Recent advances in Staphylococcus aureus infection: focus on vaccine development

Abstract

Staphylococcus aureus normally colonizes the nasal cavity and pharynx. After breaching the normal habitat, the organism is able to cause a number of infections at any site of the body. The development of antibiotic resistance has created a global challenge for treating infections. Therefore, protection by vaccines may provide valuable measures. Currently, several vaccine candidates have been prepared which are either in preclinical phase or in early clinical phase, whereas several candidates have failed to show a protective efficacy in human subjects. Approaches have also been made in the development of monoclonal or polyclonal antibodies for passive immunization to protect from S. aureus infections. Therefore, in this review we have summarized the findings of recently published scientific literature to make a concise report.

Introduction

A scenario of Staphylococcus aureus infection

Staphylococcus aureus is a common human pathogen which can colonize the skin, nose, and pharynx with anterior nares as the main reservoir.^1,2 S. aureus is one of the major disease-causing organisms due to its unique ability to escape the innate immune response such as phagocytic, complement or antimicrobial peptide (AMP)-mediated killing, which assists survival in blood and other tissue during persistent infections.³ S. aureus has been found to be associated with a high rate of health care-associated infections (HAIs) in hospitalized and immuno-compromised patients as well as community-acquired infections (CAIs).⁴ A report found the nasal colonization of S. aureus in 37.8% of adults which rose up to 54.7% when throat samplings were added for detection.⁵ In fact, the challenges of HAIs and CAIs have increased in the last two decades. This organism has acquired an ability to cause a wide range of infections, from minor infections such as skin and eye infections to major infections such as bloodstream infections (BSIs) and pneumonia.^6–8 Multi-drug-resistant S. aureus has been found to be one of the major organisms causing BSIs which are associated with high morbidity and mortality worldwide.⁹ Among BSIs, neonatal septicemia has been reported to be most commonly caused by this organism.¹⁰ Epidemiological studies found that BSIs-causing pathogen differs significantly between developed and developing countries.¹¹ A recent Europen report from a Finnish Hospital Infection Program which was conducted during 1999–2001 and 2005–2010, found that S. aureus ranked among the top three organisms causing BSIs.¹² Moreover, in another nationwide observational study conducted recently in Switzerland on all intravascular catheter (IVC) tip culture cases, S. aureus was reported as one of the most prevalent organisms causing subsequent BSIs in non-intensive care (non-ICU) and ICU patients. The findings also highlighted that particular attention should be paid if Candida albicans, S. aureus, Serratia marcescens, and Pseudomonas aeruginosa are isolated from IVC tips, as these organisms are associated with a higher frequency of subsequent BSIs than other pathogens.¹³ It has also been found that S. aureus was the leading organism causing native and prosthetic valve infection in high-income countries.¹⁴ Besides, S. aureus has also been isolated from lower respiratory tract infections such as pneumonia. Several clinical studies have highlighted its role as the predominant organism causing ventilator-associated pneumonia (VAP),^15–17 which is the single most common HAI in ICUs around the world.^18,19 A surveillance study conducted in European Union (EU) and European-Economic Area countries on health care-associated pneumonia (HAP) reported that 12% of cases were caused by S. aureus, which was the second most prevalent bacteria causing HAP, with 47% isolates resistant to methicillin.²⁰ Despite causing infections in seriously ill patients, S. aureus has also been reported as the most predominant bacterial causative agent of community-acquired pneumonia.²¹ Cystic fibrosis, a predominantly P. aeruginosa-associated disease, has also been found to be caused by S. aureus.²²

S. aureus and antimicrobial resistance

The emergence of infections caused by drug-resistant bacteria is a serious and growing global health concern. Therefore, significant efforts are being made in the development of new antimicrobial compounds with improved efficacy.^23,24 However, despite these efforts, an increasing number of multi-drug-resistant bacteria including methicillin-resistant S. aureus (MRSA), extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, and carbapenem-resistant Gram-negative bacteria are being reported continuously.^25–27 Once, beta-lactams, aminoglycosides, fluoroquinolones, macrolides, and trimethoprim-sulfamethoxazole were considered effective antibiotics to treat infections caused by S. aureus. However, its abuse and misuse have caused resistance and up to 85% of isolates have been reported to be non-susceptible to most of these antibiotics in current clinical use.^28–30

