Immunodominance of Epitopes and Protective Efficacy of HI Antigen Are Differentially Altered Using Different Adjuvants in a Mouse Model of Staphylococcus aureus Bacteremia.

HI, a fusion protein that consists of the alpha-toxin (Hla) and the N2 domain of iron surface determinant B (IsdB), is one of the antigens in the previously reported S. aureus vaccine rFSAV and has already entered phase II clinical trials. Previous studies revealed that HI is highly immunogenic in both mice and healthy volunteers, and the humoral immune response plays key roles in HI-mediated protection. In this study, we further investigated the protective efficacy of immunization with HI plus four different adjuvants in a mouse bacteremia model. Results showed that HI-mediated protection was altered in response to different adjuvants. Using antisera from immunized mice, we identified seven B-cell immunodominant epitopes on Hla and IsdB, including 6 novel epitopes (Hla1-18, Hla84-101, Hla186-203, IsdB342-359, IsdB366-383, and IsdB384-401). The immunodominance of B-cell epitopes, total IgG titers and the levels of IFN-γ and IL-17A from mice immunized with HI plus different adjuvants were different from each other, which may explain the difference in protective immunity observed in each immunized group. Thus, our results indicate that adjuvants largely affected the immunodominance of epitopes and the protective efficacy of HI, which may guide further adjuvant screening for vaccine development and optimization.

Zinc transporter SLC39A7 relieves zinc deficiency to suppress alternative macrophage activation and impairment of phagocytosis.

Macrophages are key phagocytic cells and play an important role in eliminating external microorganisms and endogenous danger signals. Dysregulation in macrophage functions have been reported in patients with asthma. Zinc homeostasis is critical in maintaining macrophage functions. The solute carrier (SLC) protein SLC39A7, a Zn2+ importer, has recently been linked to asthma. However, the roles of SLC39A7 in macrophage phagocytosis are not well understood. Here we found that phagocytosis efficiency was significantly decreased in SLC39A7-knockdown THP-1 cells, however the phagocytosis capability could be reversed with zinc supplementation. SLC39A7 deficiency skewed macrophages towards alternative activation, as indicated by increased expression of M2 activation marker CD206 and decreased expression of M1 activation marker NOS2. Consistent to this result, SLC39A7-knockdown cells produced reduced amounts of proinflammatory cytokines TNF- and IL-6. Furthermore, the mRNA level of receptor Clec4e previously known to be involved in phagocytosis of BCG was significantly reduced in SLC39A7 knockdown cells. Importantly, all these defects due to SLC39A7 deficiency could be reversed by zinc supplementation. Thus, zinc transporter SLC39A7 provide support for phagocytosis and classical macrophage activation.

Zip6 Transporter Is an Essential Component of the Lymphocyte Activation Machinery.

Zinc deficiency causes immune dysfunction. In T lymphocytes, hypozincemia promotes thymus atrophy, polarization imbalance, and altered cytokine production. Zinc supplementation is commonly used to boost immune function to prevent infectious diseases in at-risk populations. However, the molecular players involved in zinc homeostasis in lymphocytes are poorly understood. In this paper, we wanted to determine the identity of the transporter responsible for zinc entry into lymphocytes. First, in human Jurkat cells, we characterized the effect of zinc on proliferation and activation and found that zinc supplementation enhances activation when T lymphocytes are stimulated using anti-CD3/anti-CD28 Abs. We show that zinc entry depends on specific pathways to correctly tune the NFAT, NF-κB, and AP-1 activation cascades. Second, we used various human and murine models to characterize the zinc transporter family, Zip, during T cell activation and found that Zip6 was strongly upregulated early during activation. Therefore, we generated a Jurkat Zip6 knockout (KO) line to study how the absence of this transporter affects lymphocyte physiology. We found that although Zip6KO cells showed no altered zinc transport or proliferation under basal conditions, under activation, these KO cells showed deficient zinc transport and a drastically impaired activation program. Our work shows that zinc entry into activated lymphocytes depends on Zip6 and that this transporter is essential for the correct function of the cellular activation machinery.

