![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
SUMMARY INTRODUCTION Definition of S. aureus Encapsulation Biochemistry HIGHLY ENCAPSULATED SEROTYPE 1 AND 2 STRAINS Serotype 1 and 2 Capsules Impede Phagocytic Killing Role of Serotype 1 and 2 Capsules in Virulence Protection Afforded by Crude Capsule Preparations Protection Afforded by Purified CP1 SEROTYPE 5 AND 8 S. AUREUS CAPSULES Capsule Production In Vitro Capsule Production In Vivo Phagocytic Killing of Serotype 5 and 8 S. aureus Strains Interaction of S. aureus Capsules with Serum Complement Components Role of Serotype 5 and 8 Capsules in Virulence IMMUNOMODULATORY PROPERTIES OF CAPSULAR POLYSACCHARIDE CP5 AND CP8 AS PROTECTIVE ANTIGENS Protection Afforded by Whole-Cell Vaccines Protection Afforded by CP5 and CP8 Antigens Active Immunization of Humans with CP5-CP8 Conjugate Vaccines Phase I and II clinical trials. Phase III clinical trial. PASSIVE IMMUNIZATION OF HUMANS WITH CAPSULAR ANTIBODIES GENETIC ANALYSIS OF S. AUREUS CAPSULE EXPRESSION Serotype 1 Capsule Gene Cluster Serotype 5 and 8 Capsule Gene Clusters Molecular characterization of the cap8 locus. BIOSYNTHESIS OF TYPE 5 CAPSULAR POLYSACCHARIDE cap5H cap5P and cap5O cap5E, cap5F, and cap5G Other cap5 and cap8 Genes GENETIC REGULATION OF CAPSULE EXPRESSION CONCLUSIONS ACKNOWLEDGMENTS REFERENCES
| SUMMARY |
|---|
 Top NextReferences |
|---|
| INTRODUCTION |
|---|
|
TopPreviousNextReferences |
|---|
Because of the prevalence of antibiotic-resistant strains and the recent emergence of clinical isolates resistant to vancomycin (77), control of S. aureus has become increasingly difficult. Staphylococcus plays a major role in nosocomial infections and recently has been acknowledged as an important cause of community-acquired infections (11, 35, 40, 50). Communityacquired S. aureus infections often occur in otherwise healthy individuals who lack the expected risk factors for S. aureus infections, e.g., recent hospitalization or surgery, residence in a long-term-care facility, or use of injected drugs. It is postulated that strains causing these community-acquired infections have a high virulence potential because of their ability to cause disease in immunocompetent hosts (22, 39).
The bacterial components and secreted products that affect the pathogenesis of S. aureus infections are numerous (93) and include surface-associated adhesins, a capsular polysaccharide, exoenzymes, and exotoxins. This constellation of bacterial products allows staphylococci to adhere to eukaryotic membranes, resist opsonophagocytosis, lyse eukaryotic cells, and trigger the production of a cascade of host immunomodulating molecules. Because of the multifactorial nature of staphylococcal infections and the functional redundancy of S. aureus adhesins and exoproteins, it has been difficult to sort out the role that individual virulence determinants play in the pathogenic process.
In 1982 Karakawa and Vann proposed a new capsular polysaccharide typing scheme for S. aureus based upon the preparation of absorbed rabbit antiserum to prototype S. aureus strains (52). These investigators were the first to report that most S. aureus strains were encapsulated, and they described eight capsular serotypes. The heavily encapsulated strains M and Smith diffuse were assigned to serotypes 1 and 2, respectively. Strains of these two serotypes produce mucoid colonies on solid medium, and they are rarely encountered among clinical isolates (4, 49, 52, 64, 103). Isolates belonging to the remaning serotypes produce nonmucoid colonies on solid medium, and their colony morphology is indistinguishable from that of strains lacking a capsule. Some investigators have referred to nonmucoid, encapsulated S. aureus isolates as microencapsulated to distinguish them from the atypical mucoid strains.
Serotyping studies of staphylococcal isolates from diverse strain collections representing several geographic regions have revealed that serotype 5 and 8 isolates account for 
25% and 50%, respectively, of isolates recovered from humans (4, 49, 64, 103). Moreover, these two serotypes are prevalent among isolates from clinical infections as well as from commensal sources. Serotype 5 and 8 capsules are also made by S. aureus strains isolated from cows, rabbits, poultry, pigs, and horses (20, 43, 92, 104, 110). The type 5 and 8 capsules were localized to the cell surface of clinical isolates by immunoelectron microscopy with type-specific monoclonal antibodies to stabilize and visualize the polysaccharides (49, 103). In 1985, Sompolinsky et al. (103) serotyped a large collection of S. aureus isolates from different sources and included three new serotypes in their collection, bringing the number of putative capsular serotypes to 11.