In recent years, antimicrobial resistance has become a major public health issue and MRSA strains which have developed resistance to all beta-lactam antibiotics including penicillins, cephalosporins (except ceftaroline and ceftobiprole), and carbapenems have been reported to represent around 25% and even in some regions greater than 50%. The Centers for Disease Control and Prevention has reported 80,000 severe MRSA infections in the United States alone in 2011, with a rate of 11,000 deaths every year.^31,32 More than half of hospital-acquired infections are caused by S. aureus in most Asian countries.^33,34 Similarly, in 2012, MRSA was estimated to have caused infections in over 75,000 patients leading to the death of more than 9,600 in the United States.³⁵ In the EU, the proportion of fatal cases is about 50,000 caused by multi-drug-resistant staphylococci out of approximately 3 million nosocomial infection cases, as reported by the European Centre for Disease Prevention and Control.³⁶ A Chinese surveillance study reported S. aureus as one of the major pathogens causing BSIs, with more than half of the strains isolated being resistant to penicillin, erythromycin, cefazolin, and cefuroxime, whereas proportions of MRSA ranged from 30%–40%.³⁷ In another study, conducted in 26 public hospitals in Hong Kong between January 2010 and December 2012, an increasing rate of MRSA was reported.³⁸ In a recent meta-analysis report from Asia Pacific regions, the proportion of MRSA among all tested samples was reported to be up to 39% and the proportion of MRSA among all S. aureus isolates was reported to be up to 89%.³⁹ Multi-drug-resistant S. aureus, including MRSA, can easily spread from the hospital setting to the community and within the community and poses additional problems for infection control strategies.⁴⁰ However, infection control programs have been implemented recently in several countries. In the United States, Europe, and many other countries, multiple infection control “bundles“ such as allotting single rooms for MRSA-colonized or infected patients, targeted admission screening for high-risk patients and health care workers at high risk for infection with multi-drug-resistant pathogens, molecular typing of all MRSA strains, and decolonization of MRSA carriers, have been initiated and tested to control the spread. As a result of these strategies, a decreasing rate of MRSA has been reported. However, the pattern of drug resistance still remains a great challenge. Empirical treatment of presumptive S. aureus diseases with an alternative to the anti-staphylococcal beta-lactams such as clindamycin and trimethoprim-sulfamethoxazole, became widespread during the 1990s when community-associated MRSA was on the rise until 2000s.^41,42 However, due to the overuse of these antibiotics, an increasing resistance continued to be reported and currently the resistance to these antibiotics pose a great threat to the treatment of infections.^43,44 However, in a recent observational study on pediatric clinical cultures performed between 2005 and 2017 in the United States, a declining trend of MRSA from 41% to 27% over the study period, yet an increasing trend of clindamycin (from 21%–38% in MRSA and 5%–40% in MSSA) and trimethoprim-sulfamethoxazole (from 2%–13% in MRSA and relatively stable in MSSA) resistance were reported.⁴⁵ Moreover, other studies have reported an increased incidence of MRSA as well as antibiotic resistance.^46–49 Multiple factors have been implicated in the development of antibiotic resistance, such as over- and misuse of antibiotics mostly in developing countries; however, biofilm-mediated drug resistance in bacteria is another major mechanism and it has been predicted that if the current treatment practice continues unchanged, the infections caused by antibiotic-resistant bacteria would be a major cause of death in 2050 where the expected number of deaths will be around 10 million every year.⁵⁰

