Staphylococcal Vaccine Antigens related to biofilm formation

ABSTRACT

The number and frequency of multidrug-resistant (MDR) strains as a frequent cause of nosocomial infections have increased, especially for Methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis, in part due to device-related infections. The transition to antibiotic-resistance in related bacterial genes and the capability for immune escape have increased the sustainability of biofilms produced by these bacteria. The formation and changes in biofilms have been suggested as a target to prevent or treat staphylococcal infections. Thus, this study reviews the development of candidate staphylococcal vaccines by database searching, and evaluates the immunogenicity and efficacy profiles of bacterial components involved in biofilms. The literature suggests that using common staphylococcal vaccine antigens and multivalent vaccines should further enhance vaccine efficacy.

KEYWORDS:

• Candidate vaccines
• biofilm
• device-related infection
• immunotherapy
• prophylaxis
• nosocomial pathogen
• staphylococcus aureus
• staphylococcus epidermidis

I. Introduction

The Staphylococcaceae family is considered to be the etiological agents of several serious disorders, such as sepsis and endocarditis. According to preclinical models, various antigens can stop the spread of staphylococcal infections either alone or in combination with other antigens. Therefore, the development of a protective vaccine to prevent sepsis is one of the most challenging issues in pharmaceutical development.¹

S. aureus is a primary pathogen causing a range of diseases, such as mild skin and soft tissue infections (SSTIs), bacteremia, endocarditis, pneumonia, metastatic infections, sepsis and toxic shock syndrome (TSS) in patients admitted to the hospital. Although the reason for this wide range of symptoms is not yet known, it may be related to undiscovered factors making the host susceptible to colonization.² S. aureus contamination of medical devices inserted into a patient’s body might be remarkably dependent on patient health. Although staphylococcal biofilm-associated infections share some similarities, more intensive care is usually needed in patients with S. aureus involvement. Infections caused by S. epidermidis are more difficult to treat with antibiotic therapy than those caused by S. aureus.³ Moreover, medical devices act as a spreading source of several bacterial diseases to different parts of the human body. Over the past decades, the number of nosocomial infections caused by staphylococcus species, especially S. aureus, has increased.^4,5 Thus, it is essential to know what relevant factors are involved in biofilm formation from a molecular pathogenesis perspective and to discover the physiological status of these biofilms within the body to understand whether they are capable of developing aggressive behaviors. S. epidermidis, as an inhabitant of human skin, has long been considered a contamination agent when cultured from blood or tissue samples.^6,7 Because S. epidermidis is a part of the normal skin flora, it probably initiates contamination after implantation of a medical device. In recent years, S. epidermidis has been widely accepted as a leading cause of nosocomial bloodstream infections, especially in patients with prosthetic medical devices.^7,8 S. epidermidis is an opportunistic pathogen known principally to cause infection in immunocompromised patients.⁹

Coagulase negative staphylococci (CoNS), such as S. hominis, S. epidermidis, S. saprophyticus, S. warneri, S. cohnii, S. saccharolyticus, S. haemolyticus, S. capitis, and S. lugdunensis are a kind of staphylococci that inhabit on human skin. Most staphylococcus strains, including S. epidermidis, are normally harmless to their host, but they might be pathogenic in cases where the skin is injured. CoNS colonization seems to be relevant to specific sites of infection and its abundance. For instance, S. saprophyticus, which is a common inhabitant of inguinal and perineal areas, is an etiological agent of urinary tract infections.¹⁰

Even though S. aureus and S. epidermidis are a part of human flora, they can adhere to the surfaces of medical devices and develop multilayered structures known as “biofilms” which are difficult to treat.¹¹ Biofilm is defined as a complex community of bacteria attached to a surface or interface and enclosed within an exopolysaccharide matrix. In general, there is no consensus about the different steps of biofilm formation in staphylococci, but most researchers believe that biofilm is developed in four steps comprising adherence, aggregation, maturation, and dispersal.¹² Phase-specific factors are needed for each of these steps. There are two mechanisms of biofilm formation in strains of the Staphylococcaceae family including protein- and polysaccharide-dependent procedures.¹¹

There are four stages of biofilm formation:

Adherence

Adherence, the first stage of biofilm formation, is the attachment of bacteria to the cell membrane of their host by bacterial appendages, which are cell-surface components facilitating the adhesion to other cells. Matrix proteins play a critical role in adherence and the evasion of the host immune system by interacting on the biofilm formation making the matrix proteins key virulence factors in the staphylococci.

Aggregation and Maturation

The maturation phase has two main characteristics in biofilm formation: A) intercellular aggregation through a wide range of molecules including sticky macromolecules, and B) formation of the three-dimensional structure of mature biofilm.

