Immunization Against Staphylococcus aureus Infections

Abstract

Background:

Infections caused by Staphylococcus aureus continue to plague surgical patients, whether as surgical site infections or other nosocomial infections that complicate surgical care. The only meaningful methods available to decrease the risk of developing such infections are topical skin antisepsis (pre-operative skin preparation) and peri-operative antibiotic prophylaxis, neither of which offer a panacea. Alternatives to the latter are sought so as to minimize antibiotic selection pressure as a factor in the increasing problem of antimicrobial drug resistance. This review considers the possibility that immunization against S. aureus may offer a viable alternative for prophylaxis.

Methods:

Review and synthesis of pertinent English-language medical literature.

Results:

Vaccination against viral pathogens has been in successful clinical use for more than two centuries and was instrumental in the eradication of smallpox and the near-elimination of diseases such as poliomyelitis. Vaccinations against a limited number of bacterial pathogens (e.g., Bordetella pertussis, Clostridium tetanii, Corynebacterium diphtheriae, Haemophilus influenzae type b, Neisseria meningiditis, Streptococcus pneumoniae) have also been introduced with success, whereas others against bacteria are in development (C. difficile, Pseudomonas aeruginosa, S. aureus). Vaccination against S. aureus infection is in current veterinary use (e.g., to prevent mastitis among dairy cattle) but has not been successful to date in human beings despite multiple attempts, although development continues.

Conclusions:

Because of its complex microbiology, including multiple virulence factors and the ability to evade host immune surveillance, S. aureus presents numerous antigenic targets for vaccine development. Failure of two prior single-antigen vaccines in clinical trials has led to the consensus that future vaccine candidates must be directed against multiple antigens. Two distinct four-antigen vaccines are in clinical trials, but efficacy is yet to be determined.

Vaccination is the administration of antigenic material (the vaccine) to stimulate an individual's immune system to develop adaptive immunity to a pathogen, so as to prevent or ameliorate infectious disease(s). Historically and to the present day, vaccination has been directed clinically against viral pathogens with great success, but more recently vaccines against bacterial pathogens have become successful (e.g., Bordetella pertussis, Clostridium tetanii, Corynebacterium diphtheriae, Haemophilus influenzae type b, Neisseria meningiditis, Streptococcus pneumoniae), many of which are utilized regularly in acute care surgery practice. Interest in immunization (used herein synonymously with vaccination) against bacterial pathogens would be stimulated if a vaccine were to be developed against hospital-acquired pathogens that, as established infections, carry substantial morbidity and some risk of death. By contrast, inoculation refers to the induction of active adaptive immunity, which uses un-attenuated live pathogens, whereas passive immunity is the hoped-for result of administration of immunoglobulin or, experimentally, a monoclonal antibody or antibiotic–antibody conjugates.

Vaccination is the most effective method of preventing viral diseases, and in some circumstances the prevention may extend to diseases consequent to chronic infection (e.g., hepatitis B vaccination and reduced incidence of hepatocellular carcinoma [1,2]; human papilloma virus vaccination uptake is not yet sufficiently prevalent to assess the effect on cervical or oropharyngeal carcinoma incidence [3,4]. When a sufficiently large percentage of a population has been vaccinated, herd immunity results. Widespread immunity as a result of vaccination is largely responsible for the worldwide eradication of smallpox and the marked reduction of diseases such as poliomyelitis, measles, and tetanus (arguably the most successful example of vaccination against a bacterial pathogen-C. tetanii) from much of the world.

Historic Perspective

Smallpox was likely the first disease subjected to prevention by inoculation [5] and was the first disease for which a vaccine was produced. The smallpox vaccine was “invented” in 1796 by the British physician Edward Jenner and although at least six people had used the same principles years earlier, he was the first to publish evidence that it was effective and to provide advice on its production [6]. The terms “vaccine” and “vaccination” are derived from Variolae vaccinae (smallpox of the cow), the term devised by Jenner to denote cowpox. The immunization was called vaccination because it was derived from a virus affecting cows (Latin: vacca ‘cow’) [5,6]. Smallpox was a contagious and deadly disease, causing the deaths of 20%–60% of infected adults and more than 80% of infected children [7]. When smallpox was finally eradicated in 1979, it had already killed an estimated 300–500 million people [8] in the twentieth century.