Strains that do not react with antibodies to capsule types 1, 2, 5, or 8 are currently referred to as nontypeable (2, 4, 49, 103), since neither the prototype strains nor antiserum to the other putative serotypes is available.
4)-
-D-GalNAcA-(14)--D-GalNAcA-(13)--D-FucNAc-(1)n; a taurine residue is amide linked to every fourth D-GalNAcA residue (70, 80). Two other strains, D (52) and SA1 mucoid (65), produce capsules that are serologically and biochemically similar to that produced by strain M. The Smith diffuse capsule of serotype 2 has the structure: (4)-ß-D-GlcNAcA-(14)-ß-DGlcNAcA-(L-alanyl)-(1)n (44).
Type 5 and 8 capsular polysaccharides (CP5 and CP8, respectively) purified from the prototype strains Reynolds and Becker, respectively, are structurally very similar to each other and to the capsule made by strain T, described previously by Wu and Park (123). Type 5 has the structure (4)-3-O-Ac-ß-D-ManNAcA-(14)--L-FucNAc-(13)-ß-D-FucNAc-(1)n (32, 78), and type 8 has the structure (3)-4-O-Ac-ß-D-ManNAcA-(13)--L-FucNAc-(13)-ß-D-FucNAc-(1)n (33). Type 5 and 8 polysaccharides differ only in the linkages between the sugars and in the sites of O-acetylation of the mannosaminuronic acid residues, yet they are serologically distinct. The structure of CP4 was never elucidated, although the prototype serotype 4 strain 7007 has been shown to react with antibodies to CP5. CP4 is most likely a polysaccharide with a trisaccharide structure identical to that of CP5 but differing in the presence, absence, or location of the O-acetyl moieties on the N-acetylmannosaminuronic acid residues (113). The linkage(s) that anchors any of the S. aureus capsular polysaccharides to the staphylococcal cell wall remains undefined.
| HIGHLY ENCAPSULATED SEROTYPE 1 AND 2 STRAINS |
|---|
|
TopPreviousNextReferences |
|---|
To determine whether the size of the S. aureus capsule influenced virulence in vivo, our group prepared transposon-induced mutants from a highly encapsulated serotype 1 strain of S. aureus (62). Using mouse models of staphylococcal infection, we compared the virulence of the wild-type, highly encapsulated parent strain (SA1 mucoid) with that of two nonmucoid mutants, one microencapsulated (producing less capsule than the parental strain) and the other nonencapsulated. When challenged by the intraperitoneal route, strain SA1 mucoid had a 50% lethal dose (LD5o) for mice of 2.1 x 104 CFU. The microencapsulated and nonencapsulated mutant strains had comparable LD5o values (2 x 108 CFU) that were similar to those of serotype 5 and 8 isolates of S. aureus (1) and >3,000-fold higher than that of the highly encapsulated parental strain. In mice challenged intravenously with the bacterial strains, the nonmucoid mutants were cleared more readily from the bloodstream and kidneys than strain SA1 mucoid. In an in vitro assay, only the highly encapsulated strain demonstrated antibody-dependent, complement-mediated opsonophagocytosis by human leukocytes. The microencapsulated and nonencapsulated mutants were opsonized for phagocytosis by preimmune serum with complement activity that lacked antibodies to the capsule. These studies not only established the importance of the type 1 capsule in staphylococcal virulence but showed that the amount of surface-associated capsular polysaccharide was critical in the host-parasite interaction.
In contrast to the enhanced virulence of the mucoid serotype 1 strain of S. aureus in the murine lethality or bacteremia model, the same organism was poorly virulent in the rat model of catheter-induced endocarditis (82). The inoculum dose of S. aureus SA1 mucoid required to induce infection in 50% of catheterized rats (ID50) was 4 x 106 CFU, a dose 1,000-fold higher than that reported for serotype 5 and 8 isolates (7). Likewise, serotype 5 and 8 S. aureus strains were less virulent in the endocarditis infection model than were isogenic mutant strains that were devoid of capsule production. In dose-response experiments, the acapsular mutants showed ID50 values 10-fold lower than those of the parental serotype 5 and 8 strains (7). Staphylococcal adherence to the damaged heart valve is critical to initiate infection in the S. aureus endocarditis model. The inverse correlation between capsule production and infectivity in this model suggests that the capsule may be masking adhesins that have been shown to be important determinants of virulence in endocarditis (57, 79).