Detachment

The dispersal of bacteria to connect to another colonization site during the establishment of mature biofilm in staphylococci is known as detachment. It may happen by the detachment of either single cells or larger cell aggregates. Cell dispersal leads not only to embolism, sepsis, and hospital-acquired pneumonia, but also to biofilm formation at other sites.¹³

The capability of biofilm formation especially in S. aureus and S. epidermidis is a critical factor involved in pathogenesis, because the mentioned bacteria can be colonized on medical devices, making them stable not only to multiple antibiotics but also to host defenses. Biofilm formation and consistency in host immune evasion by the S. epidermidis and S. aureus make them the main concern regarding nosocomial infections occurring in hospitals.^14,15 It has been proved that biofilms are resistant to antimicrobial therapy and host defenses.¹⁶ Biofilm-associated infections are more difficult to treat by antibiotic therapy,¹⁶ and there is an urgent need to remove or replace the used implant following biofilm formation. Practical studies should be performed on putative vaccine candidates to prevent biofilm formation.¹⁷

Although there are some similarities in biofilm-associated infections caused by S. aureus and S. epidermidis, more intensive care is needed for cases with S. aureus involvement. The prevention of bacterial attachment and accumulation at biofilm-related stages caused by the presence of some common antigens in staphylococci could play an effective role in preventing staphylococcal biofilm-related infections. Then, staphylococcal targeting of macromolecules involved in the attachment and accumulation of the biofilm-forming phase (proteins and polysaccharides) and antibodies arising in response to them may be suitable options for antibody-dependent treatment of biofilms.¹⁸

II. Vaccine Strategy

Conserved surface components in Staphylococcus spp., preferably with a high rate of expression in the bloodstream and to some extent in the biofilm-forming process, stand as suitable staphylococcal candidate vaccines to decrease the number of staphylococcal infections.^5,6

Many researchers have attempted to find an immunoprophylactic agent against infections caused by staphylococci. Although staphylococcal infections do not result in protective immunity, vaccination can be plausible.⁶ Protective immunity against S. aureus infection may be gained either by active or passive immunization.⁶ Given the current problems in finding an effective vaccine for diseases associated with S. aureus, there is an essential need to shift to both therapeutic antibodies and the use of multivalent vaccines with more attentive antigen selection focused on new molecular techniques.⁶ The virulence factors are less evident in S. epidermidis. Previously, the strains were discriminated against as nonpathogenic strains; then, a vaccine was developed for S. aureus. Despite this, a new understanding of biofilm formation in S. epidermidis virulence has been achieved in recent years.

As previously mentioned, biofilms are resistant to antibiotics and can escape from the host immune system. Recently, several studies have been accomplished on the selection of antigens to eradicate biofilm-related infections. General immunization in receiving medical implants, including short-term venous catheters, seems to be more cost-efficient than the removal and replacement of contaminated devices. In the case of permanent users of medical devices, removing the contaminated device might be risky because of the long hospitalization and increased healthcare costs involved. In such cases, active vaccination can grant extensive protection that is economically preferable. The target group for active vaccination comprises patients who will receive the implants susceptible to the actual risk of staphylococcal infections, such as the joint, heart valve, lens replacement, and port-a-cath. However, to prevent morbidity and mortality, passive immunoprophylaxis and immunotherapy are recommended for those patients who cannot properly respond to a vaccine at the immediate risk of infection, such as immunocompromised critically ill patients and neonates.⁶

III. Vaccine Development Based on the Targets

S. epidermidis and S. aureus are ubiquitous colonizers of human skin that are in constant contact with the human immune system. Because anti-S. epidermidis and anti-S. aureus are normally present, whole-cell S. epidermidis vaccines are not effective. Many investigations have introduced a large number of targets for vaccine development against S. epidermidis and S. aureus, which increases the number of putative targets. In the classical approach, different targets with certain functions have been studied and evaluated as a subunit vaccine. Also, new target candidates have been suggested by reverse vaccinology and bioinformatics.^19–21 Moreover, the presence of suggested vaccine candidates among the multiple isolates has been determined using the pan-genome reverse vaccinology approach. To cover the genetic diversity of a pathogen in vaccine development strategies, its pan-genome as well as its molecular epidemiology should be analyzed. Furthermore, there is a necessity to know whether a selected antigen is possibly upregulated or downregulated during infection. It is essential to avoid the use of antigens that are downregulated in biofilms in vivo. It has to be determined whether the proteins secreted by a unique pathogen can have a critical role in an immune response in the host.^6,22 For example, the immunogenicity and vitamin D-binding protein (DBP) within the cell wall-associated proteins of S. epidermidis O-47 have been identified by applying a proteomic approach.⁷ Also, the sera derived from the individual pathogens have been screened to detect the secreted antibodies belonging to a peptide library.⁶ The epitopes of proteins with an unknown function and epitopes corresponding to the gene sequences that have not been identified yet can be determined using this approach with bioinformatics tools.²³

Antigens of S. epidermidis expressed during human infection have been determined by antigenome technology.²⁴ New vaccine targets may be provided by immunomics tools. Some of these proteins are categorized as immunome. Each protein within the pathogen can be determined⁷ after epitope mapping of T-cell and B-cell algorithms.^6,25

IV. Putative Targets for Development of a Staphylococcal Vaccine

A large number of independent proteins have been investigated as putative candidate vaccines for staphylococcus spp., especially against S. aureus and S. epidermidis, to reduce nosocomial infections, such as neonatal and biomaterial-related ones.