In common parlance, vaccination and immunization have a similar meaning. This distinguishes them from inoculation, which uses un-attenuated live pathogens, although in common usage any of the terms can refer to an immunization. Vaccination efforts have been met with some controversy on scientific, ethical, political, medical safety, and religious grounds, all largely unfounded. Early success of vaccination brought widespread acceptance, and mass vaccination campaigns have reduced greatly the incidence of many diseases in numerous geographic regions.

Principles of Vaccination

Until relatively recently, vaccine development has been empirical, with limited understanding of how the immune system is activated to elicit adaptive immunity, but, advances in immunology, microbiology, genetics, and molecular biology have led to burgeoning understanding of the interactions of microorganisms with the human immune system [9]. Modern vaccine development builds on precise knowledge of the microbiology of pathogens, their interaction with the immune system, and their capacity to counteract and evade innate and adaptive immune mechanisms, using technologies to produce highly purified antigens that provide a lower reactogenicity (the propensity of a vaccine to produce common, expected adverse reactions (e.g., fever, soreness at injection site, bruising, erythema, induration, and edema). However, it is unclear whether a higher degree of reactogenicity to a vaccine correlates with more severe adverse events; the U.S. Food and Drug Administration (FDA) has been unable to make such an association.

Attempts to improve vaccine antigen purity may result in impaired vaccine immunogenicity (the ability of a particular antigen or epitope [or antigenic determinant]; the part of an antigen molecule to which an antibody attaches itself to provoke an immune response). Distinction is made between wanted (i.e., successful vaccination) and unwanted immunogenicity (i.e., drug allergy). Some such disadvantages related to highly purified or genetically engineered vaccines can be overcome by innovative technologies, such as live vector vaccines, and DNA or RNA vaccines. Moreover, recent years have witnessed the development of novel adjuvant (in immunology, a substance used to enhance antigenicity, e.g., a suspension of minerals [alum, aluminum hydroxide, or phosphate] on which antigen is adsorbed) formulations that specifically focus on the augmentation or regulation of the interplay between innate and adaptive immune systems and the function of antigen-presenting cells. Finally, vaccine design has become more tailored, which in turn enhances the potential of extending its application to complex microbial pathogens hitherto not accessible. This review provides an overview of the key considerations and processes involved in vaccine development against S. aureus.

Staphylococcus aureus as a Human Pathogen

Staphylococcus aureus is ubiquitous and a virulent human and animal pathogen that may afflict healthy individuals or those with compromised immunity, outpatients, or those hospitalized. More than two million people become infected with S. aureus each year in the United States, with more than 100,000 deaths annually [10]. A multiplicity of infections has been observed, because the pathogen can infect almost any tissue imaginable. Surgical infections of interest caused by S. aureus include community-acquired infections, especially of skin and soft tissue; surgical site infections (in which it is the most common pathogen); and post-operative nosocomial infections [11].

Identification of the patient at risk is a crucial first step in prevention. Population-based studies have identified male gender and both extremes of age as risk factors for the acquisition of staphylococci. Dialysis dependence, diabetes mellitus, cancer, human immunodeficiency virus infection, intravenous drug use, alcohol abuse, recent surgery, critical illness, and staphylococcal nasal carriage have been identified as independent risk factors [12,13]. Individuals who are persistent nasal carriers (the primary locus, although other sites are possible) of S. aureus may represent as much as 30% of healthy people [14]. Most persistent carriers (as many as 90%) harbor a single strain of methicillin-susceptible S. aureus (MSSA), although methicillin-resistant S. aureus (MRSA) nasal carriage is recognized now in up to 10% of carriers). Patients who are persistent carriers are at higher risk of acquiring S. aureus infection. In addition to decolonization of staphylococcal carriers [15,16], other tactics being investigated include phage therapy, molecules aimed at blocking regulation of virulence determinants and surface adhesins, and vaccination.

Mechanisms of virulence and immune evasion

The high prevalence and threat of S. aureus infections in surgical patients has led to vigorous efforts to develop specific preventive and therapeutic measures directed against this pathogen, by enhanced infection control practices, use of topical antiseptics, and narrow-spectrum systemic antibacterial agents [17,18]. Prevention remains elusive despite these efforts.

