Immunization with Staphylococcus aureus iron regulated surface determinant B (IsdB) confers protection via Th17/IL17 pathway in a murine sepsis model

We have previously shown that IsdB, a conserved protein expressed by Staphylococcus aureus, induces a robust antibody response which correlates with protection in a murine challenge model. Here we investigate the role of cellular immunity in IsdB mediated protection using lymphocyte deficient SCID mice. As opposed to WT CB-17 mice the CB-17 SCID mice were not protected against a lethal challenge of S. aureus after active and passive immunizations with IsdB. Adoptive transfer of in vitro isolated lymphocyte subsets revealed that reconstituting mice with IsdB specific CD3+ or CD4+ T-cells conferred antigen specific protection while CD8 (+) T-cells, CD19 (+) B-cells and plasma cells (CD138 (high) B220 (int) CD19 (lo) ) alone were not protective. A combination of CD3 (+) T-cells plus CD19 (+) B-cells conferred protection in CB-17 SCID mice, whereas bovine serum albumin (BSA) immune lymphocytes did not confer protection. Active immunization experiments indicated that IsdB immunized Jh mice (B-cell deficient) were protected against lethal challenge, while nude (T-cell deficient) mice were not. In vitro assays indicated that isolated IsdB specific splenocytes from immunized mice produced abundant IL-17A, much less IFN-γ and no detectable IL-4. IL-23 deficient mice were not protected from a lethal challenge by IsdB vaccination, pointing to a critical role for CD4 (+) Th17 in IsdB-mediated vaccination. Neutralizing IL-17A, but not IL-22 in vivo significantly increased mortality in IsdB immunized mice; whereas, neutralizing IFN-γ did not alter IsdB-mediated protection. These findings suggest that IL-17A producing Th17 cells play an essential role in IsdB vaccine-mediated defense against invasive S. aureus infection in mice.     

Selection and Characterization of Murine Monoclonal Antibodies to Staphylococcus aureus Iron-Regulated Surface Determinant B with Functional Activity In Vitro and In Vivo

In an effort to characterize important epitopes of Staphylococcus aureus iron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed by S. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (≥50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (<50% killing in the in vitro assay) and was protective in one lethal challenge model. Neither MAb 13G11 nor MAb 1G3 exhibited OPK activity in vitro or was active in a lethal challenge model. The data suggest that several nonoverlapping epitopes are recognized by the IsdB-specific MAbs, but not all of these epitopes induce protective antibodies.The search for an efficacious vaccine or immunoglobulin (Ig) preparation to prevent invasive disease due to Staphylococcus aureus has proceeded for well over a decade (17, 22, 23, 26). However, with the recent dramatic increase in cases of methicillin-resistant S. aureus (12, 13), this effort has become more urgent. S. aureus has increasingly become a pathogen of great clinical concern over the last 3 decades (7, 12, 13, 16). The importance of S. aureus vaccine development to aid in the treatment of hospitalized individuals, as well as to reduce the economic burden on the health care system, is well established.Although extensively investigated, native protective immunity against S. aureus is poorly understood. Acute infection with S. aureus does not prevent reinfection with this bacterium (17). Preclinical and clinical data indicate that immunization with intact whole bacteria induces high immune titers to staphylococcus but does not confer protection from S. aureus disease (10, 17). Clearance of S. aureus is thought to be dependent upon antibody and complement-mediated uptake and killing by neutrophils, known as opsonophagocytic killing (OPK) (6, 11, 18, 19, 24, 33). S. aureus is a part of the normal bacterial flora of humans. As such, all individuals have antibodies to S. aureus, perhaps due to repeated subclinical infections or to carriage of the bacteria on mucosal surfaces in the nares, rectum, vagina, etc. Humans, as well as many mammals, have preexisting antibody titers to iron-regulated surface determinant B (IsdB) (15), but it is unknown whether these preexisting titers offer protection. Confirmed protection by natural antibodies to individual S. aureus antigens has been demonstrated for a single antigen, staphylococcal toxic-shock toxin 1 (17). Other antibodies to individual antigens have been proposed to correlate with natural protection, such as an immunodominant ABC transporter described by Burnie and coauthors (3) and antigens described by Clarke and coauthors (4). Several polysaccharide and protein antigens have been tested as vaccine candidates for S. aureus (reviewed in reference 26; 1, 29). Active immunization with these vaccine candidates leads to high titers of IgG which may confer protection from challenge (26). Kuklin et al. demonstrated that immunization with IsdB formulated on amorphous aluminum hydroxyphosphate sulfate adjuvant increased murine antibody titers by up to 20-fold and nonhuman primate titers by fourfold. Importantly, increased antibody titers correlated with enhanced survival in a murine lethal challenge model (15). IsdB is an antigen expressed on the cell surface of S. aureus in environments with limited iron, with a molecular mass of approximately 72 kDa. Its function is to capture and import heme iron from hemoglobin (20). Since little is known about the protective immune response to IsdB, the current study was undertaken to investigate IsdB-specific antibodies which may confer protection.In an effort to further our understanding of potentially protective IsdB epitopes, IsdB-specific murine monoclonal antibodies (MAbs) were selected and characterized. An understanding of the protective epitopes of IsdB will inform decisions on the type of antibody response necessary for protection from S. aureus challenge. Epitope-specific and protective MAbs are also important as reagents to ensure the maintenance of appropriate structural integrity of IsdB antigen during vaccine formulation. The IsdB MAbs were grouped based on recognition of similar epitope regions. The MAbs fell into three or four groups depending on the method of analysis. Several nonoverlapping epitopes were delineated by these MAbs, and two were important for in vivo protection in murine challenge models.     

