines and chemokines in the kidneysof vaccinated versus control mice after infection, The cytokines IFN-γ and IL-17A and theCXC chemokine CXCL2were measured.Results1. All recombinant fragments were cloned into the pGEX-6P-2vector. Following IPTGinduction, the recombinant fragments were expressed as soluble proteins. The purificationof the recombinant proteins from E. coli BL21cells was efficient. The recombinant proteinscorresponded to their predicted molecular masses by SDS-PAGE.2. Immunization with the chimeric bivalent antigen HI induced the strongest immunity against lethal challenge with S. aureus MRSA252. The mice immunized with the bivalentHI vaccine exhibited survival rates that were6.7%,20%,26.7%and66.7%higher than themice immunized with IH, IsdB, Hla, and adjuvant, respectively. Immunization with vaccineantigens significantly reduced both organ burden and pathology, Intriguingly, in contrast tothe immunization with a single vaccine antigen (IsdB, HlaH35L), the recombinant bivalentvaccine (HI, IH) afforded a higher level of protection against S. aureus challenge.3. Immunization with the chimeric bivalent antigen induced strong antibody response.Administration of the immunizing antigens did not alter the specific antibody titers againstIsdB or HlaH35L. Immunization with the chimeric bivalent antigen induced the strongest Tcell responses and Vaccination primed lymphocytes to produce cytokines that in turnenhanced phagocytic staphylococcal killing. the concentrations of IFN-γ, IL-17A andCXCL2were higher in the kidneys of vaccinated than control mice after infection, Further,immunization with HI or IH resulted in higher levels of IFN-γ, IL-17A and CXCL2thanimmunization with IsdB or Hla.Conclusion1. A novel subunit fusion protein MRSA vaccine based on iron-responsive surfacedeterminant B and alpha-toxin was constructed. Immunization with the chimeric bivalentvaccine induced strong antibody and T cell responses.2. The bivalent vaccine showed a stronger protective immune response than theindividual proteins. The chimeric bivalent vaccine HI has potential as a vaccine against S.aureus infection.