The Search for an Effective Staphylococcus Aureus Vaccine

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pathogenesis5,8–11, as well as on the nature of OPINION protective immunity against S. aureus infec- tion8,12–14. There seems to be a consensus Models matter: the search for an among investigators that the multiplicity and redundancy of S. aureus virulence fac- effective Staphylococcus aureus tors creates an enormous challenge for the development of an effective vaccine and that the failure of vaccine development so far vaccine is a result of not knowing the correct combination of antigens or of using a range of Wilmara Salgado-Pabón and Patrick M. Schlievert antigens that is too narrow7,9,10. Furthermore, some argue that not knowing the correlates Abstract | Staphylococcus aureus is a highly successful bacterial pathogen owing of immune protection is the greatest obstacle to its abundance of cell surface and secreted virulence factors. It is estimated that to vaccine development11,13,14. In addition, 30% of the population is colonized with S. aureus, usually on mucosal surfaces, and the protective role of antibodies in immune methicillin-resistant S. aureus is a major public health concern. There have been defence against S. aureus infections has also multiple attempts to develop an S. aureus vaccine using one or more cell surface been questioned, and it has been argued that opsonic S. aureus-specific antibodies already virulence factors as antigens; all of these vaccine trials have failed. In this Opinion exist in humans and are not sufficient to article, we suggest that an over-reliance on rodent models and a focus on targeting provide protection from infection, and an cell surface components have been major contributing factors to this failure. antibody-based vaccine is therefore unlikely to succeed14,15. More than 100 years have passed since Vaccination is widely recognized as one that protect against life-threatening S. aureus the discovery of S. aureus and the diseases of the most cost-effective public health infections, but it is hoped that vaccines that it causes, and we still know relatively interventions. Early vaccine development could be developed that protect against all little about S. aureus pathogenesis3. As efforts that targeted bacterial pathogens S. aureus infections, including the many soft Projan et al. wrote, “little knowledge is a resulted in vaccines for Clostridium tetani, tissue infections, such as abscesses and dangerous thing”5. However, the develop- Corynebacterium diphtheriae and Bordetella surgical site infections, that are associated ment of useful vaccines and immunothera- pertussis, and later efforts resulted in vac- with S. aureus. The vaccine approaches that peutic agents to target S. aureus infections cines that targeted Streptococcus pneumoniae, have been used so far have all involved tar- depends on a correct understanding of Haemophilus influenzae and Neisseria geting cell surface components, including the molecular pathogenesis of S. aureus. meningitidis. Most of these vaccines involved the polysaccharide capsule, the extracel- This requires the use of appropriate animal a single antigenic target — either a toxin or lular polysaccharides that are important for models. Mice have been the animal models the capsule — that, when targeted, provided biofilm formation, the cell wall-associated of choice for most pathogenesis studies, immune protection. The development of vac- proteins that recognize adhesive matrix owing to their low cost. However, the most cines for most remaining bacterial pathogens, molecules and aid in surface attachment and important consideration when selecting a including Staphylococcus aureus, is more dif- colonization, or ATP-binding cassette (ABC) model system for preclinical studies should ficult, as the pathogenesis of these organisms transporters, which have a role in nutrient be whether or not it closely mimics human is multifaceted. uptake and drug resistance. However, none infections. So far, mouse models of S. aureus S. aureus is the most important cause of these immunization strategies has suc- infection have failed to predict human of serious infectious diseases, such as toxic ceeded in human trials (BOX 1). The latest outcomes in clinical trials8,12. Although shock syndrome (TSS), pneumonia, sepsis S. aureus vaccine failure4 emphasizes the many investigators concede that mouse and infective endocarditis (FIG. 1), in the need to address the reasons for these models are imperfect5–7,10,12,14, in practice, United States1. The life-threatening illnesses failures and provides an opportunity to most investigators continue to dismiss these that are associated with S. aureus infec- correct