Interleukin-12 as an adjuvant for an antischistosome vaccine consisting of adult worm antigens: protection of rats from cercarial challenge

Our group previously demonstrated that a detergent extract (fraction S3) prepared from immature (4-week) Schistosoma mansoni parasites can induce partial, serum-transferable immunity to challenge infection in rats when administered as an alum precipitate. In the present study, we examined whether S3 prepared from adult (7-week) worms could similarly induce protection and whether immunity could be positively influenced by treatment with interleukin-12 (IL-12). IL-12 coadministered to Fischer rats and C57BL/6 mice at the time of S3 vaccination altered the prechallenge kinetics of S3-specific antibody titers in both species, ultimately leading to a stable enhancement of titers (relative to those in animals vaccinated without IL-12) in mice but not rats. Immunoblot analysis of prechallenge immune sera demonstrated that IL-12 treatment was associated with changes in the S3 antigen recognition profile in each species. Isotyping of specific antibodies in S3- plus IL-12-vaccinated mice prior to challenge infection revealed a moderate elevation in immunoglobulin G1 (IgG1) responses, strongly enhanced IgG2a and IgG2b responses, as well as diminished total serum IgE responses compared to those in mice given S3 only. In vaccinated rats, IL-12 profoundly suppressed specific IgG1 and enhanced IgG2b responses but did not affect IgG2a responses. S3- plus IL-12-vaccinated rats also produced less total IgE upon challenge infection. Enumeration of worm burdens revealed that vaccination with S3 plus IL-12 conferred 50% protection from cercarial challenge to rats, whereas rats given S3 only were not protected; mice were not protected by S3 vaccination regardless of IL-12 coadministration. The protection observed in S3- plus IL-12-vaccinated rats could not be transferred with serum, suggesting participation of an activated cellular component in the expression of immunity.     