In the Staphylococcaceae family (especially in S. aureus and S. epidermidis), biofilm-forming-related macromolecules are divided into polysaccharides (cell wall TAs, polysaccharide intracellular adhesion (PIA), and matrix-binding characteristics) and proteins, including two families of microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and serine-rich repeat proteins (SRRPs).²⁶ MSCRAMMs can mediate indirect binding to hosts’ plasma-covered surfaces with fibronectin (Fn), collagen (Cn), and fibrinogen (Fg) as matrix proteins. Cell surfaces are covered with a lot of different macromolecules, such as proteins (Embp, GehD, SdrG, SdrF, AtlE and Aae autolysins). Serine-aspartate repeat (Sdr) protein family members are categorized into two distinct species, but their functions are the same.^27–29 Given the first step of biofilm formation, each of the surface-located macromolecules could be considered as a putative vaccine candidate.³⁰ After devices are inserted, they become covered by host matrix proteins. The interaction between the host matrix proteins and MSCRAMMs is among the most important factors in colonization. Vaccine antigens are listed in order of general state of development and potential utility based on data to date. Some putative anti biofilm vaccine are listed in Table 1.

Table 1. Studies conducted on anti-biofilm antibodies raised by the macromolecule (s) related to biofilm formation

Name of Antigens	Properties of Native Antigens	Vaccine related Information	Status	Citations
AAP	35-kDa surface-associated protein of S. epidermidis and S. aureus	In S. epidermidis accumulation (of up to 79% and 87% higher than individual MAbs) and biofilm formation was inhibited by monoclonal antibody against (AAP).	Preclinical	^Citation81
SesC protein	S. epidermidis surface (Ses) proteins (A cell wall-anchored protein)	Elicited antibodies showed biofilm inhibition and opsonophagocytic killing activities in immunized sera.	Preclinical	^Citation82
Conjugated PNAG-ClfA	-Poly-N-acetyl-β-(1-6)-glucosamine (PNAG), also known as polysaccharide intercellular adhesion (PIA), a main aggregative macromolecule in both S. aureus and S. epidermidis. -Clumping factor A (ClfA) is an S. aureus binding protein to the C-terminal region of the fibrinogen gamma chain	Raised antibodies (in rabbits, rhesus monkeys, and goats) have shown 54% killing activity to wild type S. aureus Newman	Preclinical	^Citation80
PhnD	A transmembrane protein which has an organic phosphonate transport function	PhnD-specific antibodies blocked biofilm development at the initial attachment and aggregation stages.	Preclinical	^Citation83
Pagibaximab®	LTA (cell wall component) Pagibaximab® [Biosynexus; NCT00631800]	In animal model and more limited phase II study of very–low-birth-weight (VLBW) infants by treatment of biofilm-associated S. epidermidis sepsis, satisfactory results were obtained.	Failed in Phase III clinical trial	^Citation84
Ses proteins	S. epidermidis surface (Ses) proteins (A cell wall-anchored protein)	IgGs to SesB, M, O, Q, and R of in in vitro and in vivo models significantly reduced the formation of biofilms, and phagocytic killing was accomplished only after opsonization with SesB-specific IgGs.	Preclinical	^85
Conjugated PIA- rSesC	-Poly-N-acetyl-β-(1-6)-glucosamine (PNAG), also known as polysaccharide intercellular adhesion (PIA), a main aggregative macromolecule in both S. aureus and S. epidermidis. -75 KD truncated protein (A cell wall-anchored protein)	The immunization of mice (BalB/c) with the PIA–rSesC conjugate raised the levels of opsonic antibodies, and the vaccinated mice were protected when challenged intravenously by wild-type S. epidermidis strain 1457. The conjugated vaccine was able to eliminate S. epidermidis biofilm formation in in-vitro and in-vivo assays.	Preclinical	^Citation11

AAP: Accumulation-Associated Protein; mAb: monoclonal antibodies; JVC: jugular vein-catheterized; dPNAG-ClfA, poly-N-β-(1–6)-acetyl-glucosamine, clumping factor A(ClfA); PhnD: phosphonates-binding periplasmic protein, LTA: lipoteichoic acid.

Related Proteins in the Formation Process of Staphylococcal Biofilms

As several bacterial surface-located components are engaged in the initial phase of biofilm production, they have been evaluated as putative candidate vaccines. Furthermore, the MSCRAMMs/surface proteins have also been studied.⁶

The contributions of S. aureus surface protein C and G (SasC and SasG), fibronectin/fibrinogen-binding proteins A and B (FnBPA and FnBPB), serine-aspartate repeat-containing protein C (SdrC), clumping factor B (ClfB), and the biofilm-associated protein (Bap) have been individually implicated in biofilm matrix formation. In S. epidermidis, the accumulation-associated protein (Aap) plays a main role in both the primary attachment and the aggregation of bacterial cells in the biofilm formation phases by forming fibrils on the cell surface. Moreover, an extracellular matrix binding protein (Embp) and S. epidermidis surface protein C (SesC) can mediate the formation of proteinaceous biofilms in S. epidermidis. Additionally, two iron-regulated surface determinant proteins, the extracellular adherence protein (Eap) and the extracellular matrix protein-binding protein (Emp) of S. aureus, are capable of forming the biofilm in iron-depleted conditions.³¹

Protein-Based Putative Vaccine Candidates in S. epidermidis

SdrG (Fbe)