Slc11a1 (Nramp-1) gene modulates immune-inflammation genes in macrophages during pristane-induced arthritis in mice.

OBJECTIVE AND DESIGN: Pristane-induced arthritis (PIA) in AIRmax mice homozygous for Slc11a1 R and S alleles was used to characterize the influence of Slc11a1 gene polymorphism on immune responses during disease manifestation. Previous reports demonstrated that the presence of the Slc11a1 S allele increased the incidence and severity of PIA in AIRmax SS , suggesting that this gene could interact with inflammatory loci to modulate PIA. We investigated the effects of Slc11a1 alleles on the activation of phagocytes during PIA. TREATMENT: Mice were injected intraperitoneally with two doses of 0.5 mL of mineral oil pristane at 60-day intervals. Arthritis development was accompanied for 180 days. RESULTS: AIRmax SS mice showed differential peritoneal macrophage gene expression profiles during PIA, with higher expression and production of H2O2, NO, IL-1ß, IL-6, TNF-α, and several chemokines. The presence of the Slc11a1 R allele, on the other hand, diminished the intensity of macrophage activation, restricting arthritis development. CONCLUSION: Our data demonstrated the fine-tuning roles of Slc11a1 alleles modulating macrophage activation, and consequent PIA susceptibility, in those mouse lines.

Immunisation With Immunodominant Linear B Cell Epitopes Vaccine of Manganese Transport Protein C Confers Protection against Staphylococcus aureus Infection.

Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA) infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC), which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d) crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine.

Protective efficacy of the chimeric Staphylococcus aureus vaccine candidate IC in sepsis and pneumonia models.

Staphylococcus aureus causes serious sepsis and necrotic pneumonia worldwide. Due to the spread of multidrug-resistant strains, developing an effective vaccine is the most promising method for combating S. aureus infection. In this study, based on the immune-dominant areas of the iron surface determinant B (IsdB) and clumping factor A (ClfA), we designed the novel chimeric vaccine IsdB151-277ClfA33-213 (IC). IC formulated with the AlPO4 adjuvant induced higher protection in an S. aureus sepsis model compared with the single components alone and showed broad immune protection against several clinical S. aureus isolates. Immunisation with IC induced strong antibody responses. The protective effect of antibodies was demonstrated through the opsonophagocytic assay (OPA) and passive immunisation experiment. Moreover, this new chimeric vaccine induced Th1/Th17-skewed cellular immune responses based on cytokine profiles and CD4(+) T cell stimulation tests. Neutralisation of IL-17A alone (but not IFN-γ) resulted in a significant decrease in vaccine immune protection. Finally, we found that IC showed protective efficacy in a pneumonia model. Taken together, these data provide evidence that IC is a potentially promising vaccine candidate for combating S. aureus sepsis and pneumonia.

Identification of CD4+ T-cell epitopes on iron-regulated surface determinant B of Staphylococcus aureus.

Iron-regulated surface determinant B (IsdB) of Staphylococcus aureus (S. aureus) is a highly conserved surface protein that can induce protective CD4(+) T-cell immune response. A pivotal role of CD4(+) T-cells in effective immunity against S. aureus infection has been proved, but CD4(+) T-cell epitopes on the S. aureus IsdB have not been well identified. In this study, MHC binding assay was firstly used to predict CD4(+) T-cell epitopes on S. aureus IsdB protein, and six peptides were synthesized to validate the probable epitopes. Two novel IsdB CD4(+) T-cell epitopes, P1 (residues 159-178) and P4 (residues 287-306), were for the first time identified using CD4(+) T-cells obtained from IsdB-immunized C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice spleen based on cell proliferation and cytokines response. The results showed that P1 and P4 emulsified in Freund's adjuvant (FA) induced much higher cell proliferation compared with PBS emulsified in FA. CD4(+) T-cells stimulated with peptides P1 and P4 secreted significantly higher levels of IFN-γ and IL-17A. However, the level of the cytokine IL-4 almost remained unchanged, suggesting that P1 and P4 preferentially elicited polarized Th1-type responses. In addition, BALB/c mice just respond to P4 not P1, while C57BL/6 mice respond to P1 not P4, implying that epitope P1 and P4 were determined as H-2(b) and H-2(d) restricted epitope, respectively. Taken together, our data may provide an explanation of the IsdB-induced protection against S. aureus and highlight the possibility of developing the epitope-based vaccine against the S. aureus.