耐甲氧西林金黄色葡萄球菌IsdB活性片段的克隆、表达及抗原免疫保护性初步研究

目的探讨耐甲氧西林金黄色葡萄球菌(MRSA)抗原IsdB活性片段(IsdB2)免疫保护作用。方法利用生物信息学技术预测分析出IsdB活性片段(IsdB2),PCR扩增编码IsdB2的基因片段,亚克隆至GST标签融合表达的原核表达载体pGEX-6P-2中,将载体转化入大肠杆菌XL-1 blue,通过IPTG诱导表达IsdB2/GST融合蛋白,利用GST亲和层析初步纯化获取IsdB2蛋白。用IsdB2蛋白抗原辅以氢氧化铝佐剂对小鼠进行免疫实验,统计小鼠存活率对IsdB2抗原的免疫性进行初步研究。结果重组质粒经过BamHⅠ和NotⅠ双酶切鉴定、核酸序列测定和IPTG诱导表达IsdB2/GST及酶切获取IsdB2蛋白的SDS-PAGE分析表明,IsdB2蛋白相对分子质量大小约72 000,GST标签相对分子质量大小约26 000,与预期相符合。用IsdB2蛋白对小鼠进行3次疫苗免疫实验,IsdB2对小鼠的保护率分别为84.6%、50%和60%。结论成功构建重组表达载体pGEX-6P-2-IsdB2,利用大肠杆菌表达系统、GST亲和层析和酶切方法获得IsdB2蛋白抗原,通过3次动物疫苗免疫实验结果表明IsdB2具有免疫保护性,为研制新型有效的MRSA疫苗奠定实验基础。     

Klebsiella pneumoniae type 3 fimbria-mediated immunity to infection in the murine model of respiratory disease

Type 3 fimbriae are expressed by most strains of Klebsiella pneumoniae and facilitate adherence to the basement membrane of human respiratory tissues. The ability of these appendages to stimulate a protective immune response in vivo has not been investigated. A murine model of acute pneumonia was used to determine whether the production of type 3 fimbria-specific antibodies correlated with protection against infection by K. pneumoniae. Purified fimbriae from several strains were used to immunize mice prior to challenge with a virulent strain. The immunized mice produced high titers of specific antibody and this was associated with protection against challenge with a low dose of bacteria that was lethal in unimmunized animals. However, challenge with a high number of bacteria resulted in no protection against infection.     

Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A

The Staphylococcus aureus MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein clumping factor A (ClfA) has been shown to be a critical virulence factor in several experimental models of infection. This report describes the generation, characterization, and in vivo evaluation of a murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis revealed that MAb 12-9 recognized ClfA protein expressed by all of the clinical S. aureus strains obtained from a variety of sources. In assays measuring whole-cell S. aureus binding to human fibrinogen, MAb 12-9 inhibited S. aureus binding by over 90% and displaced up to 35% of the previously adherent S. aureus bacteria. Furthermore, a single infusion of MAb 12-9 was protective against an intravenous challenge with a methicillin-resistant strain of S. aureus in a murine sepsis model (P < 0.0001). These data suggest that anti-ClfA MAb 12-9 should be further investigated as a novel immunotherapy for the treatment and prevention of life-threatening S. aureus infections.     

Protective efficacy of protein A-specific antibody against bacteremic infection due to Staphylococcus aureus in an infant rat model.

Staphylococcal protein A (SpA) is a potent antiphagocytic component of the cell wall of most pathogenic Staphylococcus aureus strains. We studied the in vitro opsonophagocytic and in vivo protective activities of rabbit immunoglobulin G (IgG) antibody to purified SpA obtained from two unencapsulated S. aureus strains (Cowan I and 17A). Postimmune serum contained high titers of specific IgG to SpA, as measured by a modified enzyme-linked immunosorbent assay that blocked nonspecific binding of IgG to SpA. In vitro, both S. aureus strains were efficiently phagocytosed and killed by polymorphonuclear leukocytes in the presence of nonimmune sera and complement. With one strain (Cowan I), opsonophagocytosis was significantly enhanced in the presence of SpA antibody, but with the other strain (17A), killing was significantly decreased with immune serum. We then evaluated the potential protective benefit of SpA antibody in preventing S. aureus bacteremia in infant rats. Two-day-old rats received saline or various doses of SpA antiserum and were challenged subcutaneously 1 day later, but even the highest levels of antibody did not significantly reduce mortality, bacteremia or metastatic infection to lungs or liver (frequency or magnitude). This lack of protective efficacy was not related to a failure of SpA F(ab')2 to bind to cell surface-exposed epitopes, since F(ab')2 fragments prepared from hyperimmune serum bound avidly to the whole organism in an enzyme-linked immunosorbent assay.     