To cope with these multi-drug resistance problems, several anti-staphylococcal drugs such as vancomycin, teicoplanin, linezolid, tedizolid, daptomycin, tigecycline, ceftaroline, ceftobiprole, oritavancin, and dalbavancin have been approved for treating the life-threatening infections caused by multi-drug-resistant S. aureus. Moreover, currently, in some countries vancomycin and teicoplanin are the most commonly used drugs to treat MRSA infections.⁵¹ However, increased MICs and reduced susceptibility to these antibiotics, poor tissue penetration, and adverse reactions due to the use of these antibiotics, have been reported to cause a limitation of its use in clinical practice.^{43,44,52–58} Because of the emerging problem of resistance, the World Health Organization (WHO) has listed MRSA and recently emerged vancomycin-intermediate and resistant S. aureus (VRSA) as “high-priority” deadly bacterial pathogens.⁵⁹ To overcome the challenging situations in the management of multi-drug-resistant S. aureus infections, alternative therapeutic strategies are of utmost importance.

Recent advances in therapeutic strategies

The increasing resistance to conventional antibiotics is the most common health issue worldwide. To overcome this problem, many natural antimicrobial compounds have been attracting many researchers’ attention in the development of novel therapies for infections caused by the multi-drug-resistant organisms. Several such compounds with antimicrobial properties have been reported recently in many studies.

Peptides (amino acids) and their drug-conjugated derivatives

AMPs are small peptides of less than 50 amino acids with a net positive charge, possessing broad-spectrum antibacterial activity, and have attracted considerable attention.⁶⁰ These AMPs exert antimicrobial activity by pore formation in the cell membrane and disrupting the membrane integrity. Although they do not need a specific ligand to bind, they exhibit capability to inhibit the activity of certain enzymes and prevent the protein and nucleic acid synthesis in bacteria.^61,62 The antimicrobial activity of AMPs such as dicentracin-like peptide and moronecidin, against Gram-negative bacteria (such as Escherichia coli, Acinetobacter baumannii, P. aeruginosa), Gram-positive bacteria (such as S. aureus, Staphylococcus epidermidis), and Candida spp. (such as C. glabrata, C. tropicalis, C. albicans) was evaluated and high activity against S. aureus, S. epidermidis, and E. coli and a lower activity were found against other Gram-negative bacteria such as P. aeruginosa and A. baumannii clinical isolates. Moronecidin was found to exert more potency than dicentracin-like peptide against S. aureus including MRSA.⁶³ Another such peptide, Hecate conjugated with vancomycin (Van/Hec) was tested in vancomycin-resistant and susceptible strains of S. aureus, and the microscopic findings revealed the disruption of bacterial cell integrity leading to the killing of all tested strains including wild-type, MRSA, and VRSA which was not observed when vancomycin or Hecate was used alone.^64,65 Human cathelicidin (LL-37) and thrombocidin-1 (TC-1) have been found to synergize the activity of amoxicillin/clavulanic acid and teicoplanin against S. aureus.^66,67 Xanthones are a class of heterocyclic compounds possessing the oxygen moiety which is widely distributed in nature, including two major plant families, Guttiferae and Gentianaceae, and also in fungi and lichens.^68–70 The pharmacological activities of naturally occurring and synthetic xanthone derivatives have been described in several recent pieces of literature.^71–73 Antibacterial activities of synthesized xanthone conjugated amino acids were recently evaluated against Gram-positive organisms (S. aureus and Bacillus subtilis) and Gram-negative organisms (E. coli and Klebsiella pneumoniea) as well as against several fungi (Aspergillus niger, C. albicans, and Fusarium oxysporum).⁷⁴

Anti-staphylococcal phenolic compounds

Anti-staphylococcal phenolic compounds such as polyphenols (flavonols and phenolic acids) have been found to exert antimicrobial activity against several bacterial pathogens by inhibiting the activity of bacterial virulence factors, possessing a capability to interact with cytoplasmic membrane, suppressing the formation of biofilms, and can enhance the antimicrobial activity of antibiotics. The antibacterial activity of polyphenolic compounds against staphylococcal strains has been evaluated and found to exert a promising activity either alone or in combination with antibiotics.⁷⁵