them. inadequacies and still expect these models tion are dependent both on cell surface Recent discussions on the failure of to shed light on crucial aspects of S. aureus and on secreted virulence factors2,3. As the staphylococcal vaccines have focused on our pathogenesis16 and mechanisms of immune emergence of multidrug-resistant S. aureus lack of understanding of S. aureus pathogen- protection against S. aureus infection8,17. strains is predicted to continue, efforts have esis in humans5–7, on the large range of viru- It is also crucial to recognize that focused on disease prevention via vaccina- lence factors that are produced by S. aureus S. aureus causes serious illnesses, most nota- tion or immunotherapeutic approaches. As a (many of which have redundant func- bly menstrual and non-menstrual TSS18, minimum, it is desirable to develop vaccines tions) and their contributions to S. aureus lethal pneumonia19, infective endocarditis

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of the V710 vaccine (Merck) (which targeted aggregation of E. faecalis in vitro, which the cell wall-anchored iron-responsive suggests that antibody-mediated hyper surface determinant B (IsdB)), in which vac- aggregation contributes to poorer disease Pneumonia cinated patients who developed an S. aureus outcomes and increased lethality in vacci- Infective infection were fivefold more likely to die nated rabbits. However, treatment of rabbits endocarditis than unvaccinated patients who developed with monovalent, AS‑specific antibody frag- an S. aureus infection4, took the field by ments (IgG Fabs), which had been shown + Sepsis surprise, and investigators have struggled to prevent aggregation of AS E. faecalis to make sense of these findings. in vitro, effectively decreased vegetation size, Osteomyelitis However, it is well-established that cell reduced bacterial recovery from vegetations surface virulence factors facilitate host colo- and increased survival when challenged with + 27 Menstrual toxic nization via a combination of cell attachment AS E. faecalis . shock syndrome and avoidance of the innate immune system. A similar effect was recently observed S. aureus uses a range of these virulence with S. aureus19. Rabbits were vaccinated Soft tissue factors (for example, fibronectin-binding with an S. aureus multivalent surface anti- infections proteins and clumping factors) to aggre- gen preparation that was enriched for Isds, gate, forming large bacterial clumps that and were challenged intravenously with a are protected by a capsule and ‘decorated’ sublethal dose of S. aureus MW2. Vacci- with host factors7,25,26. We hypothesize that nated rabbits succumbed to infection within antibodies that are directed against surface 6 hours of MW2 challenge, whereas unvac- Figure 1 | Diseases caused by Staphylococcus components further promote S. aureus cinated rabbits, albeit sick, survived to day Nature Reviews | Microbiology aureus. S. aureus causes life-threatening dis- aggregation. These large aggregates of bac- 4 (REF. 19). Hence, these studies suggest that eases, including pneumonia, infective endocar teria, antibodies and other host factors in E. faecalis and S. aureus share the property ditis, toxic shock syndrome and sepsis, as well as the blood may not be cleared by the host of increased disease severity in the pres- minor diseases such as soft tissue infections. and could become trapped in various tissues ence of antibodies that target their surface (particularly in the lungs, but also in skin, components. In our opinion, the increased the kidneys, the liver and joints) where they incidence of multi-organ failure and death and sepsis20, via massive immune system would fix complement, recruit granulocytes in the V710-vaccinated human cohort that activation and dysregulation, and the lethal- and other phagocytes and induce the release acquired S. aureus bacteraemia4 could be ity of these diseases is dependent on the of pro-inflammatory mediators, as well as explained by a similar mechanism. These effects of superantigens (SAgs), such as TSS potentially causing septic emboli. Should effects are quite dramatic and require further toxin 1 (TSST1), staphylococcal entero- this prove to be the case, then the expected investigation in S. aureus; however, mouse toxin B (SEB), SEC, and cytotoxins, such consequences are tissue damage, ischaemia models of S. aureus infection do not reproduce as α‑toxin, on the cardiovascular system and abscess formation, which would lead to these lethal effects. (reviewed in REF. 19). Physiologists recognize multi-organ failure and shock. Hence, in our that the cardiovascular physiology of mice opinion, targeting staphylococcal surface Mice do not mimic