Influence of Staphylococcus aureus antibody on experimental endocarditis in rabbits.

To evaluate the potential protective benefit of antibody to whole cells of Staphylococcal aureus for the prevention of endocarditis, the rabbit endocarditis model was used. Methicillin-sensitive (17A) and methicillin-resistant (173) S. aureus strains were evaluated in rabbits with or without indwelling intracardiac catheters. All immunized rabbits developed significant homologous agglutinating antibody titers (the mean reciprocal titers were 15,300 to strain 17A and 1,150 to strain 173). After challenge, virtually no significant differences were observed between immunized and unimmunized animals with respect to (i) incidence of endocarditis, (ii) concentration of bacteria in infected vegetations, (iii) incidence of metastatic renal abscesses, or (iv) concentrations of bacteria in infected kidneys. The clearance of homologous S. aureus strains from blood cultures was similar for immunized and unimmunized animals at 10 to 90 min after intravenous challenge. In vivo adherence of homologous S. aureus strains to aortic valves and vegetations was similar in immunized and unimmunized animals when evaluated at 30 and 90 min postchallenge. Even without catheterization, the incidence of bacteremia and renal abscesses was the same in immunized and unimmunized rabbits. Whole-cell-induced S. aureus antibody did not prevent or modify any stage in the development of endocarditis in rabbits.     

Toxicity of recombinant toxic shock syndrome toxin 1 and mutant toxins produced by Staphylococcus aureus in a rabbit infection model of toxic shock syndrome.

Menstrually associated toxic shock syndrome (TSS) is attributed primarily to the effects of staphylococcal exotoxin toxic shock syndrome toxin 1 (TSST-1). A region of the 194-amino-acid toxin spanning residues 115 through 144 constitutes a biologically active site. Several point mutations in the TSST-1 gene in that region result in gene products with reduced mitogenic activity for murine T cells. In this study we evaluated the toxicity of recombinant TSST-1 and several mutants of TSST-1 made by transformed Staphylococcus aureus during in vivo growth in a rabbit infection model of TSS. The toxicities of the transformed strains of S. aureus for rabbits correlated with the mitogenic activities of the recombinant toxins. An isolate originally obtained from a patient with a confirmed case of TSS (S. aureus 587) implanted in a subcutaneous chamber served as a positive control. TSST-1 produced in vivo led to lethal shock within 48 h, and a TSST-1-neutralizing antibody (monoclonal antibody 8-5-7) administered to rabbits challenged with S. aureus 587 prevented fatal illness. Rabbits infected with transformed S. aureus RN4220 expressing wild-type toxin (p17) or mutant toxins retaining mitogenic activity for T cells succumbed within a similar time frame. Blood chemistries of samples obtained from infected animals before death indicated abnormalities in renal and hepatic functions similar to those induced by parenteral injection of purified staphylococcal TSST-1. Mutant toxin 135 (histidine modified to alanine at residue 135) possessed only 5 to 10% of the mitogenic activity of wild-type toxin. Rabbits challenged with transformed S. aureus RN4220 expressing mutant toxin 135 exhibited only mild transient illness. Mutant toxin 135 retained reactivity with monoclonal antibody 8-5-7 and by several criteria was conformationally intact. Toxin from a double mutant, 141.144, with alanine substitutions at residues 141 (histidine) and 144 (tyrosine), also was devoid of mitogenic activity. In this case, antibody recognition was lost. Mutant toxins 115 and 141 were found to possess approximately half-maximal mitogenic activity. Rabbits challenged with S. aureus RN4220 expressing either 115 or 141 toxin succumbed to lethal shock. We conclude that the ability of TSST-1 to activate murine T cells in vitro and its expression of toxicity leading to lethal shock in rabbits are related phenomena.     

