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Rational Design of Toxoid Vaccine Candidates for Staphylococcus aureus Leukocidin AB (LukAB).

ABSTRACT: Staphylococcus aureus (SA) infections cause high mortality and morbidity in humans. Being central to its pathogenesis, S. aureus thwarts the host defense by secreting a myriad of virulence factors, including bicomponent, pore-forming leukotoxins. While all vaccine development efforts that aimed at achieving opsonophagocytic killing have failed, targeting virulence by toxoid vaccines represents a novel approach to preventing mortality and morbidity that are caused by SA. The recently discovered leukotoxin LukAB kills human phagocytes and monocytes and it is present in all known S. aureus clinical isolates. While using a structure-guided approach, we generated a library of mutations that targeted functional domains within the LukAB heterodimer to identify attenuated toxoids as potential vaccine candidates. The mutants were evaluated based on expression, solubility, yield, biophysical properties, cytotoxicity, and immunogenicity, and several fully attenuated LukAB toxoids that were capable of eliciting high neutralizing antibody titers were identified. Rabbit polyclonal antibodies against the lead toxoid candidate provided potent neutralization of LukAB. While the neutralization of LukAB alone was not sufficient to fully suppress leukotoxicity in supernatants of S. aureus USA300 isolates, a combination of antibodies against LukAB, ?-toxin, and Panton-Valentine leukocidin completely neutralized the cytotoxicity of these strains. These data strongly support the inclusion of LukAB toxoids in a multivalent toxoid vaccine for the prevention of S. aureus disease.

SUBMITTER: Kailasan S

PROVIDER: S-EPMC6628420 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Rational Design of Toxoid Vaccine Candidates for Staphylococcus aureus Leukocidin AB (LukAB).

Kailasan Shweta S Kort Thomas T Mukherjee Ipsita I Liao Grant C GC Kanipakala Tulasikumari T Williston Nils N Ganjbaksh Nader N Venkatasubramaniam Arundhathi A Holtsberg Frederick W FW Karauzum Hatice H Adhikari Rajan P RP Aman M Javad MJ

Toxins 20190614 6

Staphylococcus aureus (SA) infections cause high mortality and morbidity in humans. Being central to its pathogenesis, S. aureus thwarts the host defense by secreting a myriad of virulence factors, including bicomponent, pore-forming leukotoxins. While all vaccine development efforts that aimed at achieving opsonophagocytic killing have failed, targeting virulence by toxoid vaccines represents a novel approach to preventing mortality and morbidity that are caused by SA. The recentl ...[more]

PMID: 31207937

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Project description:The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo. Staphylococcus aureus has a propensity to form multicellular communities known as biofilms. While growing in a biofilm, S. aureus displays increased tolerance to nutrient deprivation, antibiotic insult, and even host immune challenge. Previous studies have shown that S. aureus biofilms thwart host immunity in part by preventing macrophage phagocytosis. It remained unclear whether this was influenced solely by the considerable size of biofilms or whether molecules were also actively secreted to circumvent macrophage-mediated phagocytosis. This is the first report to demonstrate that S. aureus biofilms inhibit macrophage phagocytosis and induce macrophage death through the combined action of leukocidin AB and alpha-toxin. Loss of leukocidin AB and alpha-toxin expression resulted in enhanced S. aureus biofilm clearance in a mouse model of orthopedic implant infection, suggesting that these toxins could be targeted therapeutically to facilitate biofilm clearance in humans.

Project description:Staphylococcus aureus is a leading cause of significant morbidity and mortality and an enormous economic burden to public health worldwide. Infections caused by methicillin-resistant S. aureus (MRSA) pose a major threat as MRSA strains are becoming increasingly prevalent and multi-drug resistant. To this date, vaccines targeting surface-bound antigens demonstrated promising results in preclinical testing but have failed in clinical trials. S. aureus pathogenesis is in large part driven by immune destructive and immune modulating toxins and thus represent promising vaccine targets. Hence, the objective of this study was to evaluate the safety and immunogenicity of a staphylococcal 4-component vaccine targeting secreted bi-component pore-forming toxins (BCPFTs) and superantigens (SAgs) in non-human primates (NHPs). The 4-component vaccine proved to be safe, even when repeated vaccinations were given at a dose that is 5 to 10- fold higher than the proposed human dose. Vaccinated rhesus macaques did not exhibit clinical signs, weight loss, or changes in hematology or serum chemistry parameters related to the administration of the vaccine. No acute, vaccine-related elevation of serum cytokine levels was observed after vaccine administration, confirming the toxoid components lacked superantigenicity. Immunized animals demonstrated high level of toxin-specific total and neutralizing antibodies toward target antigens of the 4-component vaccine as well as cross-neutralizing activity toward staphylococcal BCPFTs and SAgs that are not direct targets of the vaccine. Cross-neutralization was also observed toward the heterologous streptococcal pyogenic exotoxin B. Ex vivo stimulation of PBMCs with individual vaccine components demonstrated an overall increase in several T cell cytokines measured in supernatants. Immunophenotyping of CD4 T cells ex vivo showed an increase in Ag-specific polyfunctional CD4 T cells in response to antigen stimulation. Taken together, we demonstrate that the 4-component vaccine is well-tolerated and immunogenic in NHPs generating both humoral and cellular immune responses. Targeting secreted toxin antigens could be the next-generation vaccine approach for staphylococcal vaccines if also proven to provide efficacy in humans.

Dataset Information

Rational Design of Toxoid Vaccine Candidates for Staphylococcus aureus Leukocidin AB (LukAB).

Publications

Rational Design of Toxoid Vaccine Candidates for <i>Staphylococcus aureus</i> Leukocidin AB (LukAB).

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