humans does not resemble that of humans21, and components does not seem to be a sound Resistance to lipopolysaccharide, cytotoxins in our opinion, this is a major drawback vaccine strategy. and SAgs. Mouse immune and cardiovascu- of mouse models of S. aureus infection. By Scientific evidence to support our lar systems respond differently to bacterial contrast, the cardiovascular physiology of hypothesis comes from published vac- toxins, compared with human and rabbit sys- rabbits closely resembles that of humans22,23. cination studies that were carried out in tems, which partly explains their resistance Rabbits and humans also respond simi- the Gram-positive pathogen Enterococcus to the lethal effects of lipopolysaccharide larly to SAgs and cytotoxins, whereas mice faecalis and from recent studies with (LPS; also known as endotoxin), SAgs and respond differently24. Although rabbits are S. aureus in the rabbit model of infective cytotoxins (TABLE 1). The lethal dose of LPS in more difficult to work with, owing to the endocarditis. It is important to consider that humans is approximately 0.01 μg per kg (REF. cost, the lack of inbred strains and greater S. aureus and enterococci both form aggre- 31), and the lethal dose in rabbits is approxi- government regulatory control, their gates and both cause infective endocarditis. mately 0.5 μg per kg (REF. 32). However, in immune and cardiovascular systems respond The enterococcal aggregation substance commonly used laboratory mice, the LPS to S. aureus infection in a manner that is (AS), as the name suggests, promotes the lethal dose is >80,000 μg per kg (REFS 33,34). more comparable with that of humans. formation of large bacterial aggregates and The lethal doses of cytotoxins, notably In this Opinion article, we discuss the mediates attachment to eukaryotic cells27,28. α‑toxin, in humans are unknown, but α‑toxin soundness of targeting S. aureus surface com- AS is a major contributor to E. faecalis is considered to be highly lethal when ponents, compared with secreted factors, as pathogenesis in rabbit models of infective administered intravenously, as evidenced in a vaccine strategy, as well as the use of mouse endocarditis and sepsis29,30. Vaccination with the Bundaberg, Australia, disaster in which models to study S. aureus pathogenesis and heat-killed or gentamicin-treated aggrega- 12 of 21 recipients of a diphtheria toxoid vaccine outcomes. tion substance-expressing (AS+) E. faecalis, vaccine developed fatal disease that was asso- followed by challenge with either AS+ or ciated with the production of α‑toxin by an Targeting the S. aureus surface AS− organisms, resulted in endocarditis S. aureus contaminant35. In rabbits (specifi- Most S. aureus vaccines or immunotherapies vegetations and increased lethality in AS+ cally, Dutch-belted rabbits), 0.005 μg per kg that have been tested or that are in develop- E. faecalis-infected rabbits27. AS‑specific of α‑toxin injected intravenously kills in ment target surface components. The failure antibodies were shown to promote the 24 hours and 0.5 μg per kg kills in 1 minute36.

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Box 1 | Immunization: mouse versus human Attenuated virulence. A comparison of the virulence of representative isolates of So far, two Staphylococcus aureus vaccines (StaphVAX (Nabi Biopharmaceuticals) and V710 S. aureus USA200, USA300 and USA400 (Merck)) and five passive immunization preparations (Pagibaximab (Biosynexus, Glaxo Smith Kline), clonal types has been carried out in animal tefibazumab (Aurexis; Inhibitex), Veronate (Inhibitex), Aurograb (Novartis) and AltaStaph (Nabi models of pneumonia, sepsis and infective Biopharmaceuticals)) have completed clinical trials. All seven vaccines targeted S. aureus surface components. endocarditis. The USA200 isolates colonize Pagibaximab is a humanized monoclonal antibody that is targeted against lipoteichoic acid (LTA). human mucosal surfaces and are frequently LTA is an important component of the Gram-positive cell wall and is essential for its integrity and associated with life-threatening infections, for bacterial survival84. Pagibaximab was tested in very low birthweight neonates for the such as post-influenza pneumonia, surgical prevention of staphylococcal sepsis85,86. In both studies, Pagibaximab showed a positive trend, but wound infections, infective endocarditis, the results were not conclusive. A larger study was recently completed, and Pagibaximab failed to sepsis and TSS40–43. USA200 isolates are also reduce staphylococcal sepsis in this population87. associated with almost all cases of menstrual StaphVAX is a bivalent vaccine that is composed of capsular polysaccharide 5 (CP5) and CP8 