Serine-aspartate repeat-containing protein G (SdrG), also known as Fbe, is a 119-kDa cell -surface adhesive protein which is associated with 78% of orthopedic infections and 91% of catheter-related infections caused by S. epidermidis.⁶ It belongs to the Sdr family and is anchored by LPXTG motif to the cell wall. SdrG also has a high binding affinity to fibrinogen (Fg).^32,33 The Fbe/SdrG protein consists of five regions: 1) the secretory signal sequence, 2) A region with Fg -binding activity, 3) B region with an unknown function, 4) R region comprising serine-aspartate repeats, and 5) C-terminal region that is related to the anchoring of SdrG to the peptidoglycan. Similarly, clumping factor A (ClfA) in S. aureus binds to the C-terminal region of the fibrinogen gamma chain.^32,34 ClfA and ClfB, as SdrG homologs, are major fibrinogen-binding MSCRAMMs on the cell wall of S. aureus.³⁵

The effect of SdrG surface density on the ability of different strains of S. epidermidis to attach to Fg-coated surfaces has not yet been investigated. The results of using methods such as single-molecule atomic force microscopy (AFM) have shown that S. epidermidis displays a higher density of SdrG on cell surfaces exhibiting greater adhesion to Fg-coated substrates.³³ The clinical importance of SdrG in the colonization and infection of S. epidermidis was highlighted after the injection of S. epidermidis into the bloodstream of mice when the SdrG expression was induced at higher levels than the in-vitro condition in which it was not expressed at detectable levels.³⁶ Furthermore, higher levels of antibody titers against SdrG have been found in patients after S. epidermidis infection in comparison with uninfected ones.³⁶ The possible role of SdrG in biofilm formation and virulence has been determined by taking advantage of immunological aspects of the higher antibodies and opsonic activity of SdrG compared to SdrG-deficient mutants.²⁵ The antisera of patients recovered from S. epidermidis infections demonstrated higher reactivity with recombinant SdrG than those of the healthy controls, suggesting that SdrG is likely to be expressed during an infection.³⁷ SdrG is highly present in clinical isolates, but it is not necessary for the establishment of infection.^37,38 High variations of FG-binding in the species and the vulnerability of SdrG to the sequence alteration are important reasons for the development of an SdrG-based vaccine.³⁹ Several previous in-vitro studies have shown that SdrG is not detectable on bacterial surfaces. Considering the host-specific signal induction of SdrG following an invasive infection, it is assumed that the expression of the SdrG protein is related to the infections, not to commensal life. Thus, SdrG may be a distinct marker between commensal S. epidermidis and its pathogenic strains and also a valuable candidate for a prophylactic vaccine.^6,40 The vaccination of mice with recombinant SdrG-N2N3 (consisting of two subdomains of N-terminal A domain) has been shown to decrease the number of S. epidermidis 0–47 cells in the bloodstream, whereas recombinant His-SdrG-N1N2N3 (consisting of three subdomains of N-terminal) failed to reduce the bacteria counts in the blood.⁴⁰ The protective mechanisms of the two vaccines have not yet been identified. Primary results from animal models led to the launch of INH-A21 (Veronate) (Inhibitex, Inc., USA). One study used high polyclonal immunoglobulin titers to assess S. aureus ClfA and S. epidermidis SdrG in donors’ sera. In rabbit endocarditis models, the prophylactic and therapeutic effects of INH-A21 were observed against S. aureus and S. epidermidis infections.⁴¹ Evaluation of INH-A21 in late-onset sepsis among premature infants with very low birth weights in phases I and II showed that INH-A21 could reduce the number of S. aureus infections, but it did not affect CoNS infections.⁴² In phase III of a clinical trial, no endpoints were achieved to prevent septicemia in infants with very low birth weights. S. aureus ClfA, as an adhesive protein, is involved in the mouse model of septic arthritis and in rabbit and rat models of infective endocarditis.³⁴ Basically, all S. aureus clinical strains carry the ClfA gene.³⁴ In both active and passive immunization studies, strong immune responses were triggered by ClfA, indicating that CIfA is a potentially important vaccine candidate for the prevention of invasive S. aureus infections.³⁴ The rate of arthritis in mice immunized with recombinant ClfA carrying an Fg-binding domain after challenging it with S. aureus infection was significantly lower in comparison with unimmunized mice.²⁶ Moreover, passive immunization of mice with monoclonal or polyclonal antibodies against the ClfA Fg-binding domain revealed similar results.²⁶

Results of combination therapy with Aurexis (human monoclonal antibody) have shown that ClfA was a suitable target for therapeutic strategy when high Fg-binding affinity was observed for ClfA in the presence of humanized monoclonal antibodies.²⁶

SdrF

Serine-aspartate repeat-containing protein F (SdrF), similar to SdrG, possesses features typical of MSCRAMMs including the existence of the LPxTG wall-sorting motif.⁴³ It has been suggested that SdrF plays a fundamental role in the initiation of driveline infections (DLIs) as the most common type of left ventricular assist device–associated infections (LVADIs) caused by S. epidermidis. Domain B of the SdrF protein is an attachment site adhering to the host collagen deposited on the surface of the driveline and other diseases caused by S. epidermidis, especially medical device-related infections.⁴⁴ Despite the presence of SdrF in most S. epidermidis isolates, there is a high sequence variability in the SdrF of these isolates. Furthermore, anti-SdrF antibodies have not been detected in patients recovered from S. epidermidis infection.³⁶