Staphylococcus aureus manifests a broad array of virulence factors to account for its pathogenicity, and new virulence characteristics continue to be acquired. Staphylococcus aureus is able to evade immune surveillance mechanisms as well. The genome of S. aureus consists of 75% core genes that appear in all species and 25% accessory genes that vary among strains [19]. Within the accessory component of the genome are mobile genetic elements that are transferable among S. aureus strains. Accessory components may consist of pathogenicity islands, prophages (from bacteriophages), staphylococcal cassette chromosomes (SCC), genetic islands, and plasmids [20]. Structural elements, metabolic regulators, and virulence factors of S. aureus may be common to all species (e.g., coagulases), or they may be highly specific for each strain (especially in the case of virulence factors). The exchange of mobile genetic units among strains of S. aureus and the acquisition of genetic material from other bacterial species (e.g., Enterococcus), results in fluid patterns of virulence and resistance.

Microbial virulence factors of S. aureus generally have been grouped into bacterial structural factors, secreted bacterial products and enzymes, and resistance mechanisms to antimicrobial agents [21]. Structural elements of the bacterial cell include a polysaccharide capsule that resists phagocytosis and the peptidoglycan cell wall that anchors a number of surface adhesion proteins, allowing microbial binding to the extracellular matrix of targeted host tissues [22]. The surface adhesion proteins of the cell wall are referred to collectively as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) [11,22 –24]. The MSCRAMMs are identified in many species of gram-positive organisms, of which several are unique to S. aureus.

Products secreted by S. aureus include those that enhance microbial adherence to host tissues, exotoxins that are directly toxic to host cells, and enzymes that degrade and digest the extracellular matrix. Secretable expanded repertoire adhesive molecules (SERAMs) are a group of secreted proteins that have duplicative functions of the MSCRAMMs identified above, except that they are secreted proteins, not part of the staphylococcal cell wall [25]. The SERAMs of considerable interest for S. aureus are coagulase (of which there are actually two, staphylocoagulase and von Willebrand factor binding protein), which target prothrombin and fibrinogen. The result of the action of coagulases is generation of a staphylococcal–fibrin matrix and creation of a local environment that facilitates microbial adherence to tissue and a protected environment against host defense mechanisms and antimicrobial therapy [26]. Coagulases have effects that are analogous to clumping factor of the MSCRAMMs, but they are freely secreted and unique proteins [27].

Three exotoxin families predominate in the pathogenesis of S. aureus infections and allergic responses, namely pore-forming toxins, exfoliative toxins, and superantigens. Exotoxins are those secreted products that are cytotoxic to host cells [28 –30], whereas superantigens are secreted products that provoke an exuberant inflammatory response by the host [31,32]. Individual cytotoxins have different cell targets but generally function similarly by damaging the cell membrane, creating pores, and leading to extravasation of cellular contents.

Expression of virulence

Staphylococcus aureus secretes numerous virulence factors, including the aforementioned toxins capable of membrane pore formation and cytolysis. These virulence factors include bi-component leukocidins [29], heptameric beta-barrel–forming alpha-toxin (Hla), and phenol-soluble modulins (PSMs) [33]. An array of cell wall-anchored (CWA) proteins, which number more than 25 and have substantial functional redundancy, bind to cell wall peptidoglycans [34]. Clumping factor A (ClfA) is the major fibrinogen-binding protein of S. aureus. Clumping factor A causes platelet aggregation and clumping of bacteria on plasma, and is a major factor relating to pathogen survival and dissemination in blood. By contrast, S. aureus attachment to nasal epithelial cells is facilitated by the staphylococcal surface adhesin clumping factor B (ClfB) [34]. Fibronectin-binding proteins (FnBPs) A and B enable S. aureus to adhere to and invade a host of cell types, including epithelia, fibroblasts, and osteoblasts [35]. Protein A (SpA) is a conserved, multi-functional surface protein that has a number of immunosuppressive traits, including induced apoptosis of B-lymphocytes thereby suppressing the host antibody response, and is arguably the most important mechanism of immune evasion in S. aureus [34] (see below). Iron-regulated surface proteins (Isd) A and B are part of the heme uptake system (iron is a necessary nutrient for bacteria) [34]. The Isd proteins play a role in bacterial adherence to epithelial cells and IsdA on the bacterial surface causes the bacteria to become more hydrophilic and negatively charged, facilitating cellular attachment.