Anti-biofilm compounds

Biofilm is a thick extracellular polysaccharide material produced by many organisms and its synthesis prevents many antibiotics from penetrating the bacterial cell and renders them resistant. It has been elucidated that more than 25% of infections are associated with the biofilm producing ability of the bacteria. Biofilm producing S. aureus develops the ability to grow within the biofilm and survive phagocytosis and antibiotic action.⁷⁶ Nano-scale materials such as silver nanoparticles have emerged as novel antimicrobial agents in combination with existing antibiotics and have shown the most effective antimicrobial activity in vitro.^77–79 Several recent studies have tested the efficacy of these silver nanoparticles in combination with antibiotics and they have been found to be a novel therapeutic strategy to treat infections caused by multi-drug-resistant organisms.^80–82 A synergistic effect increasing the antibiotic activity of penicillin combined with silver nanoparticles has been found against S. aureus including MRSA.^83–85 In a recent study, Manukumar et al described the efficacy of thymol-loaded chitosan silver nanoparticles (T-C@AgNPs) against biofilm producing MRSA using disc diffusion method. Using different concentrations of T-C@AgNPs from 10, 25, 50, 100, 200, and 250 μg/mL and comparing the concentration that produced 10.08±0.06 mm of zone of inhibition (ZOI) with the standard antibiotic ciprofloxacin (10 μg) that had 10.95±0.08 mm ZOI, a dose-dependent biocidal and anti-biofilm activity was found.⁸⁶ Another recent study also described the antibacterial activity of benzodioxane midst piperazine decorated chitosan silver nanoparticles (BP*C@AgNPs). In the study, using well diffusion test by loading different concentrations of synthetic BP*C@AgNPs against biofilm producing MRSA, depicted the dose-dependent membrane damage leading to bacterial killing. The study also depicted the role of BP*C@AgNPs in the inhibition of biofilm synthesis leading to the decreased adherence of bacterial cells to each other.⁸⁷

Recent developments in active immunization

Because antibiotic resistance has been found as the major issue in the treatment of infections caused by multi-drug-resistant bacteria, vaccination could provide protection against the infections caused by antibiotic resistance as well as susceptible organisms. Primarily, the vaccine development focuses on the driving of antibody response which is able to block the toxins involved in the killing of immune cells as well as helping in the opsonization of bacterial cells. Therefore, several attempts have been made in the development of safe and effective vaccines (Table 1). However, some vaccine candidates failed to show significant protection and this may be because of overreliance on the antibody-mediated protective response.⁸⁸

Table 1.

Candidate vaccines for active immunization

Target antigen	Study phase	Outcome	References
CP5 and CP8	Phase III	No significant protection against bacteremia in patients receiving hemodialysis when compared with placebo	87
CP5-CRM197, CP8-CRM197, and ClfA (SA3Ag)	Phase I	Showed significant neutralizing antibody response	91,93
CP5-CRM197, CP8-CRM197, MntC, and ClfA (SA4Ag)	Phase I	Showed robust immune response, safe, and well-tolerated and phase 2b is ongoing	91,97
Alpha-toxin and Panton-Valentine Leukocidin	Phase I	Showed a good toxin neutralizing sero-positive response	103
EsxA and EsxB	Preclinical	Showed protection with improving survival of murine model	106
Surface protein A (SpA)	Preclinical	Showed protection in mouse model	110,111
D-alanine auxotrophic Staphylococcus aureus	Preclinical	Showed protection from the formation of abscesses and improved survival in immunized mice	113
AdsA	Preclinical	Showed protection in the immunized mouse model	114
Coa (Hc-CoaR6)	Preclinical	Showed a strong T-cell response and protection in mice against lethal dose of S. aureus	118
Staphylococcal enterotoxin B	Preclinical	Showed an efficient protection in BALB/c mice	122