TSS, which develops in otherwise healthy 88 individually conjugated to recombinant exoprotein A (rETA) from Pseudomonas aeruginosa . females18. USA200 strains usually produce CP5 and CP8 are highly conserved and are produced by most pathogenic S. aureus strains89–92. CP5 α‑toxin at low levels (0.05–5.0 μg per ml)36,44 increased bacterial virulence in a mouse model of bacteraemia and arthritis, compared with a CP5‑deficient strain93. Vaccination of mice with CP5–rETA provided protection against a lethal and TSST1 at high levels (5–20 μg per ml challenge94. In humans, StaphVAX failed to reduce the incidence of S. aureus bacteraemia in in liquid culture and up to 16,000 μg per patients undergoing haemodialysis95,96. ml in biofilms) or α‑toxin, TSST1 and SEC, AltaStaph comprises pooled hyperimmune polyclonal antibodies from healthy individuals who which is also produced at high levels (up have been vaccinated with StaphVAX97. Treatment of mice with AltaStaph significantly reduced to 100 μg per ml in liquid culture)45. In the bacteraemia and deep tissue infections after sublethal challenge94. Treatment of rats with rabbit rabbit pneumonia model, USA200 isolates CP5–rETA-specific antibodies lowered the prevalence of infective endocarditis and decreased cause 90% lethality in 48 hours at doses of bacterial counts in vegetations, blood and kidneys98. In humans, AltaStaph failed to reduce the 6.5 × 108 colony-forming units (CFUs) per 97 incidence of S. aureus bacteraemia in low birthweight neonates or to improve the outcome of kg46. Furthermore, in a rabbit infective endo- patients with persistent bacteraemia99. carditis or sepsis model, USA200 isolates Tefibazumab (which comprises humanized monoclonal antibodies) and Veronate (which is a effectively cause lethal sepsis, with the LD pooled human hyperimmune preparation) target clumping factor A (ClfA). ClfA is a cell wall 50 protein that binds to fibrinogen and fibrin and promotes bacterial clumping in plasma and (dose lethal to 50% of animals tested) calcu- 8 adherence to blood clots. Mutational analysis provided evidence for the role of ClfA in S. aureus lated at 0.3–1.5 × 10 CFUs per kg (REF. 47). pathogenesis in a mouse model of septic arthritis and a rat model of indwelling catheter-infective By contrast, in the mouse pneumonia model, endocarditis100,101. Vaccination of mice with recombinant truncated ClfA led to less severe arthritis USA200 strains inoculated at a dose of after S. aureus challenge. In humans, tefibazumab failed to prevent S. aureus bacteraemia relapse, 1.0 × 1010 CFUs per kg fail to induce lethal- complications or death102; Veronate failed to reduce the incidence of late-onset sepsis in ity in a 48 hour time period and exhibit a premature neonates103. greatly reduced capacity to induce lethality Aurograb is a single-chain fragment of the immunoglobulin variable region that is specific for the in a sepsis model48. S. aureus ATP-binding cassette (ABC) transporter GrfA. Human sera from 26 patients with epidemic USA300 isolates are commonly methicillin-resistant S. aureus 15 (EMRSA‑15) septicaemia contained high antibody titres against three different S. aureus ABC transporter peptides104. In a mouse sepsis model, treatment with community-associated methicillin-resistant human antibodies to the individual peptides significantly reduced the recovery of EMRSA‑15 from S. aureus (CA‑MRSA) strains, which are the kidneys, liver and spleen105. Aurograb failed to show efficacy when tested as an add‑on therapy frequently associated with skin and soft tis- in patients with severe, deep-seated staphylococcal infections6,106. sue infections and necrotizing pneumonia49. V710 is a monovalent vaccine that is composed of the iron-responsive surface determinant B The USA300 LAC strain produces α‑toxin (IsdB), which is a cell wall-anchored protein that is conserved among diverse S. aureus strains and is at high levels (~150–500 μg per ml)50 and expressed during iron limitation4,107. The vaccine provided protection against nasal carriage in a the SAgs SE-like X (SEl‑X) (~200 ng per ml cotton rat model, increased survival in a mouse sepsis model and induced high-level antibody in liquid culture), SEl‑Q and SEl‑K at low 16,108,109 titres in mice . In humans, V710 failed to reduce the incidence of post-operative S. aureus levels (<30 ng per ml)20. In the rabbit pneu- bacteraemia and major chest wound infection, and it was associated with an increased incidence monia model, LAC inoculated at 6.5 × 108 of multi-organ dysfunction and increased mortality in members of the vaccinated cohort who developed S. aureus bacteraemia4,110. CFUs per kg induces 90% lethality 24 hours post-infection; whereas, in the mouse pneumonia model, 80–90% lethality is reported with an inoculum that is tenfold higher on By contrast, in BALB/c mice, the lethal mini-osmotic pumps causes 100% lethality24. a per-kilogram basis than that used for rab- dose of α‑toxin is >200,000 μg per kg (P.M.S, In mice, continuous exposure to TSST1 at bits51. Furthermore, an intravenous dose unpublished observations). 