Vaccine candidates for cellulitis from Staphylococcus aureus and Streptococcus pyogenes – In silico approach

1 Introduction

Skin is the largest organ in the body with multiple functions; its breakdown leads toseveral diseases (Tortora and Grabowski, 1993). The global burden of diseases estimated in 2013associated 1.79% of skin disorders withsignificant morbidity and mortality; the numbers were especially high for cellulitis (Karimkhani et al. 2017). Considering the disability-adjusted life years, skin illnesses contributed to 1.79% of the worldwide burden of disease, among the 306 diseases (DALY’s). Skin and soft tissue infections are majorly caused by bacteria, with skin inflammation (Karimkhani et al. 2017).

Cellulitis is a form of bacterial skin infection that can damage the dermis. It is caused by Staphylococcus aureus and Streptococcus pyogenes, which cause inflammatory immune responses (Dennis and Bryant, 2016). These bacteria are present on the skin, and when the skin in damaged, they enter in through the damaged regions resulting in infection of the skin. The disease-causing bacteria are more virulent during the winter months in cold countries (Olafsdottir et al. 2015). The main symptoms of such infections include skin redness, skin colonizing, skin maceration, and redness of the skin within a few days after infection. The entrance point may not be obvious, and the breach can include modification in the superficial skin or could manifest as more intrusive infections depending on the type of bacteria. More than 95% of infections occur below the knee (Vary and Connor, 2014). Diabetic patients and elderly people are majorly affected by this disease (Karimkhani et al. 2017). S. aureus is overrepresented in the skin microbiome, and the local microbiome and immunity influence the relationship between the microbiome and cellulitis (Christensen and Bruggemann, 2014).

“Virulence” is an organism’s capacity to infect its host and produce sickness. Virulence factors are components that help the bacteria establish an infection in the host at the cellular level. These elements either have a secretory, membrane-related, or cytosolic origin (Aditya Kumar et al., 2017). Virulence factors have contributed to the discovery of significant virulence traits in microbes, considerably advancing our understanding of microbial pathogenesis (Arturo and Pirofski, 2009).

When the pathogens enter into the break in the skin, they cause deep dermal and subcutaneous infection and cutaneous barrier disruption. Low surface pH, low temperature of the skin surface, and the occurrence of common microbes on the skin surface reduce colonization by pathogens (Raff and Kroshinsky, 2016). Heavy neutrophil infiltration around the blood vessels and dermal edema lymphatic dilation are important histological features of this disease (Burian et al., 2021). The primary strategy to combat cellulitis is vaccination. Peptide-based vaccines are novel and promising for the treatment of mild to chronic cellulitis (Wang and Walfield, 2005).

The UK National Health Service (NHS) conducted an analysis for cellulites diseases using the secure Anonymized information linkage databank, which compiles population-scale, individual-level anonymized linked data from a variety of sources, including 80% of primary care general practices in Wales (Humphreys et al., 2023) The number of general practice visits and in-patient stays were tracked for all patients linked to the pertinent codes in primary care settings for the last twenty years. The Welsh and UK NHS would experience significant financial savings through initiatives to assist patients and healthcare providers in identifying early indicators and dangers of cellulitis, enhance the accuracy of the initial diagnosis, avoid cellulitis recurrence, and improve evidence-based treatment pathways (Humphreys et al., 2023). In response to these discoveries, Wales has created the ground-breaking National Lymphoedema Cellulitis Improvement Program, which offers a proactive approach to cellulitis management (Humphreys et al., 2023).

Therefore, this study aimed to identify possible B-cell and T-cell epitopes from the pathogenic membrane proteins that could stimulate immune responses, both cellular and humoral (Hajighahramani et al. 2017).

2 Materials and methods

2.1

2.1 Selection of virulence proteins

Virulence factors for cellulitis were selected from the membrane proteins of S. aureus and S. pyogenes. Five proteins were selected from S. aureus – sak, tst, isdA, clfB, and cna (Fig. 1) and three proteins from S. pyogenes – SPy, scpA, and hylpL (Fig. 2). The antigenic properties of these proteins were predicted.

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Fig. 1

3D structures of selected S. aureus membrane proteins.

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Fig. 2

3D structures of selected S. pyogenes membrane proteins.