In vivo administration of monoclonal antibody to the NK 1.1 antigen of natural killer cells: effect on acute murine cytomegalovirus infection

Monoclonal antibody to the NK 1.1 antigen, found on the natural killer cells of a number of strains of mice, specifically suppresses NK cell function when given in vivo. Using this monoclonal antibody, we have examined the effects of specific suppression of natural killer (NK) cells in vivo on acute murine cytomegalovirus (MCMV) infection in C57BL/10ScN mice. Administration of antibody to NK 1.1 substantially lowered the resistance of C57BL/10ScN mice to lethal virus challenge. In addition, antibody administration prior to intraperitoneal infection significantly increased MCMV replication in salivary glands, lungs, and spleens. In C3H/HeN mice, a strain that lacks the NK 1.1 antigen, antibody to NK 1.1 had no effect on virus replication or lethal infection. Thus, in vivo administration of monoclonal antibody to NK 1.1 alters the course of acute MCMV infection. These findings further substantiate the role of NK cells in defense against acute MCMV infection.     

A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.     

Nonspecific resistance to Escherichia coli in mice.

Nonspecific cell-mediated immunity to a relatively virulent strain of Escherichia coli was studied in mice infected with Staphylococcus aureus and elicited with specific antigens. The infected and elicited mice were protected against as intraperitoneal challenge by E. coli for an observation period of 7 days, whereas normal mice, given the same number of bacteria, died within 18 to 24 h. However, the amount of time elapsing between elicitation and challenge greatly affected the rate of protection. Little or no protection was observed in mice injected with S. aureus but not elicited or in mice injected with staphylococcal antigens but not infected with staphylococci.     

Role of neutrophil leukocytes in cutaneous infection caused by Staphylococcus aureus

Despite the high prevalence of cutaneous infections, little is known about the role of host immune responsiveness during Staphylococcus aureus dermatitis. We have recently described a murine model of infectious dermatitis induced by superantigen-producing S. aureus. To assess the role of neutrophils in staphylococcal dermatitis, mice were given granulocyte-depleting monoclonal antibody prior to and on several occasions following intracutaneous inoculation with staphylococci. The granulocyte-depleted mice that had been intradermally inoculated with S. aureus developed crusted ulcerations which tended not to heal, whereas animals injected with control monoclonal antibody displayed only minor and transient skin lesions. The finding of severe ulcerations in neutropenic mice correlated with a significantly higher burden of bacteria in the blood and skin during the early phase of the infection. Importantly, while mice with an intact granulocyte population showed only limited skin infection, bacteremia occurred in the great majority of the neutrophil-depleted animals. As a consequence, the latter individuals exhibited significantly increased levels of the proinflammatory cytokine interleukin-6 and specific antibodies to staphylococcal cell wall components and toxic shock syndrome toxin-1 in the serum. Our data point to a crucial protective role of granulocytes in S. aureus dermatitis.     

No priming of the immune response in newborn brown norway rats dosed with ovalbumin in the mouth

BACKGROUND: Other researchers have reported that the specific immune response to subsequent antigen challenge is primed in newborn mice or rats dosed orally by gavage. We wanted to investigate if priming of a subsequent specific IgE response could be achieved by dosing newborn rats orally with ovalbumin and if this method could be used in an animal model for food allergy. METHODS: Newborn Brown Norway rats were dosed with ovalbumin in the mouth (100 microg or 6 mg). As young adults, the animals were dosed by gavage for 35 days with 1 mg ovalbumin/day or once intraperitoneally with 100 microg. Control groups were dosed by gavage or intraperitoneally but not as neonates. Additionally, young adult rats were dosed with 1 mg ovalbumin/day in the mouth for 35 days. Sera from individual animals were analysed for specific IgE and specific IgG. RESULTS: In all experiments with neonatal rats the specific IgE and IgG responses were decreased compared to the control groups, however, not always reaching statistical significance. A statistical significant decrease in the specific immune response was found in young adult rats dosed in the mouth as compared to by gavage. CONCLUSIONS: Dosing Brown Norway rats with ovalbumin in the mouth as neonates do not prime the specific immune response. The decrease in immune response found in our experiments when dosing newborn animals in the mouth in opposition to the priming seen by others when dosing by intragastric intubation may be explained by a dissimilar antigen presentation when dosing includes both oral mucosa and gut.     