GehD

GehD lipase as an extracellular 43-kDa protein probably mediates the adherence of S. epidermidis to immobilized collagens through attachment to collagen types I, II, and IV. Some previous studies have confirmed that lipases have an important role in localized infections, like abscesses. Using in-vitro expression technology (IVET), it has also been indicated that these proteins are highly expressed in murine models of infection.^45,46 It is assumed that the bacterial resistance of GehD to secreted fatty acids of skin might be related to its pathogenicity.⁴⁶ Moreover, the bacterial resistance of GehD is essential for bacterial skin colonization.⁴⁷ GehD lipase is probably a bifunctional molecule with glycerol ester hydrolase and collagen-binding activities. Bowden et al. found that the recombinant mature GehD and anti-GehD antibodies inhibited the attachment of S. epidermidis to immobilized collagen. However, there was no significant attachment to the collagen (Cn)-binding protein when the recombinant GehD was expressed on the cell wall of Lactococcus lactis.^45,48 Furthermore, the levels of anti-GehD IgG titer were higher among patients infected with S. epidermidis than uninfected ones.²⁴ Although GehD may be an interesting vaccine candidate against staphylococcal infections, further studies are needed to determine its potential role in S. epidermidis virulence and biofilm formation.⁶

Embp

Extracellular matrix-binding protein (Embp) is a fibronectin‐binding protein mediating biofilm formation in the icaADBC and aap isolates of S. epidermidis that leads to the escape of bacteria from phagocytosis by macrophages.⁴⁹ Embp also has some intercellular and extracellular matrix binding properties.⁵⁰ Some studies have reported that the frequency of the Embp gene among S. epidermidis isolated from prosthetic joint infections and septicemia was around 92% and 87%, respectively.⁶ Embp antibodies injected in rats did not alter the opsonization capacity and phagocytosis of S. epidermidis by alveolar macrophages.⁵¹ Further investigations should be done to determine the potency of Embp protein as a vaccine candidate.

AtlE and Aae

Autolysin E (AtlE) is a 60-kDa protein mediating the primary adherence of S. epidermidis to polymeric materials and vitronectin (VN), which is an extracellular matrix (ECM) protein. This protein exists on the surface of all gram-positive bacteria including S. epidermidis isolates. As the AtlE mutant of S. epidermidis is less infectious in a rat model of central venous catheter (CVC)-associated infection, it probably has a role in S. epidermidis pathogenesis.⁴⁴ AtlE may be a suitable target for immunotherapy and drug development.⁵² A novel autolysin/adhesin (Aae) is a 35-kDa surface-associated protein of S. epidermidis consisting of vitronectin, fibrinogen, and fibronectin-binding activity.⁵³ Pourmand et al. experimented on the antigens expressed during human infections with S. epidermidis and showed that the reactive immunoglobulin G titers against the repeat sequence and mature amidase domains of AtlE, ScaB, and GehD increased significantly in both infected patients and healthy individuals.²⁴ The presence of a highly conserved domain in the Aae makes it a promising target for vaccine development.^24,54,55 It is noteworthy that the contrast effect may be induced by various epitopes of Aap. Moreover, Aap can be a part of a multivalent vaccine but not a solo one, as it is not the only possible factor involved in biofilm accumulation.⁶

Bhp

Bhp as a Bap homologue protein is known as a putative proteinaceous intercellular adhesive protein that has been shown to contribute to the biofilm formation of S. epidermidis and could be considered as a vaccine candidate.⁵⁶ Bap exists in some human isolates of S. epidermidis. Despite the role of Bap in biofilm accumulation, Lasa et al. have indicated that the disruption of the Bhp gene in two clinical strains does not change the capacity for biofilm formation, suggesting that in some strains, other factors may compensate for the absence of Bhp in biofilm formation.⁵⁷ Bhp encodes the downregulation of the genes in a PIA-negative S. epidermidis strain as well.⁵⁸ It is only involved in the development of 9% of bacteremia and 13% of skin colonization.²¹

Reverse Vaccinology and the Search for Other Surface-Located Targets

Bowden et al. determined the 11 genes involved in cell-wall protein synthesis using bioinformatics analysis. Cell wall-anchored proteins, such as Aap, Bhp, SdrF, and SdrG, were encoded by four of these genes, and the remaining genes have not yet been characterized.¹⁸ S. epidermidis surface (Ses) proteins are structurally similar to the cell wall proteins of S. aureus and gram-positive cocci.²¹ Because high antibody titers against some Ses proteins were detected in patients recovered from S. epidermidis infection, the expression of Ses proteins is likely to be induced by S. epidermidis infection.²¹ Shahrooei et al. concluded that biofilm formation in S. epidermidis was inhibited by rabbit polyclonal antibodies against sesC protein by reducing the primary attachment and biofilm formation ability of S. epidermidis.⁵⁹ They also suggested that anti-sesC antibodies could be utilized for the immunotherapy of biofilm-related infections with S. epidermidis.⁵⁹ It has been shown that all clinical isolates of S. epidermidis contain the sesC gene, while it was absent in some S. epidermidis isolated from healthy individuals.^21,60 According to these studies, sesC may be a vaccine candidate for the prevention and treatment of S. epidermidis biofilms. The sesI gene was not detected in any isolate of healthy individuals, but it was highly expressed in clinically infected patients. Thus, the sesI protein may play a key role in the pathogenesis and invasive capacity of S. epidermidis.⁶¹