Immune evasion

When innate immune mechanisms are insufficient to clear a bacterial infection, adaptive immune mechanisms are activated [33 –35]. The adaptive immune response to S. aureus requires an integrated response involving T-helper type 1 (T_H1), T_H17, and humoral antibody (IgG) responses, generating the production of interleukin (IL)-1 and IL-17A to promote abscess formation, which in turn is required for the clearance of bacteria via phagocytosis and oxidative burst. Staphylococcal infections in human beings are known to elicit only modest, transitory antibody responses [36,37], two tangible manifestations of which are the nasal carrier state (see above) and recurrent acute infections but also begging the question as to whether active immunization against staphylococcal infections will be of benefit. Staphylococci can exist as human commensal flora because several mechanisms to “evade” detection by immune surveillance mechanisms (e.g., complement deposition, neutrophil killing) have evolved [36]. Elaborated enterotoxins act as potent T-lymphocyte superantigens, which bind major histocompatibility complex class II (MHC-II) determinants on the surface of antigen-presenting cells, but the multiplicity of these many toxins, both in number and functional redundancy of staphylococcal enterotoxins and toxic shock syndrome toxins (TSSTs), has prevented development of a superantigen vaccine (see below).

Also during infection, S. aureus produces adenosine (via adenosine synthase A-AdsA) from high-energy phosphates [38], which may be the most potent immunosuppressive signaling molecule. Adenosine receptor-mediated signaling impedes phagocytosis by neutrophils (staphylococci that express wild-type AdsA are capable of surviving neutrophil phagocytosis) and may degrade adaptive host immune responses by impairing antigen presentation to macrophages and dendritic cells. The B-cell superantigens staphylococcal protein A (SpA) [39] and S. aureus binder of immunoglobulin (Sbi) [40] (both of which harbor multiple immunoglobin-binding domains) inhibit opsonophagocytic clearance of the pathogen, and may inhibit normal B-lymphocyte functioning, possibly by induction of clonal expansion leading to B-call apoptosis, or by inhibiting receptor interaction of complement factor C3. The SpA protein mediates binding of IgG in an incorrect orientation on the bacterial surface, leading to decreased neutrophil binding and consequent evasion of phagocytosis.

Staphylococcus aureus as a Target for Passive Immunization

Passive immunization against S. aureus has been unsuccessful to date [41]. No efficacy was seen from infusion of enriched human serum (IgG infusion) from unvaccinated subjects [42, cited in Janssen et al. (41)]. Neither was efficacy demonstrated from either of two trials of immune serum collected from subjects who received a capsular polysaccharide (CP) CP5/CP8/Pseudomonas exotoxin conjugate vaccine [43,44 (cited in Janssen et al. (41),45]. More recently, a phase 2 trial failed to prevent S. aureus ventilator-associated pneumonia after pre-treatment of patients with heavy airway colonization by S. aureus with an anti-staphylococcal monoclonal antibody (ASN100) [46]. ASN100 is a combination of two monoclonal antibodies (ASN-1 and ASN-2) that together neutralize six S. aureus cytotoxins. ASN-1 neutralizes α-hemolysin and four of the five leukocidins (HlgAB, HlgCB, LukED, and LukSF, the latter also known as Panton-Valentine leukocidin or PVL). ASN-2 neutralizes the fifth leukocidin, LukGH (also known as LukAB). LukGH, a potent immune evasion factor of S. aureus, is expressed by the majority of all clinical isolates and contributes substantially to S. aureus-mediated phagocyte killing.