Host defenses in experimental scrub typhus: role of cellular immunity in heterologous protection.

The relative contributions of cellular and humoral immunity in scrub typhus infections were studied in inbred mice employing paired strains of Rickettsia tsutsugamushi differing in virulence. An infectious dose (100 MID50) of the less virulent Gilliam strain resulted in heterologous immune protection against an otherwise lethal challenge (1,000 MLD50) of the virulent Karp strain. Partial heterologous protection against lethal Karp challenge was observed in animals preimmunized with the Gilliam strain as early as 3 days prior to challenge, whereas complete protection against illness and death existed in animals immunized at least 7 days prior to challenge. In the heterologous protection provided by prior Gilliam infection, the role of humoral immunity was not of primary importance for the following reasons: (i) significant levels of complement-fixing antibody against R. tsutsugamushi were not detectable until long after animals were solidly immune; (ii) antibody eventually appearing after Gilliam immunization exhibited a consistently low complement-fixing titer against the immunizing homologous (Gilliam) strain and contained no detectable activity against the heterologous challenge (Karp) strain; and (iii) passive transfer of large quantities of serum from Gilliam immune mice, themselves immune to Karp challenge, failed to protect recipients against a similar challenge. However, protection was afforded by the passive transfer of serum containing antibody against Karp, suggesting a major role for antibody in protection against homologous infection. This heterologous challenge system was particularly useful because it minimized the role of humoral immunity, at least early in the course of infection, and allowed a definitive examination of the cellular response. Cell-mediated immunity played a major role in the heterologous protection observed after Gilliam immunization. This was evidenced by the significant protection against Karp challenge afforded by the passive transfer of spleen cells from animals immunized with Gilliam 7 to 63 days previously. Of the immune spleen cells, only those which were nonadherent, presumably lymphocytes, were capable of transferring passive heterologous protection. This protective effect of nonadherent cells could be ablated by depleting the cell population of thymus-derived or T cells with anti-theta serum and complement prior to transfer but not by use of anti-immunoglobulin serum and complement, which selectively removes bone marrow-derived or B cells. These results suggested that the cell in immune spleens capable of conferring heterologous protection was a T lymphocyte.     

Evidence for macrophage-mediated protection against lethal Candida albicans infection

Systemic infection of mice with a Candida albicans strain (PCA-2) incapable of yeast-mycelial conversion conferred protection against a subsequent intravenous challenge with the pathogenic strain of the parent organism, strain CA-6. Protection was nonspecific since it was also detected upon challenge of mice with Staphylococcus aureus. Moreover, the PCA-2 organisms had to be viable, their effects being most evident when they were given intravenously at a dose of 10(6) cells 7 to 14 days prior to microbial challenge. Thus, all mice pretreated with PCA-2 and challenged 14 days later with viable CA-6 cells lived through a 60-day observation period, whereas all control mice not treated with PCA-2 died within 3 days. In an attempt to correlate the immunostimulatory effects observed in vivo with possible modifications in in vitro functions, it was found that administration of PCA-2 was accompanied by an increase in the number of peripheral blood polymorphonuclear cells and by the activation in the spleen of cells with highly candidacidal activity in vitro. Moreover, the adoptive transfer of plastic-adherent cells from PCA-2-infected mice into histocompatible recipients conferred considerable protection against subsequent CA-6 challenge.     

Protection of C3H/HeJ mice from lethal Salmonella typhimurium LT2 infection by immunization with lipopolysaccharide-lipid A-associated protein complexes.