Proteomics

Sellman et al. identified the cell-wall-associated proteins of S. epidermidis by two-dimensional (2D) gel electrophoresis, and they detected them in the sera obtained from rabbits immunized with live S. epidermidis 0–47 or biotin-labeled serum proteins eluted from the surface of bacteria grown in rabbit serum. From 29 serum-binding proteins identified by mass spectrometry, 27 were constructed as recombinant proteins and used to immunize mice.⁷ The results showed that five proteins, i.e. cysteine synthase, Na⁺/H⁺ antiporter, lipoate–protein ligase, acetyl-CoA acetyltransferase, and alanine dehydrogenase, induced an immune response that led to a significant reduction in the number of bacteria recovered from the spleen and bloodstream of the infected mice. These proteins were recognized as a putative vaccine candidate.⁶

Adhesin proteins

In a vast majority of S. epidermidis strains, biofilm formation is mediated by a bifunctional cell wall-anchored protein called the accumulation-associated protein (Aap), which is needed either for commensal or lifestyle of biofilm formation in the bacteria.⁶² Because of the Aap frequency in both invasive and commensal S. epidermidis strains, it has been determined as a good putative vaccine candidate.⁶³ Aap as an LPXTG protein includes a terminal A domain which promotes binding to corneocytes enabling skin colonization. Additionally, the B-repeat regions that remain after stimulating intercellular adhesion by proteolytic cleavage are caused by the biofilm accumulation (supra).⁶² Biofilm formation in aap-positive and icaADBC-negative S. epidermidis strains is inhibited through the polyclonal (except for anti-Aap domain B antiserum) antibodies raised against the Aap. This finding supports the hypothesis that other macromolecules are involved in biofilm production.⁶⁴ Single monoclonal antibodies (mAbs) against Aap reduce the biofilm accumulation in the ICA-positive S. epidermidis RP62A strain by more than 66%; in contrast, the mixture of two mAbs reduced S. epidermidis accumulation up to 79–87%.⁶⁵ The results of a study on mice vaccinated by Aap indicated that passive immunization by Aap and LTA mice monoclonal antibodies cannot be effective on catheter-induced infections by PIA-dependent biofilm formation of S. epidermidis in the mice model. Also, the bacterial adherence to the implants significantly increased in mice which received an injection of anti-LTA. Anti-Aap and anti-LTA had no opsonic activity and did not protect the mice against biomaterial-associated infection, but they had a binding activity to S. epidermidis.⁶⁶ In recent research, three mAbs against the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5) were generated and compared concerning their role in in-vitro biofilm accumulation and planktonic cell aggregation. The results showed that mAb (18B6) impeded biofilm formation by S. epidermidis RP62A; nonetheless, mAb (25C11) and mAb (20B9) promoted biofilm accumulation. In addition, 18B6 recognized an epitope that is present in all 12 B-repeats within Aap through epitope mapping. Another epitope that only exists in the six B-repeats resulted in a flawless inhibition of Aap dimerization. Not only Aap expression, but also extracellular polymeric substance biosynthesis in S. epidermidis was influenced by all three mAbs, which also enhanced cell accumulation.¹⁷

Polysaccharide antigens

Ribitol and glycerol teichoic acid (RI-TA and Gly-TA) and PIA in both S. aureus and S. epidermidis and capsular polysaccharide (CP) with amino uronic acid contents in S. aureus are considered as main polysaccharides in staphylococcal biofilm formation. The mentioned macromolecules play the main role in the primary attachment and accumulation of bacterial cells in the biofilm-forming process, and they may be good vaccine candidates. PIA and TA are two common related polysaccharides in the biofilm formation process of S. epidermidis and S. aureus.^11,67,68

PIA is also known as poly-N-acetyl-glucosamine and is synthesized by the enzymes encoded on the icaADBC operon.⁶⁹ Accumulation can occur independently from the ica, instead of relying on the expression of surface proteins such as Aap in S. epidermidis and S. aureus surface protein G (SasG) in S. aureus.⁶⁹

Polysaccharide-Based Putative Vaccines

PIA as a Polysaccharide-Based Vaccine Candidate

Surface polysaccharide poly-N-acetyl-β-(1-6)-glucosamine (PNAG), also known as polysaccharide intercellular adhesion (PIA), is produced in-vitro by either S. aureus or S. epidermidis with high levels of acetate substituting for amino groups that lead to the generation of opsonic and protective antibodies.⁷⁰ The positive charge of PIA has a great effect on staphylococcal infections and evasion of the host immune system.

PIA has been extensively evaluated as a putative candid for vaccine development. Biofilm formation is classified as either PIA-independent or PIA-dependent based on the presence of PIA as the main macromolecule in the exopolysaccharide matrix.