Mice immunized with polysaccharide antigens extracted from cells of Smith diffuse were not protected against challenge with 108 CFU of the homologous strain in saline (25). However, if the staphylococcal inoculum was mixed with 5% hog gastric mucin, the mouse-lethal dose of strain Smith diffuse was reduced from 108 to 105 CFU. Under these experimental conditions, immunization with polysaccharide antigens extracted from S. aureus cells (25) or heat-stable culture supernatants (31) was protective against lethality induced by the homologous strain. The protection could be passively transferred to naive animals by the injection of immune serum (25, 30). However, no protection against infection by the Smith diffuse strain was elicited by immunization with whole cells or culture extracts prepared from two heterologous strains of S. aureus. Likewise, mice immunized with the Smith diffuse strain were not protected against a lethal inoculum of a heterologous S. aureus isolate (strain Foggie; capsular phenotype unknown) (24).
|
|
Capsular antibodies were also protective in a sublethal model of S. aureus bacteremia and renal abscess formation. Mice were immunized with killed strain SA1 mucoid or CP1, as described above, and then challenged intravenously with one of three S. aureus strains with various amounts of cell-associated capsule (67). Immunization with killed cells of the homologous strain protected mice against infection with each of the three S. aureus isolates (67). Mice immunized with CP1 were protected when challenged intravenously with either the highly encapsulated strain SA1 mucoid or the nonmucoid microencapsulated mutant, but not with the acapsular mutant. Protection against infection with the encapsulated S. aureus correlated with capsular antibody levels in the immunized animals. Immunization with a heterologous strain lacking CP1 was not protective against challenge with strain SA1 mucoid. Naïve mice passively immunized with antiserum raised to strain SA1 mucoid or CP1 had significantly fewer CFU of strain SA1 mucoid in their blood and kidney samples than did mice given preimmune serum (67). The results of these experiments indicate that capsular antibodies are protective against infection by the prototype serotype 1 strains. Moreover, these studies provided the foundation for subsequent investigations to test whether the more clinically relevant serotype 5 and 8 capsules could serve as the target of protective antibodies.
| SEROTYPE 5 AND 8 S. AUREUS CAPSULES |
|---|
|
TopPreviousNextReferences |
|---|
CP5- and CP8-producing staphylococci are often referred to as microencapsulated. This terminology is used to distinguish these isolates from mucoid serotype 1 or 2 strains and was based on visualization of scant capsular material produced by staphylococci harvested from poorly aerated or logarithmic-phase broth cultures (1). It is noteworthy that serotype 5 and 8 S. aureus cells cultivated on agar plates or in well-aerated overnight broth cultures produce a substantial capsule, as visualized by immunoelectron microscopy (Fig. 3).
|
Poutrel et al. (91) analyzed CP5-specific monoclonal antibody binding to 30 different serotype 5 S. aureus strains by flow cytometry. Their results confirmed that the growth medium affects surface expression of CP5 and revealed intraisolate heterogeneity of cell-associated CP5 expression. Flow cytometry analysis provides information on the expression of capsular polysaccharide by thousands of individual cells, and the results revealed large variations in the amount of CP5 produced by individual bacteria in a population. There was also striking strain-to-strain variability in the response of S. aureus capsular polysaccharide production to environmental culture conditions (90, 91). The results of most studies, however, have indicated that little capsular polysaccharide is produced in the logarithmic phase of growth and that maximal capsule production occurs during the postexponential growth phase (15, 17, 88, 90). This observation is consistent with the fact that capsular polysaccharide expression has been shown to be positively controlled by the global regulator agr (18, 72, 88). The agr locus is a complex multigene operon in S. aureus that acts as a two-component quorum-sensing system. Genes under the control of agr include secreted virulence factors and adhesins that are regulated in a growth phase-dependent manner.