Capsular polysaccharides (CPs) as vaccine candidates

Bacterial capsule is an extra-cellular material, which can be microscopically visualized using special techniques, covering the bacterial cells. Several bacteria have been found to possess the capsules such as E. coli, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae as well as S. aureus. Bacterial capsules are composed of long polysaccharide chains known as CPs. Capsules are the bacterial structure first recognized by the immune system, therefore, encapsulated bacteria have developed an immune evasion property which is exploited in the development of vaccines.⁸⁹ The CPs have been targeted as an effective vaccine candidate for the protection from many bacterial infections such as S. pneumoniae, H. influenzae, and N. meningitidis.⁹⁰ As many as eight different serotypes of capsules such as CP 1–8 (CP1 to CP8) have been found in S. aureus; however, the majority of the isolates causing diseases possess CP5 and CP8 which are the major effective vaccine targets.^91–94 The expression of these CPs can be dynamic during infection, therefore, additional protein antigens are required for adequate protection.⁹⁵ In 2002, the first S. aureus vaccine StaphVAX, developed by Nabi Biopharmaceuticals, consisting of CP5 and CP8 conjugated to recombinant P. aeruginosa exoprotein A, was used as a vaccine candidate in patients receiving hemodialysis in its initial phase III clinical trials. However, the study failed to show a significant protective effect compared with placebo in a follow-up period of 3–54 weeks post-vaccination. It was suggested that it may be due to many reasons such as the population targeted, production of the sub-optimal conjugate, or varying conjugate manufacture between trials; however, partial protection with a significant reduction in the S. aureus bacteremia number in the follow-up period of 3–40 weeks post-vaccination was found in a subsequent trial.⁹⁶ Based on this partial protection, Fattom et al conducted a similar study using StaphVAX in the same patient population receiving hemodialysis. The assessment of the protective efficacy in vaccine recipients vs placebo up to 35 weeks after receiving a single dose or up to 60 weeks after receiving one or two vaccine doses suggested no protection against S. aureus bacteremia.⁹⁷ The failure of this vaccine containing two single-antigens suggested that a multi-antigen vaccine containing several antigens might be successful. As a result, the first generation of multi-antigen vaccine containing three-antigens (S. aureus three-antigen [SA3Ag]) such as CP5, CP8 conjugated to the CRM₁₉₇ and ClfA was designed.⁹⁸

Recently, two types of vaccines namely, SA3Ag vaccine possessing CP5, CP8, and ClfA and S. aureus four-antigen (SA4Ag) vaccine possessing CP5, CP8, ClfA, and recombinant P305A developed from a lipoprotein manganese transporter C (MntC) have been successfully developed by the researchers, which have exhibited superior immunogenicity compared to previous vaccines.^96,98–104 The studies have revealed that the previous vaccines generated anti-staphylococcal antibodies capable of binding with S. aureus leading to the uptake by phagocytic cells while the multi-antigen vaccines (SA3Ag and SA4Ag) are capable of inducing high level of anti-staphylococcal antibodies that lead to the killing of S. aureus by increasing the phagocytosis of bacteria and were concluded to be safe with no significant increase in systemic adverse effects or local adverse effects in healthy adults.^96,104,105 The partial success of the first phase trial encouraged the researchers to design a novel multi-antigen vaccine (SA4Ag) containing CP5 and CP8 conjugated with CRM₁₉₇ (CP5-CRM₁₉₇ and CP8-CRM₁₉₇) together with MntC and ClfA antigens.¹⁰⁶ A multicenter phase I/II trial study conducted in the United States evaluated the immunogenicity, safety, and tolerability of SA4Ag vaccine in healthy adult volunteers of 18–64 years of age when injected as a single intramuscular dose.¹⁰⁶ The findings of a recent animal model study demonstrated that this vaccine could elicit cytokine production by naive peripheral blood mononuclear cells leading to the induction of anti-staphylococcal antibodies and memory B-cell response.¹⁰⁷ A phase II/III study to evaluate the efficacy of the SA4Ag vaccine for the prevention of invasive S. aureus disease in patients between 18–85 years of age who have had elective spinal surgery is under way.^108,109 This vaccine was shown to be safe and well tolerated in the early stage of clinical trials inducing high levels of bacterial killing antibodies.¹⁰¹

Alpha-toxin and Panton-Valentine Leukocidin (PVL)