PIA has an important effect on the biofilm production and pathogenicity of S. epidermidis. In the exponential growth phase, the ica operon is responsible for PIA synthesis by utilizing the assembled UDP-N-acetylglucosamine.⁷¹ According to epidemiological studies, the icaADBC operon is a typical feature of nosocomial infections by S. epidermidis, S. aureus, S. caprae, and S. lugdunensis strains.^72,73 There are similar reports regarding the presence of the ica operon in other gram-positive and gram-negative human pathogens, including E. coli, Yersinia pestis, aggregatibacter actinomycetemcomitans, and Bordetella spp.⁷⁴ Other animal studies have reported the effect of the ica gene cluster on S. epidermidis virulence.⁶ PIA plays a substantial role in staphylococcal biofilm formation and also has an effect on the evasion of the immune system. This polymer is considered the first and an important target in the development of vaccine candidates for biofilm inhibition. Several studies have been conducted on the development of a PIA-based vaccine supporting PIA as a successful target for vaccine development. Its importance in the multifactorial process of S. epidermidis invasiveness has also been confirmed. Interestingly, this polymer is produced by other pathogens, such as S. aureus, Escherichia coli, Yersinia spp., and Bordetella pertussis, which increases the application of this PIA-based vaccine.⁶ Based on recent studies, the production of PIA by S. epidermidis is an essential factor for the colonization of S. epidermidis in skin. Isolates prepared from most healthy subjects do not possess the icaADBC operon, but the existence of this operon has been confirmed in medical-device related infections.⁷⁵ Findings have demonstrated that arisen antibodies to immunized animals with a deacetylated PNAG (≤15% acetyl) labeled by diphtheria toxoid (DT) as a carrier protein decreased three strains of S. aureus and the PIA-dependent biofilm formation of S. epidermidis M187.⁷⁰ Although both PNAG-DT and dPNAG-DT vaccines were immunogenic in rabbits and raised antibodies to these vaccines mediated the opsonic killing, dPIA-DT were more efficient than anti-PIA sera in killing the PIA produced by S. epidermidis and S. aureus strains in-vitro and in the clearance of S. aureus in–vivo.⁷⁰ There are polysaccharides in other organisms, including S. aureus, as well as gram-negative human pathogens that are similar in terms of PIA and have fundamental functions in biofilm formation.⁷⁴ Moreover, PIA and other related polysaccharides are generally synthesized during biofilm formation in the vast majority of bacterial genera.⁷³

The icaB gene in the ica cluster, as a critical gene to PIA-dependent biofilm formation, has an undeniable role in the synthesis of PIA deacetylation that is bound on the cell surface. The ica Luci without the icaB gene has been shown to decrease the ability of biofilm formation in S. epidermidis. It has been proved that the overexpression of icaB gene (S. aureus 10833) enhances the capability of biofilm formation by increasing the PIA/PNAG containing a higher amount of deacetylation of N-acetylglucosamine. This strain was utilized assuming that dPNAG-specific antibodies are important for opsonophagocytosis. Antibodies raised against the strains with overexpressed icaB had higher killing activity in a PNAG-specific opsonophagocytic assay.⁷⁴ Thus, the surface-associated icaB protein can be studied as a valuable putative vaccine target. During biofilm formation, PIA mainly interferes in the accumulation phase after bacterial adhesion to the material surface. Further investigations into the efficacy of PIA-specific antibodies are still needed.

Targeting Teichoic Acid

Teichoic acids (TA), as one of the main gram-positive bacteria adhesive macromolecules, include the glycerol and ribitol phosphate copolymer with phosphodiester bonds. They seem to extend to the surface of the peptidoglycan layer and can be covalently linked to N-acetylmuramic acid (MurNAc) or a terminal D-alanine cross-linked by tetrapeptide between N-acetylmuramic acid units of the peptidoglycan layer; they can also be anchored in the cytoplasmic membrane with a lipid anchor. Serologic response to staphylococcal teichoic acids happens in various staphylococcal infections. Wall teichoic acids (WTAs) have been introduced as the main macromolecules to the primary attachment and accumulation phase in the biofilm formation of S. epidermidis,⁷⁶ and they are chiefly important in the inflammation and immune evasion (supra); they have already been used as a target for vaccination. Gly-TA structured by N-acetylglucosamine residue is a main factor in the biofilm formation process. Pagibaximab, a chimeric (murine/human) monoclonal antibody, was developed by Biosynexus Inc. (MD, USA) to prevent late-onset sepsis that commonly occurs in low birth weight infants. The efficacy of mAb was determined as >90% against the CoNS of clinical isolates. Up to 90% of bacterial killing activity was detected at doses of < 10 μg/ml as an apt opsonophagocytic result preventing related infections in animal models.

Results obtained after phase I clinical trials based on the pharmacokinetic and safety properties of pagibaximab in populations at high risk of developing CoNS infection support pagibaximab as a safe and tolerable antibody to both healthy adults and high-risk neonates.^77,78 The results of a randomized, double-blind, placebo-controlled study on infants hospitalized in intensive care units (ICU) who had received 90-mg/kg pagibaximab infusions showed that staphylococcal sepsis occurred followed by the phase II clinical trial.⁷⁹ Results of evaluations of the safety, efficiency, and pharmacokinetics (PK) of pagibaximab (100 mg/kg/dose) compared to the placebo after a phase IIb/III, randomized, double-blind, multicenter, placebo-controlled study revealed that prevention of staphylococcal sepsis in VLBW infants (600–1200 gr) who intravenously received 100 mg of pagibaximab/Kg did not decrease the sepsis (ClinicalTrials.gov Identifier: NCT00646399). In addition, Broekuizen et al. vaccinated mice by S. epidermidis cell wall passive immunization through the monoclonal antibodies against the Aap and LTA in an experimentally biomaterial-associated infection and showed that these arisen antibodies had no biofilm inhibitory effects.