Herbert et al. (48) first reported that CO2 was an environmental signal that regulated CP5 expression both in vitro and in vivo. Although CP5 was expressed under normal air conditions (0.03% CO2), CP5 expression by three different serotype 5 strains was inhibited by cultivation in air supplemented with 1 to 5% CO2 (47, 48). CO2 was shown to affect cap5 gene expression at the transcriptional level (47). In contrast, quantitative detection of CP8 on different S. aureus strains cultivated in the presence of CO2 yielded conflicting results (47). Of nine strains tested, four showed decreased CP8 expression in the presence of 5% CO2, in four strains the effect of CO2 was marginal, and strain Becker demonstrated enhanced CP8 expression in the presence of CO2. The authors employed reporter gene fusion studies to determine whether specific differences in the sequence of the cap5 or cap8 promoter would influence the pattern of CO2 regulation among different S. aureus strains. Promoters amplified from five different strains were fused to the reporter gene xylE; each genetic construct was then transformed into two different S. aureus strains, the type 5 strain Newman and the type 8 strain Becker. XylE activity associated with each construct was evaluated in both genetic backgrounds. The results indicated that the genetic background of the individual S. aureus strain rather than the promoter sequence determined the regulation of capsular polysaccharide expression by CO2. XylE activity was always upregulated by CO2 in strain Becker and was always negatively regulated by CO2 in strain Newman. The authors postulated that trans-acting regulatory molecules such as transcriptional activators or sigma factors may be differentially expressed in different staphylococcal strains.
We also evaluated capsule production in a mouse model of S. aureus nasal colonization (54). Staphylococci recovered from the nares of the colonized mice (without subculture) expressed CP5 in vivo, and a capsule-defective mutant showed reduced persistence in the nares compared to the parent strain (54). Hensen et al. (45) demonstrated in vivo expression of S. aureus CP5 in experimental bovine mastitis by immunochemical staining of tissue sections obtained from cows with acute or chronic mastitis.
In contrast, CP5 expression was not detected in vivo in several other staphylococcal infections. Minimal expression of CP5 was observed in either lung tissue or nasal polyp tissue obtained from two cystic fibrosis patients infected with S. aureus (48, 75). Similarly, when rats were challenged with a serotype 5 S. aureus strain in the granuloma pouch model, 
5% of the cells harvested from the pouch exudates were CP5 positive (48). In both of these tissues, the absence of CP5 expression correlated with elevated CO2 levels (
4%). As discussed above, CO2 has been shown to be an environmental signal that downregulates CP5 expression in vitro (47, 48).
In contrast, our laboratory performed similar experiments but obtained very different results. Serotype 5 and 8 isolates grown to logarithmic phase were opsonized for phagocytosis by either preimmune serum with complement activity or heat-inactivated serum raised to type 5, type 8, or nonencapsulated S. aureus strains (121). Our results indicated that antibodies to the capsule or to cell wall components other than the capsule were opsonic for serotype 5 and 8 staphylococci. However, because these experiments were performed with staphylococci harvested in the logarithmic phase of growth, little to no capsular polysaccharide was expressed (15, 88). When the experiments were repeated after growing the staphylococci under conditions of optimal capsule expression, the bacteria resisted opsonophagoctyic killing in the presence of either complement or antibodies. The serotype 5 strain Reynolds was efficiently killed by human polymorphonuclear leukocytes only after opsonization with both specific capsular antibodies and complement (109). These results confirmed the initial observations reported by Karakawa et al. (51) and underscore the influence of culture conditions on the biological properties of S. aureus serotype 5 and 8 isolates.
Studies on the interaction between complement components and the more clinically relevant capsular types of S. aureus were only recently reported (15, 16). Cunnion et al. (15) challenged control and C3-depleted mice intravenously with 107 CFU of a serotype 5 S. aureus strain. Whereas only 8% of the control mice succumbed to the infection, 64% of the complement-depleted animals died. The investigators also evaluated the in vitro parameters of C3 binding to serotype 5 and 8 isolates of S. aureus. At a high serum concentration (20%, similar to whole blood), the alternative pathway was active, contributing 90% of the total C3 binding to S. aureus. At a lower serum concentration (2%) that might be present in tissues, the classical complement pathway predominated.
In a comparison of bacterial strains grown on solid medium, a CP5 strain bound 42% fewer C3 molecules than its isogenic capsular polysaccharide-negative mutant. Both C3b and iC3b fragments of C3 bound to S. aureus cells (15, 16), and about one third of the bound C3b was shed from the bacterial surface as iC3b regardless of the capsular polysaccharide phenotype of the strain. CP5 was shown to mask C3 fragments deposited on the organism from binding to complement receptor 1 (16). The observation that encapsulated S. aureus organisms interfere with opsonization by complement is consistent with the fact that these cells are poorly phagocytosed in normal human serum (109). Ongoing studies by our group are aimed at determining whether purified CP5 or CP8 activates complement in human serum.