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3 Results

3.4

3.4 Pepitope cluster

Among the 58 epitopes identified from S. aureus, only the two best clusters, LKYGPKFDK and MTFDDKNGK, were present on the outer surface of the protein (Fig. 3).

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Fig. 3

Epitopes of S. aureus predicted using Pepitope.

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Five best epitopes of S. pyogenes were shortlisted from among the 64 epitopes. Among them, FKIEPDTTV, MTPSERLDL, and LKFKPAATV (Fig. 4) were present on the outer surface of the protein. The red regions are the epitopes present on the outer surface of the protein.

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Fig. 4

Epitopes of S. pyogenes predicted using Pepitope.

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IC₅₀ values of 0.01 to 5000 nM indicate that the sequence is eligible; < 5000 nM indicates low affinity, < 500 nM indicates intermediate affinity, and < 50 nMindicates high affinity. The epitopes were selected as vaccine candidates based on the IC₅₀ values for both alleles, DRB1*0101 and DRB1*0401 (Table 4), the most-prevalent MHC alleles in mammals. Further experimental validation of the vaccine candidates is necessary. Among the two epitopes from S. aureus, MTFDDKNGK exhibited an intermediate binding affinity for DRB1*0101 with a IC₅₀ value of 84.33, which is slightly higher than the threshold value of 50 nM; it had a low affinity for DRB1*0401. LKYGPKFDK exhibited low affinity for both alleles. In case of S. pyogenes epitopes, both YTNSDKGGS and MTPSERLDL had a high binding affinity for DRB1*0101; the former had an intermediate and the latter had a low affinity for DRB1*0401. FKIEPDTTV had intermediate binding affinity for DRB1*0101 and low binding affinity for DRB1*0401. Both VKTDDQQDK and LKFKPAATV showed low affinity for both alleles.

Table 4 Validation of epitopes from membrane proteins and MHCPred values.

S.No	Protein name	T-cell epitopes	IC50values of DRB1*0101 (MHCPred)	IC50 values of DRB1*0401 (MHCPred)
Staphylococcus aureus
1.	tst	LKYGPKFDK	944.06	2697.74
2.	cna	MTFDDKNGK	84.33	1729.82
Streptococcus pyogenes
1.	SPy	YTNSDKGGSFKIEPDTTV	33.81268.53	597.04672.98
2.	scpA	MTPSERLDLVKTDDQQDK	29.85620.87	1945.36571.48
3.	hylpL	LKFKPAATV	706.32	3118.89

4 Discussion

The two most common bacteria that cause cellulitis are S. aureus and S. pyogenes (Bennett et al., 2010); they are frequently encountered and clinically challenging (Sullivan and Barra, 2018). Methicillin-resistant Staphylococcus aureus (MRSA) in the population increased skin and soft tissue infections from 1993 to 2005 (Pallinet al., 2008). In a recent studyincluding40 locations and 9 countries encompassing 7477 chronic oedema patients, 16% had experienced cellulitis in the previous 12 months with a prevalence of 37% (Burian et al., 2021).

To avoid the risk factors associated with cellulitis, it is essential to identify an effective vaccine; this strategy is widely used to tackle the incidence of diseases with high risk (Oscherwitz, 2016). A vaccine for S. aureus against bacteremia and pneumonia is available (Adhikari et al. 2012). This study aimed to predict a vaccine for cellulitis.

Both the humoral response involving B-cells and cell-mediated immunity involvingT-cells are important for adaptive immunity. Pathogens as a whole are not recognized by B- and T-cells; however, molecular components known as antigens are recognized (Paul et al., 2015). Epitopes as vaccine candidates have no undesirable effect; therefore, they are preferred over whole protein vaccines (Gallimore et al., 1998). The research of Correia et al. (2014) supports the epitope-focused vaccine design employed in this study.

