Summary
Staphylococcus aureus (S. aureus) is one of six nosocomial ESKAPE pathogens, showing concerning levels of multidrug resistance (MDR). It causes Staphylococcus aureus bacteremia (SAB), a predominant cause of bloodstream infection, leading to 20-30 cases per 100,000 per year. While SAB causes a global health burden, its impact on low- and middle-income countries (LMIC) remains underrepresented. Currently, there is no vaccine effective against this major pathogen. The objective of this project is to pinpoint novel protein vaccine targets effective against both local and global S. aureus clinical isolates, with the goal of providing cross protection against diverse strains, including antibiotic-resistant variants. This will provide the initial steps of effective broad-spectrum vaccine through obtaining reliable candidates directly from clinical isolates. We will implement pan-genome reverse vaccinology to screen and characterize staphylococcal vaccine targets. Our preliminary data reveals six primary categories of vaccine candidates identified through our reverse vaccinology approach, conducted on a limited number of isolates. These categories include cell-surface adhesins, secretion systems, siderophores, exotoxins, cell wall-localized proteins, and proteins involved in immune evasion. This approach will establish a solid foundation for us to seek additional significant funding to clone promising candidates and to evaluate the immunogenicity and protective efficacy, thereby enabling the proposed workflow to be further implemented to screen potential vaccine candidates among a wide range of MDR pathogens and prevent the dissemination and emergence of novel MDR pathogens in LMIC.
Project Outcomes
The current project aims to address the issue of multidrug-resistant (MDR) S. aureus infections by applying a reverse vaccinology approach. The study addressed the reasons behind the failure of previous S. aureus vaccine trials by implementing a comprehensive workflow, called AntiPan. Here we represent the in-silico vaccine discovery pipeline, AntiPan, which augments pangenome estimation, reverse vaccinology, and immuno-informatics to predict subunit vaccine antigen candidates against MDR S. aureus. By considering S. aureus genomic diversity, AntiPan offers a tailored approach and lay the foundation for global vaccine design utilizing broader genome datasets. The AntiPan tool suite is a command-line interface designed to offer a high throughput and user-friendly platform for discovering vaccine targets. It is applicable to multidrug-resistant pathogens, and is publicly available at https://github.com/ComputationalBiologyLab/AntiPan.
AntiPan highlights antigens central to Staphylococcus aureus pathogenesis, immune evasion, and virulence, while addressing several limitations of previous subunit antigen prediction methods. In this project, three distinct S. aureus genome datasets were analyzed:
- A set of 198 Egyptian clinical isolates previously deposited in GenBank;
- A newly generated dataset of 156 Egyptian S. aureus isolates, sequenced specifically for this project.
- A collection of 572 global S. aureus genomes, also publicly available in GenBank.
This diverse genomic representation enhances the predictive power and global applicability of the AntiPan pipeline.
The reverse vaccinology analysis of the first set of 198 Egyptian clinical S. aureus isolates yielded 29 antigen candidates, 24 non-toxin and 5 toxin antigens, which were implicated in host invasion, nutrient acquisition, and immune evasion. Epitope mapping revealed overlapping B- and T-cell epitopes. The functional annotation of these antigens showed their role in secretion systems, adhesion, and cytolytic activity. The top surface-exposed/secreted and highly-immunogenic potential antigen candidates (PACs) included EbpS, AmiA, TagH, SspB, IsdC, EssA, SirA, EsxA, HlgC, and HlgB were shortlisted for future experimental validation. Molecular docking analysis showed strong binding of candidate antigens to critical residues in TLR1/TLR2 and TLR4/MD-2 heterodimer complexes. Notably, IsdC, AmiA, and TagH demonstrated strong and complementary binding to both TLR heterodimer complexes, marking them as top vaccine candidates.
The second set of 354 Egyptian S. aureus genomes, which included the abovementioned 198 genomes in addition to 156 newly sequenced genomes, showed 33 antigen candidates after reverse vaccinology filtration, 28 non-toxin and 5 toxin antigens involved in the abovementioned virulence mechanisms including bacterial pathogenesis, host cell invasion, nutrient acquisition, and hemolysins. The immune-informatics analyses shortlisted 10 surface-exposed/secreted and highly-immunogenic antigens comprising the previously mentioned PACs with IsdD replacing AmiA antigen. These results underline the significance of iron-regulated surface determinant proteins (Isd family) and TagH as promising subunit vaccine targets.
The reverse vaccinology analysis of the third set of 572 global S. aureus genomes showed 39 antigen candidates, 33 non-toxin and 6 toxin antigens involved in various virulence mechanisms including bacterial pathogenesis, nutrient acquisition, adherence, host invasion, and immune evasion. The functional annotation of the beforementioned antigens highlighted key functions and domains such as heme uptake, ABC transporters, iron transport, proteolytic activity, hydrolases, lipase activity, anchorage and adherence, fibrinogen binding, immunoglobulin binding, secretion system VII, LPXTG cell wall anchor, etc. A total of 14 surface-exposed/secreted highly-immunogenic antigen candidates were predicted from the immune-informatics analyses. The data showed a consensus of 8 PACs between the three analyzed S. aureus genomic datasets as represented in the venn diagram below. These global antigens, which included SirA, TagH, EsxA, EbpS, HlgB, HlgC, IsdC, and EssA, are promising targets for formulating an effective vaccine against S. aureus with global protection. Future work would entail protein expression followed by in vitro and in vivo validation of the immunogenic characteristics and safety of the identified PACs for the purpose of developing a successful vaccine formula.