S. aureus alpha-toxin is a highly conserved toxin that disrupts the tissue and endothelial barrier and enhances bacterial penetration.¹¹⁰ PVL is a pore-forming protein exhibiting a cytotoxic nature which destroys leukocytes and causes tissue necrosis.¹¹¹ A reduced risk of sepsis in adult patients with invasive S. aureus infection has been found with a higher level of IgG antibody against alpha-toxin.¹¹² A recent phase I study was conducted by Landrum et al in healthy adults with an age range of 18–55 years old to evaluate the safety and immunogenicity of recombinant alpha-toxoid (rAT) and recombinant PVL (rLuks-PV) either monovalent or bivalent. The subjects were injected with monovalent form and followed-up on days 7, 14, 28, and 84 and those injected with bivalent form received a second dose on day 84 and were followed-up on days 98 and 112. A sero-positivity for toxin neutralizing antibody was found in a high proportion of subjects against rAT and rLuks-PV. As a result, both the rAT and rLuks-PV vaccine formulations were found to possess a favorable safety profile, were well-tolerated, and had high immunogenicity with neutralizing antibody when administered either alone or in combination in healthy adults.¹¹³

Secretory proteins EsxA and EsxB as a vaccine model

The bacterial secretion system helps the bacteria to transport the virulence factors in the host cells. The type VII secretion system is the best-characterized system in S. aureus. Early secreted antigenic target-6 kDa (ESAT-6) secretion system (ESS) is a specialized secretion system similar to the Esx-1 secretion system described in Mycobacterium tuberculosis, also identified in S. aureus. ESS in S. aureus consists of 12 proteins including highly conserved EsxA and EsxB closely related with ESAT-6 and CFP-10 respectively of M. tuberculosis.¹¹⁴ In 2005, these proteins were identified and verified to be secreted and implicated in the development and persistence of staphylococcal abscess formation in the murine model.¹¹⁵ In a recent study, the attenuated Salmonella typhimurium SPI-1 T3SS was utilized to translocate the secretory proteins EsxA and EsxB fused with N-terminal domain of SipA (1–169 amino acids) into the host cells of BALB/c mice. The mice were immunized orally with three doses of S. typhimurium strains N19, N20, and vector control strain N106 on Day 1, Day 8, and Day 22 and 5×10¹⁰ CFU of freshly cultured and PBS-washed bacterial cells, and the vaccinated mice were intravenously challenged with 5×10⁷ CFU of S. aureus USA300 strain or Newman strains after 10 days of secondary booster dose. The immunogenicity study showed that the mice immunized with N19 strain generated a high level of EsxA-specific IgG1 and IgG2a antibody, indicating Th1/Th2-type immune response and a Th2-biased response against the EsxB antigen protecting the N20 vaccinated mice while improving the survival rate in N19 vaccinated mice.¹¹⁶

Surface protein A as a vaccine candidate

SpA is an abundant surface protein and a virulence factor which is released during normal cell division. SpA is able to interact with the Fc portion of IgG and suppresses the adaptive immune response by limiting the antibody production by B-cells whereas it enhances the immune response if it binds with B-cell receptor allowing the activation of B-cells.^117–119 Therefore, suppression of the IgG binding effects of SpA could be able to mount the immune response. In a study, Kim et al, when immunizing a mouse model with non-toxigenic protein A withg substitutions Gly₉Lys, Gln₁₀Lys, Asp₃₆Als, and Asp₃₇Ala in the D-domain of the Ig binding region (SpA-D_KKAA), found rising antibody titers and protective efficacy against MRSA and MSSA infection.¹²⁰ Another recent study depicted the efficacy of the combined vaccine containing recombinant S. aureus surface protein A (SasA) and the internal heavy chain translocation domain C-fragment of tetanus neurotoxin (TenT-Hc). The combined vaccine conferred complete protection to the mouse against lethal intra-peritoneal challenges with 3×10⁹ CFU of MRSA USA300 strains.¹²¹