As a result, passive immunization not only cannot eradicate biomaterial-related infections, but they also can increase these infections by binding to the implants.

Common Antigens in Both S. aureus and S. epidermidis

Aae, PIA, and TA are considered as common macromolecules in both biofilm-forming S. aureus and S. epidermidis.^11,68,80 Taking advantage of a common protein such as Aap in both S. aureus and S. epidermidis that plays an essential role in the attachment and aggregation of biofilm phases and its conjugation to a confirmed protective polysaccharide such as PIA^11,80 could eliminate the biofilm formation process by inducing cellular immunity-related immunoglobulin subtypes (IgG2a, IgG2b) to activate memory cells.¹¹

Selection of Vaccine Antigens Based on Biofilm Formation

Considering the role of biofilms in host surveillances and the pathogenesis of S. aureus and S. epidermidis as well as the related macromolecule similarities in the mentioned bacteria, polysaccharide and protein polyvalence vaccines can potentially increase the potency of putative vaccines to the prophylaxis of biofilm-related infections. A polysaccharide vaccine must be boosted because of its poor immunogenicity, and it should be mentioned that using a polysaccharide-based vaccine is limited in infants and the elderly due to the mentioned features. All of the mentioned antigens have achieved a preclinical proof-of-principle for being a candidate vaccine antigen that could be developed.

Conjugated PIA-SesC as a Vaccine Candidate against Biofilm Formation of Staphylococcaceae

Mirzaei et al. have revealed that the conjugation of PIA with a specialized superficial protein of S. epidermidis as a carrier enhances the function of raised antibodies both in in-vivo and in-vitro experiments.¹¹ Choosing a protein carrier is a critical key for the construction of an efficient conjugate vaccine. Results of the research showed that SesC protein in S. epidermidis has a 61.5% similarity in function to the 341-aa fragment of ClfA in S. aureus. ClfA, as a fibrinogen-binding microbial surface molecule, can recognize adhesive matrix molecules (MSCRAMM). In-vivo and in-vitro assays have shown that PIA-SesC was effective when targeted mice groups received 100 µg/ml of the conjugate on days 0, 7, 14, and 28 of the experiment. The arisen antibodies had the proper effectiveness in biofilm inhibition so that about 90% of bacterial killing in opsonophagocytosis and survival likelihood occurred following the intravenous challenges by S. epidermidis 1457. The presence of enhanced IgG2, an isotype in the immunized sera, using the conjugate leads to more opsonic activity and has been predicted to be a suitable candidate vaccine for high-risk patients. Findings have revealed that the purification of specific anti-PIA IgG2a is also a possible approach to inhibiting medical device-associated infections caused by S. epidermidis and S. aureus.¹¹

Accordingly, based on the macromolecule’s role in biofilm formation, choosing common antigens as a putative vaccine candidate could be promising to prevent biofilm-related infections. As mentioned before, PIA as a common polysaccharide in accumulation phases must be considered as one part of a multivalent vaccine, as the mixture or conjugation of a protein such as Aae. This construct will be effective for the prevention of biofilm formation in both S. aureus and S. epidermidis.

Conclusion

Vaccine development against staphylococcal infections is still at the stage of showing feasibility. Understanding staphylococcal virulence, including the multifactorial biofilm formation process and its regulation, the application of new technologies such as reverse vaccinology, epitope mapping, and anti-genome, and immune analyzes would result in the development of a large number of vaccine candidates. Irrefutably, more studies on staphylococcal virulence factors and immune evasion are needed to achieve a complete understanding of virulence mechanisms. Since there are numerous changeable infection-related factors and they are also expressed in the staphylococcus species, target multiple stages of biofilm formation vaccines consisting of several antigens related to different infection stages are needed. Cell-biomaterial interaction and biofilm formation should be eliminated by a suggested vaccine. Conjugated recombinant vaccines consisting of MSCRAMM and proteinaceous intercellular adhesion would be more effective in comparison with univalent vaccines and may also cause a broad cross-protection against different infectious staphylococci. Thus, there is a need for the identification of a suitable epitope combination for vaccine development with high protection to combat staphylococcal infections.

Declarations

Ethics approval and consent to participate: This study was approved by the Pasteur Institute of Iran Ethics Committee. All researches were performed on the enlarged ethical statement B9008 meeting number in Pasteur Institute of Iran. In this study, all ethical issues, including consent to participate forms from participants’ parents, were collected in this research.

Availability of data and material

All results of this study have been classified and maintained by the dissertation in the Pasteur Institute of Iran. We have indeed provided all raw data on which our study is based.

Disclosure of potential conflicts of interest

The authors declare no competing interests. All authors have read and approved the manuscript.

Acknowledgments

The authors wish to acknowledge the Zanjan University of Medical Sciences for providing funding for this study. The authors are grateful for the support of their colleagues in the Microbiology Department of the Pasteur Institute of Iran. Special thanks go to Mrs. Fatemeh Mohammadi for her continued cooperation as well.