Attempts continue to develop effective passive anti-staphylococcal immunity. Although S. aureus is an extracellular bacterium, it can evade host defenses by adapting as an intracellular organism, where it can evade the action of antibodies but also antibacterial agents and in so doing, serve as a nidus for dissemination. A novel antibody–antibiotic conjugate is being developed, designed to be activated specifically inside phagocytes that have engulfed S. aureus [47,48]. The antibody is directed against cell wall component teichoic acid (against which approximately one-third of innate anti-staphylococcal antibodies are directed) [47], whereas the antibiotic chosen was the rifamycin derivative rifalogue, which is bactericidal in the acidic milieu of the phagolysosome and active against non-replicating bacteria and antibiotic-tolerant persister cells. The antibody recognizes cell-free S. aureus, leading to opsonization and phagocytosis. Once intracellular, rifalogue is cleaved by cathepsins and exerts its effect. Preliminary work showed that the conjugate is ineffective against planktonic S. aureus and that the conjugate does not penetrate mammalian cells because of the size of the antibody.

Staphylococcus aureus as a Target for Active Immunization

Prevention of S. aureus infection by an effective vaccine would save lives and cost, be antibiotic sparing, and hinder the development of antibiotic resistance [49,50]. If an effective vaccine could be targeted for prevention when the risk of becoming infected is highest (e.g., in the short-term aftermath of surgery or some other invasive procedure), success might be achieved, because the need to provide immunity would exist for just a limited time. An ideal vaccine would have three main attributes: Induction of antibodies to neutralize toxins causing inflammation and tissue necrosis; induction of antibodies against CWA proteins believed to be important in the initial attachment of bacteria to host ligands; and the ability to induce a robust cytokine-mediated response to promote neutrophil recruitment and effective bacterial clearance. A successful anti-staphylococcal vaccine would not have to prevent infection by MRSA specifically, because putative vaccine targets could be independent of elements responsible for antibiotic resistance.

Development of a vaccine directed against several strains will require biologic finesse. Although anti-staphylococcal vaccines have been raised against a variety of targets, multiple efforts thus far have failed to demonstrate efficacy, including in phase 3 trials. In a double-blind trial involving patients with end-stage renal disease who were undergoing hemodialysis, Shinefield et al. [51] evaluated the safety, immunogenicity, and efficacy of a vaccine with S. aureus CP5 and CP8 capsular polysaccharides conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin A. A total of 1,804 adult patients (73 hemodialysis centers) were assigned randomly to receive a single intramuscular injection of either vaccine or saline. Immunoglobulin G (IgG) antibodies to both capsular polysaccharides were measured, and episodes of S. aureus bacteremia (primary end point, assessed at one year) were recorded. The capsular polysaccharides elicited an antibody response of at least 80 mcg/mL (the estimated minimal level conferring protection) in 80% of patients for CP5 and in 75% of patients for CP8. Between weeks 3 and 40 after vaccination, S. aureus bacteremia developed in 11 of 892 evaluable patients in the vaccine group, compared with 26 of 906 patients in the control group (vaccine efficacy [VE] estimate 57%; 95% confidence interval (CI), 10%–81%; p = 0.02). However, the efficacy during weeks 3 to 54 was only 26% (p = 0.23). Reactions to the vaccine were generally mild-to-moderate, and most resolved within two days. The authors concluded that the vaccine conferred partial immunity against S. aureus bacteremia for approximately 40 wks, after which protection waned as antibody concentrations decreased.

In view of the failure of that trial, a second study in hemodialysis patients evaluated the VE of two vaccine doses (rather than the single dose in the prior study) [52]. In a double-blind trial, 3,359 patients were randomly assigned (1:1) to receive vaccine or placebo at week 0 and 35. The VE in preventing S. aureus bacteremia was assessed between 3 and 35 wks and 3 and 60 wks post-dose 1. Serious adverse events (SAEs) were recorded for 42 d post-vaccination and deaths until study end. No substantial difference in the incidence of S. aureus bacteremia was observed between weeks 3–35 post-dose 1 (VE −23%, 95% CI: −98%–23%, p = 0.39) or at 3–60 wks post-dose 1 (VE −8%, 95% CI: −57%–26%, p = 0.70). Serious adverse events were reported by 24% and 25% of vaccine and placebo recipients, respectively. These data did not show a protective effect of either one or two vaccine doses against S. aureus bacteremia in hemodialysis patients despite a robust immune response and an acceptable safety profile. The authors suggested a need to expand the antigen composition of the vaccine among possible explanations for the failure of the trial.