4,000,000 μg per kg in mini-osmotic pumps of 6.5 × 108 CFUs per kg in rabbits results The difference between mice, rabbits is not lethal24. To induce susceptibility to in 100% mortality in <18 hours, whereas a and humans in their susceptibility to SAgs is SAgs in mice, investigators have used potent five-times higher dose is required to obtain even more striking. Humans who have been sensitizing agents, such as the hepatotoxin comparable lethality (90%) in mice in injected intravenously with SEA at doses d‑galactosamine24; however, these mice 24 hours44,52. as low as 0.001 μg per kg develop fever and develop what seems to be fulminant liver The USA400 strain MW2 is a CA‑MRSA hypotension (which are symptoms of TSS)37. failure. This condition is not observed in that was isolated from a child with lethal In rabbits, intrapulmonary inoculation of humans or rabbits with TSS38,39. Therefore, necrotizing pneumonia. MW2 produces TSST1 or continuous exposure to TSST1 at standard mouse models have reduced moderate amounts of α‑toxin (~50 μg 0.05 μg per kg implanted subcutaneously in sensitivity to LPS, cytotoxins and SAgs. per ml)53 and encodes an array of SAgs,

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Table 1 | Comparison of specific characteristics of humans, mice and rabbits Animal Property Human Rabbit Mouse LPS lethality 0.013 μg per kg 500*–0.5‡ μg per kg >80,000 μg per kg α‑toxin lethality NA 0.005 μg per kg in 24 hours >200,000 μg per kg Superantigen lethality 0.0013 μg per kg 50*–0.05‡ μg per kg Not lethal at 4 × 106 μg per kg Similarity of cardiovascular physiology NA Similar to humans Not similar to humans to that of humans Similarity of fever response to that of NA Similar to humans Not similar to humans humans LPS, lipopolysaccharide; NA, not applicable. *Young adult (2–3 kg) rabbits. ‡8 month-old rabbits. including SEC and SEl‑X. SEC is the seems to be hypervirulent (as it produces on mouse models, and it may have made dominant SAg in MW2 — it is produced at α‑toxin at high levels, even though it makes the most important contribution to the ~100 μg per ml in liquid culture — and is SAgs at low levels). There are likely to be failure of S. aureus vaccine development. required for the development of necrotiz- many additional reasons for the differen- ing pneumonia and lethal sepsis in rabbits46. tial responses of mouse models compared Basis for divergent responses MW2 doses of 6.5 × 108 CFUs per kg are with rabbit models and humans. However, The divergent responses of mice compared 100% lethal in rabbits 48 hours after infec- it is clear that mouse resistance to SAgs with humans and rabbits result from at least tion. Strikingly, a tenfold increase in the and α‑toxin produces virulence outcomes three major differences. First, the mouse MW2 dose on a per-kilogram basis results in that are not consistent with those that are transcriptional response to inflammatory only 20% lethality in mice and 36% lethality observed in humans and rabbits. In our stimuli (such as LPS) differs considerably in rats in the same 48 hour period54,55. opinion, the reliance on mouse models from that of humans. Second, the effects of Overall, the mouse models seem to has led to confusion and has perpetuated cytokines on the vasculature and the central mostly reflect the effect of high levels of misconceptions in the staphylococcal field nervous system in response to LPS and SAgs α‑toxin production, as they completely lack of research, including our understanding are different in mice compared with humans. sensitivity to SAgs and to lower levels of of the virulence of strains relative to one Third, there are differences in the composi- α‑toxin (FIG. 2). Therefore, S. aureus strains another, the role of various staphylococcal tion of the gut and vaginal microbiota in such as MN8 seem to be avirulent in mice proteins in disease and the identification of mice, rabbits and humans. (as TSST1 has no effect and this strain staphylococcal molecules that have poten- produces low levels of α‑toxin), MW2 tial as human vaccine candidates. The iden- Transcriptional response: endotoxemia. It seems to have greatly reduced lethality tification of staphylococcal molecules that has been known since the early 1960s that (although it produces high levels of SEC have potential as vaccine candidates is the mice are highly resistant to the effects of and moderate levels of α‑toxin) and LAC most important consequence of the reliance Gram-negative LPS56. A key recent study by Seok et al. investigated this difference at a b the molecular level and showed that mouse 100 100 inflammatory responses to infectious agents LAC in general, and to LPS specifically, are sig- LAC MW2 MN8 21 80 80 nificantly different from those of humans . Gene expression profiling of C57BL/6J mice and humans during endotoxemia showed a 60 60 Newman low correlation between the human genes and mouse orthologues and vice versa. In 40 40 % Lethality