C3H/HeJ mice were immunized intraperitoneally (i.p.) with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes or with purified protein-free LPS prior to lethal i.p. or intravenous Salmonella typhimurium LT2 challenge. Our results demonstrated that these Salmonella-hypersusceptible mice can be effectively protected against 1,000 100% lethal doses of S. typhimurium LT2 (i.e., 1,000 viable bacteria) administered by intravenous challenge when previously immunized with LAP-LPS complexes. In contrast to these results, immunization with LPS afforded markedly less protection regardless of the route of challenge, thus suggesting that the LAP portion of LAP-LPS complexes may be necessary for inducing protection against Salmonella infections. For most experiments, antigens were emulsified in complete Freund adjuvant (CFA); however, the CFA portion of the vaccine was suggested not to be an essential component for the induction of immunity to Salmonella infections, since equivalent levels of protection were obtained when it was omitted from the vaccine. The induction of immunity to murine salmonellosis by prior immunization with CFA-LAP-LPS was demonstrated not to be a transient phenomenon, since C3H/HeJ mice were still protected against lethal S. typhimurium LT2 challenge as late as 225 days postimmunization.     

Molecular basis for preferential protective efficacy of antibodies directed to the poorly acetylated form of staphylococcal poly-N-acetyl-beta-(1-6)-glucosamine

Poly-N-acetyl-glucosamine (PNAG) is a staphylococcal surface polysaccharide influencing biofilm formation that is also under investigation for its vaccine potential. Antibodies that bind to PNAG with either low (<15%) or high (>90%) levels of acetate are superior at opsonic and protective activity compared with antibodies that bind to PNAG with only high levels (>70%) of acetate. PNAG is synthesized by four proteins encoded within the intercellular adhesin (ica) locus icaADBC. In Staphylococcus epidermidis, icaB encodes a deacetylase needed for the surface retention of PNAG and optimal biofilm formation. In this study, we confirmed that icaB plays a similar role in Staphylococcus aureus and found that an icaB mutant of S. aureus expressed significantly less surface-associated PNAG, was highly susceptible to antibody-independent opsonic killing that could not be enhanced with antibody raised against deacetylated PNAG (dPNAG), and had reduced survival capacity in a murine model of bacteremia. In contrast, an icaB-overexpressing strain produced primarily surface-associated PNAG, was more susceptible to opsonophagocytosis with antibody to dPNAG, and had increased survival in a murine bacteremia model. The highly acetylated secreted PNAG was more effective at blocking opsonic killing mediated by a human monoclonal antibody (mAb) to native PNAG than it was at blocking killing mediated by a human mAb to dPNAG, which by itself was a more effective opsonin. Retention of dPNAG on the surface of S. aureus is key to increased survival during bacteremia and also provides a molecular mechanism explaining the superior opsonic and protective activity of antibody to dPNAG.     

Dual role for macrophages in vivo in pathogenesis and control of murine Salmonella enterica var. Typhimurium infections

Salmonella spp. are regarded as facultative intracellular bacterial pathogens which are found inside macrophages (Mphi) after i. v. infection. It is generally assumed that Mphi restrict the replication of the bacteria during infection. In this study we examined the in vivo activities of Mphi during experimental S. typhimurium infections, using a selective liposome-based Mphi elimination technique. Unexpectedly, elimination of Mphi prior to infection with virulent S. typhimurium decreased morbidity and mortality, suggesting that Mphi mediate the pathology caused by S. typhimurium. Removal of Mphi) during vaccination with attenuated S. typhimurium did not affect protection against challenge with virulent S. typhimurium, suggesting that Mphi are not required for the induction of protective immunity and that other cells must function as antigen-presenting cell to elicit T cell-mediated protection. However, Mphi appeared to be important effectors of protection against challenge infection since elimination of Mphi from vaccinated mice prior to challenge infection with virulent S. typhimurium significantly decreased protection. These results enhance our understanding of the control of S. typhimurium growth in vivo, and moreover suggest that Mphi play a major role in the pathology of virulent S. typhimurium infections. As such, these cells may present a novel target for therapeutic intervention.