Current and Future Clinical Applications of Zinc Transporter-8 in Type 1 Diabetes Mellitus.

OBJECTIVE: To evaluate the utility of zinc transporter-8 (ZnT8) in the improvement of type 1 diabetes mellitus (T1DM) diagnosis and prediction, and to explore whether ZnT8 is a potential therapeutic target in T1DM. DATA SOURCES: A search was conducted within the medical database PubMed for relevant articles published from 2001 to 2015. The search terms are as follows: "ZnT8," "type 1 diabetes," "latent autoimmune diabetes in adults," "type 2 diabetes," "islet autoantibodies," "zinc supplement," "T cells," "ß cell," "immune therapy." We also searched the reference lists of selected articles. STUDY SELECTION: English-language original articles and critical reviews concerning ZnT8 and the clinical applications of islet autoantibodies in diabetes were reviewed. RESULTS: The basic function of ZnT8 is maintaining intracellular zinc homeostasis, which modulates the process of insulin biosynthesis, storage, and secretion. Autoantibodies against ZnT8 (ZnT8A) and ZnT8-specific T cells are the reliable biomarkers for the identification, stratification, and characterization of T1DM. Additionally, the results from the animal models and clinical trials have shown that ZnT8 is a diabetogenic antigen, suggesting the possibility of ZnT8-specific immunotherapy as an alternative for T1DM therapy. CONCLUSIONS: ZnT8 is a novel islet autoantigen with a widely potential for clinical applications in T1DM. However, before the large-scale clinical applications, there are still many problems to be solved.

Phase IIa study of the immunogenicity and safety of the novel Staphylococcus aureus vaccine V710 in adults with end-stage renal disease receiving hemodialysis.

Bacteremia is the second leading cause of death in patients with end-stage renal disease who are on hemodialysis. A vaccine eliciting long-term immune responses against Staphylococcus aureus in patients on chronic hemodialysis may reduce the incidence of bacteremia and its complications in these patients. V710 is a vaccine containing iron surface determinant B (IsdB), a highly conserved S. aureus surface protein, which has been shown to be immunogenic in healthy subjects. In this blinded phase II immunogenicity study, 206 chronic hemodialysis patients between the ages of 18 and 80 years old were randomized to receive 60 µg V710 (with or without adjuvant), 90 µg V710 (with adjuvant), or a placebo in various combinations on days 1, 28, and 180. All 201 vaccinated patients were to be followed through day 360. The primary hypothesis was that at least 1 of the 3 groups receiving 2 V710 doses on days 1 and 28 would have a ≥2.5 geometric mean fold rise (GMFR) in anti-IsdB IgG titers over the baseline 28 days after the second vaccination (day 56). At day 56, all three groups receiving 2 doses of V710 achieved a ≥2.5 GMFR in anti-IsdB antibodies compared to the baseline (P values of <0.001 for all 3 groups), satisfying the primary immunogenicity hypothesis. None of the 33 reported serious adverse experiences were considered vaccine related by the investigators. V710 induced sustained antibody responses for at least 1 year postvaccination in patients on chronic hemodialysis.

The immunogenicity and safety of different formulations of a novel Staphylococcus aureus vaccine (V710): results of two Phase I studies.