The virulence of the serotype 5 strain Reynolds in a mouse bacteremia model of infection was reexamined by inoculating mice with organisms cultivated on solid medium, which supports optimal capsule expression (109). The wild-type strain showed an LD50 value that was 10-fold lower than that of the capsule-negative mutant. Likewise, strain Reynolds sustained a significantly higher level of bacteremia and was cleared from the bloodstream of the infected animals less readily than the CP5-deficient mutant strains. We attributed this to the antiphagocytic nature of CP5, because in vitro assays indicated that the parental strain was susceptible to phagocytic killing by human polymorphonuclear leukocytes only in the presence of specific capsular antibodies (109). Capsule-deficient mutant strains (or the parental strain grown in poorly aerated broth cultures, where little capsule is expressed on the bacterial surface) were opsonized for phagocytic killing by nonimmune serum with complement activity.
A serotype 5 S. aureus strain was shown to be more virulent than an isogenic, acapsular mutant in a mouse model of renal abscess formation and in a subcutaneous abscess model of infection (89). Likewise, the type 5 strain was more virulent than its capsule mutants when compared in a murine model of septic arthritis (83). Mice inoculated with the prototype strain Reynolds developed more frequent and severe arthritis than mice inoculated with the CP5 mutant strains. Furthermore, mice challenged with the wild-type strains had a higher mortality rate and more pronounced weight loss than mice inoculated with the mutants. In vitro assays indicated that mouse macrophages were not able to phagocytose the parental strain Reynolds as efficiently as a capsule-defective mutant. Once phagocytosed, the CP5-positive strain was less efficiently killed than the mutant strain (83).
As noted above in the studies related to the serotype 1 capsule, capsular polysaccharide production attenuates staphylococcal virulence in the rat model of catheter-induced endocarditis (82). In a comparative study, we evaluated the virulence of serotype 5 and 8 S. aureus strains and their capsule-deficient mutants in the endocarditis model (7). Catheterized animals were challenged with inocula ranging from 102 to 106 CFU/rat. Mutants derived from serotype 5 and 8 S. aureus strains that were deficient in capsule production had ID50 values 10-fold lower than those of the parental strains (7). These observations are consistent with the inverse relationship that has been demonstrated between encapsulation and bacterial adherence to or invasion of host tissues (36, 106, 105). The capsule may be masking adhesins that have been shown to be important virulence determinants in endocarditis (57, 79).
The influence of capsule production on in vitro adherence of the staphylococcus to endothelial cells was recently reported (88). The data from that study indicate that S. aureus adherence to endothelial cells is maximal during the logarithmic phase of growth, when CP5 production is minimal. In the stationary phase of the bacterial growth cycle, the organisms are poorly adherent, and staphylococcal capsule expression is maximal. A mutation in the regulatory agr locus diminished CP5 production and led to increased staphylococcal adherence to endothelial cells in culture. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence. An acapsular mutant showed significantly greater adherence to endothelial cells than did the parental serotype 5 strain. Complementation experiments restored capsule production and reduced adherence to a level similar to that of the parental strain. The results from these studies suggest that the binding domain of the S. aureus adhesin for endothelial cells is masked by CP5. Similarly, we have observed that expression of both CP5 and CP8 diminishes S. aureus clumping factor A-mediated binding to fibrinogen (J. C. Lee, unpublished observations).
| IMMUNOMODULATORY PROPERTIES OF CAPSULAR POLYSACCHARIDE |
|---|
|
TopPreviousNextReferences |
|---|
Structural studies of CP8 revealed that it has a zwitterionic charge motif conferred by the negatively charged carboxyl group of N-acetylmannosaminuronic acid and free amino groups available on partially N-acetylated fucosamine residues. Chemical modifications that neutralized the charged groups on CP8 also abrogated its ability to provoke abscesses in vivo, indicating that this charge motif was critical for CP8 biological activity. As shown in Table 1, animals treated subcutaneously with CP8 24 h before challenge with homologous or heterologous zwitterionic polysaccharides were protected against abscess formation (112). Likewise, treatment with CP8 protected against challenge with viable S. aureus strains that produced CP5 or CP8.