Locating possible B- and T-cell epitopes using computational techniques, such as immunoinformatics reduces the time and lowers the cost involved in pathogen gene product investigation in the laboratory. (Davies and Flower, 2007). Mondal et al. (2019) showed that the virulence factor and outer membrane proteins are promising candidates for designing epitopic vaccines. Therefore, the virulence factor-based membrane proteins, such as sak, tst, isdA, clfB, and can from S. aureus and SPy, scpA, and hylp1 from S. pyogenes were selected using VaxiJen server with a VaxiJen antigenicity cutoff score of ≥ 0.7, with 70–89% prediction accuracy (Verjovski-Almeida et al., 2003). According to Tong et al., (2009), the majority of investigations use linear epitopes, similar to that predicted in this study, because these B-cell epitopes interact with B-cell receptors to form antigenic determinants on the surface of pathogens. Chen et al. (2007) suggested that utilizing the three tools, BCPred, AAPPred, and ABCpred will increase the accuracy of epitope prediction. B-cells provide long-term protection against pathogens and harmful molecules (Jespersen et al., 2019); therefore, 20-mer B-cell epitopes (Singh et al., 2013) were predicted using BCPred and AAPred, and 16 mer-epitopes (EL-Manzalawyet al., 2008) were predicted using ABCpred. The number of accepted B-cell epitopes were 11, 28, 37, 136, and 85 from sak, tst, isdA, clfB, and cna of S. aureus and 42, 90, and 38 fromSPy, scpa, and hylpL of S. pyogens.

A vaccine delivers antigenic substances that trigger immune reactions to provide an efficient degree of protection against a disease (Kreikemeyeret al., 2017). Our earlier research concentrated on identifying conserved epitopes in several S. pyogenes membrane proteins. Therefore, it is possible to develop conserved peptide-based streptococcal vaccines against human illnesses (Ebrahimi and Mohabatkar, 2018). Clinical application of these peptide-based vaccines against S. pyogenes requires further studies and experimental validation.

Numerous S. aureus strains are resistant to common clinical drugs, including vancomycin (Howdenet al., 2010), daptomycin (Fowler et al., 2006), mupirocin (Cadillaet al., 2011), and linezolid (Locke et al., 2009). There is ongoing research for developing novel molecules, especially vaccines, to fight these infections. An important strategy to combat S. aureus is creating potent monoclonal antibodies and vaccines. However, traditional inactivated vaccines and capsular polysaccharide vaccines are not the best options for providing immunoprotection. Therefore, the B-cell epitope, ²⁷²GYTEDEIV²⁷⁹ is promising for eliciting a potent immunoprotection against S. aureus infection.

Predicting T-cell epitopes requires the identification of MHC-binding peptides because T-cell recognition of antigenic peptides requires their association with MHC (Tomar and De, 2010). Short peptides of 9–11 amino acids can bind to MHC I; therefore, the 9-mer T-cell epitopes, which bind to MHC I and MHC II with high intensity were selected for predicting the affinity for the human MHC alleles, DRB1*0101 and DRB1*0401. The frequency of the two selected alleles, DRB1*0101 and DRB1*0401 within MHC class-II ranges between 20 and 50%. They are among the most common HLA-DR alleles; therefore, these two HLA molecules were chosen during MHCPred analysis (Panigada et al., 2002; Saha et al., 2017). It is essential for a peptide to bind to more than one HLA allele to be considered as a potential peptide for vaccine development. All five epitopes assessed in this study, two from S. aureus and three from S. pyogenes, were present on the surface, as indicated by the results of epitope mapping.

5 Conclusion

Cellulitis is a typical bacterial skin infection that causes pain, swelling, and redness in the infected region. We aimed to develop a new vaccine candidate from virulence-associated membrane proteins, through epitope identification, that is selective against the pathogens and invokes effective immune responses against this skin disease. Several steps of screening were performed to identify the appropriate vaccine candidates. The epitope, YTNSDKGGS, identified from fctA of S. pyogenes is promising as a prospective vaccine candidate, considering the binding affinity and high antigenic values. In addition, the epitopes, MTPSERLDL from scpA of S. pyogenes and MTFDDKNGK from cna of S. aureus, are present on the outer surface of their respective proteins. They exhibited better affinity for the two MHC alleles; therefore, they are promising as vaccine candidates as well. Future studies should focus on in vitro and in vivo evaluations focused onverifying the effectiveness of these vaccine candidates.

Funding

Researchers Supporting Project number (RSP2023R360) King Saud University, Riyadh, Saudi Arabia.

Ethical statement

This research work does not contain any human/animal sample.