Merck V710 is a novel vaccine that contains the highly conserved Staphylococcus aureus iron surface determinant B (IsdB) protein. V710 has induced positive immune responses in healthy subjects. The purpose of the two studies described herein was to evaluate the immunogenicity and safety of two different formulations of V710. Both studies were randomized, controlled, double-blind, parallel-group trials. Study 1 compared liquid, aluminum-adjuvanted V710 (30 µg) with liquid, non-adjuvanted V710 (30 µg) in a 1:1 ratio in 64 healthy adults (18-70 years). Study 2 compared non-adjuvanted lyophilized V710 (60 µg) with saline placebo in a 4:1 ratio in 51 healthy adults (18-80 years). Blood was collected at screening and up to Day 360 postvaccination in Study 1, and at screening and up to Day 84 postvaccination in Study 2. Sera were analyzed for IsdB-specific antibodies using a total IgG assay. The primary endpoints in Study 1 were the proportion of patients with a positive immune response (≥2-fold rise in IsdB-specific IgG antibody level) the geometric mean concentration (GMC), and the geometric mean-fold rise (GMFR), all from baseline at Day 14. The primary endpoint in Study 2 was the GMFR in IsdB-specific IgG antibody concentration from baseline at Day 14. In Study 1, 84.4% responded in the adjuvanted V710 group, and 71.9% in the non-adjuvanted V710 group. The GMC was 115.4 µg/mL in the adjuvanted group and 99.1 µg/mL in the nonadjuvanted group. The GMFR in antibody concentration in the group receiving aluminum-adjuvanted V710 was 4.5 and the GMFR in the group receiving non-adjuvanted V710 was 4.0. In Study 2, the GMFR in antibody concentration in the V710 group was 5.3, and 80.5% had a positive immune response. None responded in the placebo group. Positive immune response was seen in the active treatment groups over the full duration of each study. There were no serious adverse experiences (AE) in either study, and no patients discontinued due to an AE. There were no clinically meaningful differences in AEs between groups in either study. In conclusion, V710, both with and without aluminum adjuvant, and in both liquid and lyophilized formulations, was immunogenic within 14 days of vaccination. All treatments showed similar safety profiles.

A novel triple mix radiobinding assay for the three ZnT8 (ZnT8-RWQ) autoantibody variants in children with newly diagnosed diabetes.

BACKGROUND AND AIMS: Autoantibodies against the zinc transporter 8 (ZnT8A) are common in type 1 diabetes (T1D). ZnT8A analyses are complicated by the fact that there are three variants of the autoantigen at amino acid position 325 representing ZnT8-R (Arginine), ZnT8-W (Tryptophan) and ZnT8-Q (Glutamin). The aims of the study were: 1) to develop an autoantigen triple mix Radio-Binding Assay (RBA) for ZnT8A; 2) to identify the individual ZnT8-R,-W,-QA reactivity and 3) to validate the triple mix ZnT8A RBA in children with newly diagnosed T1D. METHODS: Serum samples were obtained from 2664 (56% males, n=1436) patients in the Swedish nationwide Better Diabetes Diagnosis (BDD) study representing patients with T1D (97%, n=2582), T2D (1.7%, n=46), MODY (1.0%, n=28) and secondary diabetes (0.3%, n=8). cDNA coding for the C-terminal end of each variant was prepared by site-directed mutagenesis and subcloned into a high efficiency in vitro transcription translation vector. The ZnT8 variants were labeled with 35S-methionine and used in a standard RBA separating free from autoantibody-bound autoantigen with Protein A-Sepharose. RESULTS: ZnT8-TripleA was detected in 1678 (65%) patients with T1D, 4 (9%) T2D, 3 (11%) MODY and in none (0%) of the patients with secondary diabetes. Among the T1D patients ZnT8-RA was detected in 1351 (52%) patients, ZnT8-WA in 1209 (47%) and ZnT8-QA in 790 (31%) demonstrating that 1661 (64%) had one or several ZnT8A. The ZnT8-TripleA assay showed a false positive rate of 1.9% (n=49). Only 1.2% (n=32) of the T1D patients were false negative for ZnT8-TripleA compared to 0/46 (0%) of the T2D patients. The precision (intra assay CV) and reproducibility (inter assay CV) of the ZnT8-TripleA assay did not differ from the RBA of the individual ZnT8 variants. CONCLUSION: We conclude that the ZnT8-TripleA assay had low false positive and false negative rates. The ZnT8-TripleA assay would therefore be highly suitable not only to analyze patient with newly diagnosed diabetes but also for screening the general population since this assay demonstrated high sensitivity and very high specificity.