Several additional anti-staphylococcal vaccines have reached clinical development, using various antigenic targets (Table 1) [53,54]. Immunization against several antigenic targets has heretofore been unsuccessful (e.g., lipotechoic acid, alpha-toxin), as have single-antigen immunization approaches against plausible biologic (protein) targets. Pfizer (Pearl River, NY) is developing a four-antigen (SA4Ag) vaccine that targets CP5 and CP8 (both conjugated to a mutant non-toxic diphtheria toxin carrier protein [CRM197]), a mutated recombinant ClfA protein (rmClfA), and a recombinant form of the manganese transporter A protein (MntC), an essential metabolic regulator (manganese is essential for detoxification of the neutrophil respiratory burst) (Table 1). These targeted virulence factors have the shared attribute that they are deployed early (experimentally, within one to six hours) during infection and elicit robust functional antibody responses. In a preliminary safety and efficacy trial [55], 100 participants were vaccinated with a single dose of SA4Ag. SA4Ag was well tolerated, with a satisfactory safety profile. On day 29, antibody titers had increased 69.2-fold (for CP5) and 28.9-fold (for CP8) and 19.6-fold (for MntC) and 12.3-fold (for ClfA), respectively. The majority of participants achieved the pre-defined biologically relevant thresholds: CP5, 100%; CP8, 97.9%, ClfA, 87.8%; and MntC, 96.9%. In phase 2/2a studies conducted in healthy adults aged 18–64 yrs [56] and 65–85 yrs [57], SA4Ag elicited rapid, robust immune responses to each of the antigens, sustained for at least 12 mos. Meta-analysis of these trials [58] confirmed the robust safety profile.

Table 1.

Anti-Staphylococcal Vaccines in Clinical Development

Vaccine designation	Developer	Antigenic targets	Trial phase
SA4Ag	Pfizer	CP5, CP8, ClfA, MntC	2b (ongoing)
Unknown	GSK	CP5, CP8, AT, ClfA	1 (completed)
NOV-3A	NovaDigm	Als3p	1 (completed)
4C-Staph	GSK (Novartis)	FhuD2m Csa1A, EsxAB	1 (completed)
STEBVax	IBT/NIAID	SEB	1 (completed)
Unknown	Nabi	AT/PVL	2 (completed)
Unknown	GSK	CP5, CP8, AT	Pre-clinical

Data derived from Mohamed et al. [53] and Bagnoli et al. [54].

Als = Candida albicans agglutinin-like sequence protein (has three dimensional structural similarity to clumping factor [Clf] A); AT = alpha-toxin; CP = capsular polysaccharide; Csa = conserved staphylococcal antigen; EsxAB = fusion protein of secreted proteins ess extracellular A and B; FhuD2 = ferric hydoxamate uptake lipoprotein D2, involved in iron-hydroxamate uptake; GSK = GlaxoSmithKline, Middlesex, United Kingdom; Mnt = manganese transporter protein; NIAID = National Institute for Arthritis and Infectious Diseases; PVL = Panton-Valentine leukocidin; SEB = staphylococcal enterotoxin B.

Arguably the most promising approach for anti-staphylococcal vaccination would be to vaccinate prior to the at-risk period (e.g., prior to elective surgery). Not only would soft tissue infections be targeted (e.g., surgical site infection), but a vaccine-induced antibody response would provide antibodies as well as human matrix proteins access to surgical implants (e.g., prosthetic joints, surgical mesh), and so potentially would, in theory, preclude bacterial adhesion to the implant, biofilm formation, and subsequent dissemination. Eventually, any type of elective operation that is plagued by staphylococcal infection could be a candidate for pre-operative vaccination, but an ongoing trial (Staphylococcus aureus Surgical Inpatient Vaccine Efficacy [STRIVE]) is targeting the vaccination of patients undergoing spine surgery with multi-level instrumentation from 10 to 60 d prior to surgery [59]. Future studies might target infra-inguinal vascular surgery with insertion of a prosthetic graft, open incisional hernia repair with insertion of mesh, or other high-risk (for staphylococcal infection) elective operations.

Footnotes

Author Disclosure Statement

Doctor Barie is a consultant for and has received honoraria from Arsanis, Inc. and Pfizer, Inc. Doctor Narayan has nothing to disclose. Doctor Sawyer is a consultant for and has received honoraria from Pfizer, Inc.

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