|
| CP5 AND CP8 AS PROTECTIVE ANTIGENS |
|---|
|
TopPreviousNextReferences |
|---|
Several studies evaluated the protective efficacy of antibodies to CP5 and CP8 in experimental models of S. aureus infection. The first report evaluated the protective efficacy of capsular antibodies in a rat model of catheter-induced staphylococcal endocarditis (82). Rats were actively immunized with killed serotype 5 S. aureus cells or passively immunized intravenously with CP5 antiserum. Control animals were injected with saline or passively immunized with normal rabbit serum. Despite having elevated levels of capsular antibodies, the immunized animals were susceptible to staphylococcal endocarditis, and immunized and control animals had similar numbers of bacteria in the blood and vegetations. Likewise, antibodies to teichoic acid failed to protect the animals against staphylococcal endocarditis in this study (82) and in that reported by Greenberg et al. (42). These initial studies with whole killed S. aureus cells as immunogens did not offer promise that capsular antibodies alone would protect against staphylococcal infection.
Fattom et al. (27) conjugated S. aureus CP5 and CP8 to nontoxic recombinant exotoxin A from Pseudomonas aeruginosa and administered this preparation to mice. The mice developed serum antibodies to the polysaccharides after two injections; the third injection stimulated a booster response. Differences in the carrier proteins and the chemical methods used to couple the proteins to the polysaccharide affected the magnitude of the immune response in mice, but these variables did not affect the distribution of IgG subclasses detected in immune serum (26). Use of monophosphoryl lipid A as an adjuvant enhanced the immunogenicity of the conjugate vaccines and induced a shift in the IgG subclass composition toward the more opsonic IgG2a and IgG2b subclasses in the immunized mice.
The protective efficacy of antibodies to the CP5-recombinant exotoxin A conjugate vaccine was tested in a mouse model of lethality and disseminated infection (29). Immunization with CP5-recombinant exotoxin A protected mice against lethality induced by a serotype 5 S. aureus strain. Ten days after bacterial challenge, 33 of 45 mice immunized with the conjugate survived, compared with 4 of 30 mice injected with phosphate-buffered saline. Similarly, passive immunization with immune IgG protected mice against S. aureus-induced lethality. Passive immunization of mice with immune IgG also resulted in a reduction in the level of bacteremia at 6 and 24 h after intraperitoneal inoculation of mice with a sublethal dose of serotype 5 S. aureus. In addition, fewer animals given immune IgG showed metastatic infection by S. aureus in their livers, kidneys, and peritoneal lavage fluids (Table 2).
|
|
Phase I and II clinical trials. The CP8- and CP5-recombinant exotoxin A conjugate vaccines were evaluated for safety and immunogenicity in 70 healthy adult volunteers (27). Neither conjugate caused significant local or systemic reactions in the volunteers. The conjugate vaccine induced capsular polysaccharide-specific antibodies of both the IgM and IgG classes. A second injection 6 weeks later did not have a booster effect. The authors suggest that due to low levels of prevaccination capsular antibodies in these subjects, the initial vaccination behaved more like a booster than a primary dose.
Nabi conducted a phase II, double-blinded, placebo-controlled clinical study of StaphVax in 230 chronic ambulatory peritoneal dialysis patients, individuals who are at high risk of staphylococcal disease. The patients were actively immunized with StaphVax, and their antibody responses and infection rates were monitored. The vaccine was shown to elicit only mild local or systemic symptoms. The results of this trial indicated that the vaccine dose of 25 µg of each capsular polysaccharide in the conjugate formulation was suboptimal in these patients. Their antibody responses to the vaccine were weak, and they had infection rates similar to those of nonimmunized patients.
In subsequent phase II clinical trials, 32 volunteers with end-stage renal disease and 29 healthy controls were injected twice (6 weeks apart) with 25 µg of CP5-recombinant exotoxin A or the bivalent conjugate vaccine (25 µg each of CP5 and CP8 linked to recombinant exotoxin A). The vaccines elicited only mild local or systemic symptoms in both populations (119; G. Horwith, personal communication). Four weeks after the second dose of vaccine, 23 of 24 healthy volunteers and 14 of 17 patients in one study responded to the immunization with a 4-fold rise in preimmunization IgG and IgM antibody levels. However, the IgG and IgM levels of the patients were only 50% of those achieved by the healthy controls at all postimmunization intervals. The monovalent and bivalent vaccines did not contain any adjuvant. Data from animal studies indicate that enhanced immunogenicity may be achieved by incorporating an adjuvant such as monophosphoryl lipid A (26).