D-alanine auxotrophic strain of S. aureus as a vaccine model

D-alanine is an essential component of the bacterial cell wall polysaccharide. Lacking a gene involved in the D-alanine biosynthesis makes the strains attenuated.¹²² In a recent study an attempt was made to assess the impact of D-alanine auxotrophy on protection from the parental strains. The S. aureus 132 strain lacking the gene involved in D-alanine biosynthesis was allowed to grow on media supplemented with exogenous D-alanine. The infection with D-alanine auxotrophic strain elicited a protective immune response and generated cross-reactive antibodies which provided protection following administration of different doses of its parental strain in immunized BALB/c mice. The D-alanine auxotroph vaccine exhibited a reduction in the measured bacterial load in vital organs such as kidney, spleen, heart, liver, and lung. The vaccine protected against the formation of abscesses and survival of the immunized mice was enhanced following infection with the parental strain.¹²³

AdsA

AdsA is a cell wall anchored enzyme which plays an important role in immune evasion.³ AdsA deficient strains have been found to be labile after engulfment by polymorphonuclear leukocytes, while wild-type strains remain stable. In a study, active immunization of 6-week-old female BALB/c mice with 25 μg of rAdsA protein by intramuscular injection and subsequent infection with S. aureus Newman or USA300 strain was performed. As a result, a high level of anti-AdsA IgG and a reduced abscess size with little or no dermonecrosis was seen in the mice vaccinated with rAdsA when compared with the control mice. The anti-AdsA antibody was found to promote the killing of S. aureus by immune cells and reduced the intracellular as well as the extracellular number of S. aureus in macrophages of mice.¹²⁴ Therefore, AdsA is an important antigen candidate for vaccine or therapeutic approach against the S. aureus infection.

Coa as a vaccine model

S. aureus Coa is a protein with enzymatic action which activates prothrombin to convert fibrinogen into fibrin threads via its N-terminal D1-D2 domain. The fibrin threads generate a protective shield on the surface of S. aureus through its C-terminal R domain. The monoclonal antibody against the R domain was found to promote the phagocytosis of S. aureus by immune cells, suggesting its role in the enhancement of bacterial killing and protection of the host.^125–127 Regarding these findings, a recent study evaluated the protective efficacy of the R domain of Coa (CoaR6) fused with the carrier protein (Hc), a 66 C-terminal fragment of the heavy chain of tetanus neurotoxin (TT) in a peritonitis mouse model challenged intra-peritoneally with 2×10⁹ CFU of MRSA252 or 1×10⁹ CFU of USA300 4 weeks after the third immunization with Hc-CoaR6 combined with alum and CpG. The TT was used to increase the immunogenicity of the so-called Hc-CoaR6 vaccine. The results suggested that the Hc-CoaR6 vaccine could improve immunogenicity when compared with the immunogenicity elicited by the CoaR6 alone. The findings also suggested that a strong T-cell response and protection of mice against the lethal dose of S. aureus could be elicited by the Hc-CoaR6 vaccine model.¹²⁸

Staphylococcal enterotoxin B (SEB)

SEB is a stable toxin which exerts powerful effects in humans at a very low dose. When inhaled, SEB can induce several symptoms ranging from headache, myalgia, increased heartbeat, coughing, enteric dysfunction (nausea, vomiting, and diarrhea) to life-threatening toxic shock syndrome.^129,130 A previous study used the formalin treated SEB toxoid vaccine, and although it demonstrated some degree of protection of the animal models, it was not approved for use in humans.¹³¹ Owing to this protective efficacy, a recent study also evaluated the protection in a mouse model immunized with mutant SEB vaccine candidate produced by site-specific mutagenesis. A substantial level of toxin neutralizing antibody response was elicited, which provided efficient protection to the BALB/c mice against a lethal dose of SEB challenge.¹³²