A fully human monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB) with functional activity in vitro and in vivo.

A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.

Safety and immunogenicity of a novel Staphylococcus aureus vaccine: results from the first study of the vaccine dose range in humans.

Merck V710 is a novel vaccine containing the conserved Staphylococcus aureus iron surface determinant B shown to be protective in animal models. A phase I, multicenter, double-blind study of the dose range was conducted to assess the immunogenicity and safety of an adjuvanted liquid formulation of V710. A total of 124 adults (18 to 55 years of age) were randomized 1:1:1:1 to receive one 0.5-ml intramuscular injection of V710 (5 µg, 30 µg, or 90 µg) or saline placebo. A positive immune response was defined as at least a 2-fold increase in IsdB-specific IgG levels from baseline levels. Local and systemic adverse events were assessed for 5 and 14 days, respectively, following vaccination. Positive immune responses were detected in 12 (67%) of the 18 subjects in the groups receiving 30 and 90 µg V710 tested at day 10. At day 14, a significantly greater proportion of subjects manifested a positive immune response with higher geometric mean concentrations in the V710 30-µg (86%; geometric mean concentration of 116 µg/ml) and 90-µg (87%; geometric mean concentration of 131 µg/ml) dose groups than in the V710 5-µg (29%; geometric mean concentration of 51 µg/ml) or placebo (4%; geometric mean concentration of 23 µg/ml) groups. Immune responses were durable through day 84. Subjects <40 and ≥40 years of age had comparable immune responses. The most common adverse events were injection-site pain, nausea, fatigue, and headache, usually of mild intensity. No immediate reactions or serious adverse events were reported. In this first study of V710 in humans, a single 30-µg or 90-µg dose was more immunogenic than the 5-µg dose or placebo. Immune responses were evident by 10 to 14 days after vaccination in most responders.

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.

Adjuvant activity of ambient particulate matter in macrophage activity-suppressed, N-acetylcysteine-treated, iNOS- and IL-4-deficient mice.

In previous studies, we have shown strong adjuvant activity for Ottawa dust (EHC-93) after coexposure of the BALB/c mouse to EHC-93 and ovalbumin. Mice were intranasally sensitized at days 0 and 14 with 200 microg ovalbumin and 150 microg EHC-93, and challenged with ovalbumin at days 35, 38, and 41 with 200 microg ovalbumin. Mice were autopsied at day 42. This adjuvant activity was shown for the antibody response to ovalbumin (immunoglobulins E, G1, and G2a), histopathological lesions in the lung, cytokines, and the numbers of eosinophils in lung lavages. To study the mechanisms of this adjuvant activity, mice (BALB/cC.D2-Vil6) with natural-resistance-associated macrophage protein (Nramp1s), BALB/c mice pretreated with the antioxidant N-acetylcysteine (NAC), mice (B6.129P2-Nos2tmLau) deficient in inducible nitric oxide synthase (iNOS), and mice with interleukin-4 (IL-4) deficiency (BALB/cIl4< tm2Nnt) were coexposed to ovalbumin and EHC-93. Our studies have shown that the adjuvant activity induced after such coexposure does not change if the macrophage activation of the mice is disturbed or if the mice have been pretreated with N-acetylcysteine. In addition, the adjuvant activity does not develop through the pathway in which inducible nitric oxide synthase is involved. Because the histopathological lesions are statistically significant less in the IL-4 knockout strain in comparison with the wild type, we conclude that interleukin-4 might play an important role in the adjuvant activity caused by EHC-93.