Phase III clinical trial. Between 1998 and 2000, Nabi conducted a double-blind clinical trial to evaluate the safety, immunogenicity, and efficacy of StaphVax for prevention of bacteremia in 1,800 patients with end-stage renal disease receiving hemodialysis (99). These patients are at high risk for staphylococcal infection, with 3 to 4 of every 100 patients infected with S. aureus per year. Half of the patients in the trial were administered a placebo, and the other half were immunized with a single injection of Nabi's bivalent StaphVax (100 µg each of CP5 and CP8 conjugated to an equal weight of nontoxic recombinant exotoxin A). Efficacy was estimated by comparing the incidence of S. aureus bacteremia in the patients who received the vaccine with the incidence in control patients over weeks 3 to 54 following immunization.
During this time interval, the vaccine reduced the incidence of bacteremia in the study population by only 26% (not significant, P = 0.23) (99). However, when the data were analyzed to include only the time period between weeks 3 and 40, the vaccine efficacy was estimated to be 57% (P = 0.02). During this time period, S. aureus bacteremia developed in 11 of 892 patients in the vaccine group, compared with 26 of 906 patients in the control group. After 40 weeks the antibody levels in the vaccinated patients declined, mirroring the decline in efficacy of the vaccine. There were no significant differences in the number of deaths in the vaccine and control groups, and none of the deaths were considered related to the vaccine.
Many new questions were raised as a result of this clinical trial. Does immunization reduce S. aureus nasal colonization in these patients? Why was there no obvious correlation between capsular antibody levels induced by vaccination and susceptibility to staphylococcal bacteremia? Does impaired phagocyte function in these patients (about half are diabetic) explain why the estimated protective level of protective antibodies (>80 µg/ml) is so high? Can a vaccine that only targets the capsular polysaccharide protect patients against a bacterium with such a multitude of virulence determinants (adhesins, exoenzymes, and exotoxins)? Since CP5 and CP8 are not expressed in vitro during the logarithmic phase of bacterial growth or under all experimental conditions, does this contribute to the lack of efficacy of StaphVax in the clinical trial? Nabi plans another phase III clinical trial of their CP5-CP8 vaccine in hemodialysis patients. It is hoped that continued evaluation of StaphVax and other multicomponent S. aureus vaccines will lead to answers to some of these important questions.
| PASSIVE IMMUNIZATION OF HUMANS WITH CAPSULAR ANTIBODIES |
|---|
|
TopPreviousNextReferences |
|---|
| GENETIC ANALYSIS OF S. AUREUS CAPSULE EXPRESSION |
|---|
|
TopPreviousNextReferences |
|---|
Molecular characterization, mutagenesis, and transcriptional analysis of the cap1 locus showed that the 15 cap1 genes were transcribed in the same orientation, resulting in a single 15.5-kb transcript (71, 85). Five internal promoters within the cap1 operon were identified but were much weaker than the primary promoter located upstream of the first gene, cap1A, as measured by gene fusion experiments with Pseudomonas xylE as the reporter (85). Nevertheless, the internal promoters were demonstrated to be biologically functional. Ouyang and Lee (85) proved that the internal promoters were sufficiently active for CP1 expression by creating a CP1-negative mutant strain in which the promoter upstream of cap1A was deleted. CP1 expression was restored to the mutant by integrating a single copy of a DNA fragment comprising the promoter upstream of cap1A together with cap1A through cap1E at the phage L54a attB site in the chromosome (remote from the cap1 locus). This construct physically separated the primary promoter together with the first five genes from the downstream cap1 genes. The recombinant strain was mucoid and produced a level of CP1 similar to that of the wild-type strain, indicating that transcription of the downstream cap1 genes by internal promoters was sufficient for capsule synthesis.