Recent developments in passive immunization

Anti-staphylococcal monoclonal antibodies as prophylactic agents for patients with a high risk of developing severe S. aureus infections are considered a novel anti-staphylococcal approach. A potential advantage of increasing the effectiveness of the conventional antibiotic treatment has been suggested of the anti-staphylococcal antibody. As alpha-toxin is expressed by the majority of S. aureus strains, the monoclonal antibody against the alpha-toxin may be effective in protecting against infections caused by S. aureus, including MRSA. Several studies have claimed the protective role of anti-alpha-toxin antibody from the S. aureus infections.^133–135 A phase II trial of the monoclonal antibody has evaluated the efficacy and safety of a single dose of the human anti-staphylococcal monoclonal antibody against the S. aureus α-toxin under the project entitled “human monoclonal antibody against S. aureus α-toxin in mechanically ventilated adult subjects”. However, the results of this study and whether this approach can have a positive impact on treatment of staphylococcal diseases remain to be evaluated.¹³⁶ In another recent study, an attempt was made to evaluate the efficacy of anti-staphylococcal antibodies by injecting 200 μL of rAdsA immunized rabbit antisera into the tail vein of 8-week old BALB/c mice 24 hours prior to challenge with S. aureus. As a result, passive immunization with the AdsA-specific antisera reduced the S. aureus Newman or USA300 infection in the mouse model. The AdsA-specific antiserum was found to promote the killing of S. aureus by immune cells while decreasing the infection severity in a different mouse model.¹²⁰ In a study conducted by Varshney et al, the natural antibody against Staphylococcus protein A (514G3) was found to promote the opsonophagocytic killing of S. aureus by human blood cells, and protected the bacteremia mouse model from the lethal intravenous challenge of 3×10⁷ CFU of MRSA.¹³⁷ The protective role of passive immunotherapy with polyclonal antibodies against recombinant autolysin (r-autolysin) was recently evaluated by Kalali et al. As a result, the addition of anti-r-autolysin was found to promote the phagocytosis of S. aureus and the number of viable bacterial cells was decreased over 66.5% after 90 minutes compared with the control group; and in the mouse model of sepsis, the addition of anti-r-autolysin IgG fraction significantly enhanced the survival of the animals.¹³⁸ The role of hemolysin-alpha (Hla)-specific and Hla-leukocidin cross-neutralizing monoclonal antibodies was evaluated for their efficacy in protection from pneumonia. In the study, 6–8 week old female BALB/cJRj mice were intra-nasally challenged with a lethal dose of 8×10⁸ CFU CA-MRSA clones USA300-0114 at 24 hours post-immunization with the monoclonal antibodies and survival was monitored daily for 10 days after post-challenge. The result exhibited a protective efficacy in the induced murine pneumonia model.¹³⁹ A similar study conducted by Stulik et al, also depicted the prophylactic efficacy of anti-Hla monoclonal antibody in a lethal rabbit pneumonia model challenged with MRSA and MSSA.¹⁴⁰ MRSA exhibits methicillin resistance which is conferred by the acquisition of a mobile genetic element, mecA, which encodes an altered protein involved in the cell wall synthesis (PBP2a). Active immunization of mice with recombinant PBP2a (rPBP2a) significantly induces specific antibodies.¹⁴¹ It was assumed that the antibodies against rPBP2a might exhibit a protective activity if used for passive immunization. Naghshbandi et al conducted a study to elucidate the efficacy of passive immunization with anti-rPBP2a IgG fraction in MRSA challenged mice. In the study, the mice were passively immunized with 500 μL of IgG fraction 2 hours before and 24 hours after infection with a lethal dose of 5×10⁵ CFU of MRSA, and were monitored for survival until 30 days after inoculation. As a result, passive immunization was found to play a considerable role in the protection which enhanced the survival of the experimental mice.¹⁴² However, despite several vaccine candidate developments, there is a possibility of immune evasion. Recently it was described that the presence of the bacteriophage DNA encoding a TarP protein in MRSA can modify the bacterial cell wall polymers, inhibiting the recognition by the host adaptive immune response, which could make the bacteria resistant to being recognized by the antibodies. Thus, the evasion of bacteria of the immune system might be able to cause severe infections.¹⁴³

Conclusion

The wide-spread infections caused by multi-drug-resistant S. aureus have demanded priority in the development of an effective therapeutic approach. Although some vaccine candidates have shown protective efficacy in preclinical phase or early clinical phase studies, so far, no vaccine has been approved for human use. In addition to active immunization, the use of novel antibody-based passive immunization strategies might offer hope, as they have shown promising efficacy in the preclinical phase of evaluation.

Author contributions

All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.