% Lethality this study, the human response to endotoxemia was threefold greater than that of 20 20 MW2 mice and was poorly predicted by the mouse model. A comparison of the human and MN8 mouse pathways that are activated or sup- 0 0 pressed during endotoxemia showed that -toxin -toxin α α humans activate Fcγ receptor-mediated phagocytosis in macrophages and monocytes, interleukin-10 (IL‑10) signalling, integrin Figure 2 | Lethality induced by various Staphylococcus aureus strains as a function of the signalling, B cell receptor signalling and animal model used (mouse versus rabbit pneumonia model) andNature their Reviewslevel of |α Microbiology‑toxin pro‑ duction. a | Inoculation of the S. aureus strains LAC, MW2 and MN8 at 6.5 × 108 CFUs per kg in the Toll-like receptor signalling. In mice, these rabbit pneumonia model shows that they are equally virulent 48 hours after infection. b | Inoculation signalling pathways are poorly activated, of the S. aureus strains LAC, Newman, MW2 and MN8 at 6.5–10 × 109 CFUs per kg in the mouse pneu- particularly the Toll-like receptor signalling monia model shows that virulence correlates with the level of α-toxin production. Data taken from pathway21. Most of the signalling pathways REFS 46,48,50,51. that are suppressed during endotoxemia in

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humans are activated in mice, particularly the concentrations is highly pro-inflammatory35) on the immune system. On this basis, one calcium-induced T cell apoptosis pathway21. have major roles in pneumonia, sepsis and would expect that mice could predict disease Similar results were obtained by analysis of infective endocarditis, and their effects are in non-human primates, whereas rabbits the genomic response of humans and mice mediated by the induction of acute inflam- predict disease in humans. to other severe acute inflammatory diseases, mation19,50,54. Together, these studies clearly including sepsis and acute respiratory dis- indicate the differential production of pro- Vaccination is possible tress syndrome. This study provides evidence inflammatory cytokines by mice compared Targeting secreted virulence factors. Vac- that, at the molecular level, mouse models with rabbits and humans in response to cination with purified SAgs or SAg toxoids poorly reflect the molecular mechanisms of either LPS or SAg stimulation. alone or in combination with cytotoxins has human inflammatory diseases. proven to be a successful strategy to protect Gut and vaginal microbiota. Interestingly, rabbits against lethal doses of USA100, Cytokine response: fever and hypotension. the susceptibility of humans and rabbits to USA200, USA300 and USA400 S. aureus SAgs cause fevers and life-threatening SAgs correlates with the presence of Gram- clonal types and provides sterilizing immu- illnesses in humans, owing to overstimula- negative bacteria that produce endotoxin nity82. Vaccination with TSST1, SEC or SEB tion of the immune system, which leads shock-inducing LPS69,70 (that is, E. coli in protects rabbits from lethal challenge with to the massive production of IL‑1β, IL‑6, humans and Pasteurella multocida in rab- CA‑MRSA TSST1+ USA200, SEC+ USA400 interferon-γ (IFNγ), tumour necrosis bits) in the intestinal and vaginal tracts71–74. or SEB+ USA400 strains, respectively46. factor (TNF) and lymphotoxin-α57,58 (which Rodents75 and non-human primates76 have Three different TSST1 toxoids produce is commonly known as a cytokine storm). fewer of these organisms. Young adult rab- TSST1-neutralizing antibodies at high levels Cytokines, such as IL‑1β and IL‑6, are bits (~3 months old) succumb to TSST1 at and protect rabbits from a TSST1 lethal chal- endogenous pyrogens (most of the endog- doses of 50 μg per kg (REF. 39) and to LPS lenge in an LPS enhancement model. A tri- (REF. 32) enous pyrogens that cause fevers in humans at 535 μg per kg . At 8 months old, valent vaccine that is composed of TSST1G31S are primarily produced by immune cells) rabbits are colonized at a greater density by (or TSST1S32P), SEC and α-toxinH35L provides and subsequently initiate a fever cascade that P. multocida and become 