Recently, a 50-kb region comprising the cap1 gene locus and flanking region was sequenced (74). From this analysis, Luong et al. proposed that the cap1 operon is located within a staphylococcal cassette chromosome (SCC) element similar to the type III SCCmec island associated with methicillin resistance in S. aureus. The cap1 operon comprises more than half the size of the 27.4-kb SCCcap1 element (Fig. 4). The right boundary of SCCcap1, nearly identical to that of other SCCmec elements, had an attR site located approximately 10 kb downstream from the cap1 operon. The left boundary of SCCcap1, located 1 kb upstream of the cap1 cluster, contained direct and inverted repeats that matched the SCCmec consensus for att sites. The authors observed a high occurrence of mutations, deletions, and rearrangements within SCCcap1 and its left flanking region. Notably, only the cap1 genes and the enterotoxin gene located in the DNA region flanking SCCcap1 were intact and functional. The SCCcap1 element carries defective recombinase genes required for mobilization events (74), and this defect likely prevents the element from moving horizontally. This recombinase defect may at least partially explain why serotype 1 strains are so rare among clinical isolates of S. aureus.
|
Switching between the mucoid and nonmucoid phenotypes of a serotype 1 strain has been shown to occur upon animal passage. When nonmucoid mutants of strain M were injected intraperitoneally into mice, only mucoid colonies were recovered from the peritoneal washes of mice that succumbed to challenge (98). In contrast, loss of the mucoid phenotype was observed in a sublethal model of renal abscess formation. Organisms isolated from mouse kidneys early in the infection (prior to day 10) were all mucoid. However, by day 24, the majority of colonies cultured from the kidneys of infected animals were no longer mucoid, even though they were otherwise phenotypically identical to the mucoid challenge strain (J. C. Lee, unpublished observations). Thus, serotype 1 and 2 capsules, although not regulated at the transcriptional level, may be regulated by mutation and reversion in the structural genes required for capsule production.
17.5-kb region of the chromosome; each contains 16 closely linked genes, cap5A (cap8A) through cap5P (cap8P), transcribed in one orientation (Fig. 5). Both CP5 and CP8 are composed of the same three sugar residues, ManNAcA, L-FucNAc, and D-FucNAc. Therefore, it is not surprising that 12 of the 16 genes in the two gene clusters are nearly identical (96). The type-specific genes are located in the central region of the loci (comprising cap5H, cap5I, cap5J, and cap5K and the same genes for cap8), and they showed little homology between the two gene clusters. Wann and coworkers (118) transduced cap5HIJK into the type 8 strain P1; the genes were integrated into the chromosome by homologous recombination, with the reciprocal loss of cap8HIJK. The resultant strain produced CP5, indicating that cap5HIJK was responsible for CP5 serotype specificity.
|
Fusion studies with xylE as the reporter gene indicated that the promoter upstream of cap8A was the principal promoter, although weak internal promoters were observed in front of most of the downstream cap8 genes. Analysis of the cap8 gene sequence revealed several inverted and direct repeats upstream of the primary promoter. Ouyang et al. (86) performed deletion analyses and site-directed mutagenesis to demonstrate that one of these repeats, a 10-bp inverted repeat located 14 bp upstream of the cap8 promoter, was essential for promoter activity. Mutations within the 10-bp repeat reduced CP8 production to an undetectable level. The inverted repeat could serve as a DNA binding site for an activator that regulates cap8 gene transcription. Efforts to identify such a DNA-binding activator are under way in the laboratory of C. Y. Lee.
Luong and Lee recently replaced the native cap8 reporter of strain Becker with the strong constitutive promoter of the cap1 operon from strain M (73). The resultant strain, CYL770, synthesized approximately sevenfold more cap8-specific mRNA than wild-type strain Becker, and the strain produced about 80-fold more CP8. The CP8-overproducing strain CYL770 was more resistant to in vitro opsonophagocytic killing by human polymorphonuclear leukocytes. In a mouse infection model of bacteremia, strain CYL770 persisted longer in the bloodstream, liver, and spleen than the parent strain Becker. These results were the first to show that CP8 promoted S. aureus virulence in an animal model of infection, since previous studies had employed serotype 5 strains. The results of these studies also confirm those performed with serotype 1 strains (62) that demonstrated a positive correlation between increased capsule production and bacterial virulence.
| BIOSYNTHESIS OF TYPE 5 CAPSULAR POLYSACCHARIDE |
|---|
|
TopPreviousNextReferences |
|---|
A comparison of the amino acid sequences of the putative cap5 and cap8 gene products with sequences found in the databases allowed us to predict functions for 15 o
ccr gene complex of three open reading frames conserved among SCCmec elements. A novel enterotoxin gene (ent) and a truncated transposase gene (trp*) identified outside of the SCCcap1 are indicated by short arrows. The DNA region to the left of the ent gene is homologous to a gene region found in NCTC 8325, but many of the open reading frames within this region contain mutations. Data are adapted from Luong et al. (