1,000 times more complete protection against a lethal chal- + + begins with the production of prostaglan- susceptible to LPS (LD100 0.5 μg per kg); lenge with S. aureus MNPE (TSST1 , SEC din E2 by the hypothalamus59,60. It has been concomitantly, the susceptibility of rab- and α‑toxinHI) in the rabbit pneumonia known for decades that BALB/c mice do not bits to the lethal effect of TSST1 increases model47. Furthermore, a pentavalent vaccine respond to LPS with fever and instead show 1,000‑fold (LD100 0.05 μg per kg). Enhance- that contains TSST1G31S (or TSST1S32P), SEC, a dose-dependent reduction in body tem- ment of LPS shock by TSST1 was shown in α-toxinH35L, β‑toxin and γ‑toxin protects rab- perature59, whereas even minute doses young adult rabbits that were administered bits from infective endocarditis and lethal of LPS cause high fevers both in humans TSST1 at increasing concentrations before sepsis when challenged with a lethal dose and in rabbits32,59. High fevers, such as those LPS treatment32. TSST1 increases the sus- of S. aureus MNPE47. Studies in rabbits thus seen in patients with TSS and a variant ceptibility of rabbits to LPS lethality by up provide evidence that a multivalent vaccine form of TSS known as extreme pyrexia to 106-fold32; this effect has been observed targeting secreted staphylococcal virulence syndrome61, can participate in lethality. with all SAgs that have been tested32,77. The factors that are produced by nearly all The major pathway by which micro- SAg-induced increase in LPS lethality results S. aureus isolates protects against life- organisms that express LPS or SAgs cause from impaired LPS clearance by the liver78, threatening staphylococcal diseases82. These fevers depends on interactions of these toxic possibly via direct liver cytotoxicity and studies suggest that the neutralization of molecules with immune cells via Toll-like the synergistic effects of SAgs and LPS crucial SAgs and cytolysins effectively inca- receptors62,63 or SAg cross-bridging of on the production of TNF33. Endotoxemia pacitates S. aureus such that phagocytic T cell receptors and major histocompatibil- occurs in humans during TSS and in rabbits elimination of the organism occurs, similarly ity complex class II (MHC II) molecules19, that have been injected with TSST1. This to what would be expected for coagulase- either peripherally or within the central has led to the hypothesis that animals with negative staphylococci (coagulase-negative nervous system59. As mentioned above, mice a gut and vaginal microbiota that includes staphylococci produce fewer virulence do not activate Toll-like receptor signalling Gram-negative bacteria expressing LPS that factors than other S. aureus strains and are in response to LPS21. It is also known that is capable of inducing endotoxin shock are eliminated by innate immune mechanisms, mouse T cells show decreased responses to susceptible to SAgs plus LPS79,80, whereas such as the alternative complement cascade SAgs, whereas both human and rabbit T cells animals such as rodents and non-human and neutrophil opsonization). (and antigen-presenting cells) respond primates, in which Gram-negative bacteria comparably to SAgs33,56,64. The lack of suscep- that express toxic LPS are rarely detected, are Conclusions tibility of mice to SAgs has led to attempts resistant76,81. Although synergism between The virulence of many bacterial pathogens to humanize mice, particularly for MHC II TSST1 and LPS does occur in mice and that cause major human diseases is often (REFS 65–67). However, mice that are human- rhesus macaques that have been injected multifactorial. The diseases that these organ- ized for MHC II still do not respond to SAgs with the toxins, the susceptibility to the isms cause are becoming increasingly dif- with all of the signs of TSS68. SAg–LPS combination never reaches that of ficult to treat and require new thinking in Furthermore, massive production of TNF rabbits33. However, it supports the idea that vaccination strategies. So far, researchers and lymphotoxin-α has major effects on the the resistance or susceptibility of mice and have typically used rodents — particularly human and rabbit cardiovascular systems, monkeys versus rabbits and humans results mouse models — to study human diseases which cause capillary leak, hypotension not only from differences in cardiovascular that are caused by these bacterial pathogens, and shock. SAgs and α‑toxin (which, at physiology but also from the presence of not because mouse infections mimic human high concentrations is cytotoxic but at low endogenous LPS and combined toxin effects illnesses, but rather owing to costs, the

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