Mechanisms of NDV-3 vaccine efficacy in MRSA skin versus invasive infection.
ABSTRACT: Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge development of an effective vaccine targeting Staphylococcus aureus. This study evaluated the efficacy and immunologic mechanisms of a vaccine containing a recombinant glycoprotein antigen (NDV-3) in mouse skin and skin structure infection (SSSI) due to methicillin-resistant S. aureus (MRSA). Compared with adjuvant alone, NDV-3 reduced abscess progression, severity, and MRSA density in skin, as well as hematogenous dissemination to kidney. NDV-3 induced increases in CD3+ T-cell and neutrophil infiltration and IL-17A, IL-22, and host defense peptide expression in local settings of SSSI abscesses. Vaccine induction of IL-22 was necessary for protective mitigation of cutaneous infection. By comparison, protection against hematogenous dissemination required the induction of IL-17A and IL-22 by NDV-3. These findings demonstrate that NDV-3 protective efficacy against MRSA in SSSI involves a robust and complementary response integrating innate and adaptive immune mechanisms. These results support further evaluation of the NDV-3 vaccine to address disease due to S. aureus in humans.
Project description:Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI) in humans. Moreover, the high frequency of recurring SSSI due to S. aureus, particularly methicillin-resistant S. aureus (MRSA) strains, suggests that infection induces suboptimal anamnestic defenses. The present study addresses the hypothesis that interleukin-17A (IL-17A) and IL-22 play distinct roles in immunity to cutaneous and invasive MRSA infection in a mouse model of SSSI. Mice were treated with specific neutralizing antibodies against IL-17A and/or IL-22 and infected with MRSA, after which the severity of infection and host immune response were determined. Neutralization of either IL-17A or IL-22 reduced T cell and neutrophil infiltration and host defense peptide elaboration in lesions. These events corresponded with increased abscess severity, MRSA viability, and CFU density in skin. Interestingly, combined inhibition of IL-17A and IL-22 did not worsen abscesses but did increase gamma interferon (IFN-?) expression at these sites. The inhibition of IL-22 led to a reduction in IL-17A expression, but not vice versa. These results suggest that the expression of IL-17A is at least partially dependent on IL-22 in this model. Inhibition of IL-17A but not IL-22 led to hematogenous dissemination to kidneys, which correlated with decreased T cell infiltration in renal tissue. Collectively, these findings indicate that IL-17A and IL-22 have complementary but nonredundant roles in host defense against cutaneous versus hematogenous infection. These insights may support targeted immune enhancement or other novel approaches to address the challenge of MRSA infection.
Project description:Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI). The high frequency of recurring SSSI due to S. aureus, including methicillin-resistant S. aureus (MRSA) strains, despite high titers of specific antibodies and circulating T cells, implies that traditional adaptive immunity imparts incomplete protection. We hypothesized that innate immune memory contributes to the protective host defense against recurring MRSA infection. To test this hypothesis, SSSI was induced in wild-type and rag1-/- mice in the BALB/c and C57BL/6 backgrounds. Prior infection (priming) of wild-type and rag1-/- mice of either background afforded protection against repeat infection, as evidenced by reduced abscess severities and decreased CFU densities compared to those in naive controls. Interestingly, protection was greater on the previously infected flank than on the naive flank for wild-type and rag1-/- mice. For wild-type mice, protective efficacy corresponded to increased infiltration of neutrophils (polymorphonuclear leukocytes [PMN]), macrophages (M?), Langerin+ dendritic cells (LDC), and natural killer (NK) cells. Protection was associated with the induction of interleukin-17A (IL-17A), IL-22, and gamma interferon (IFN-?) as well as the antimicrobial peptides CRAMP and m?D-3. Priming also protected rag1-/- mice against recurring SSSI, with increased M? and LDC infiltration and induction of IL-22, CRAMP, and m?D-3. These findings suggest that innate immune memory, mediated by specific cellular and molecular programs, likely contributes to the localized host defense in recurrent MRSA SSSI. These insights support the development of targeted immunotherapeutic strategies to address the challenge of MRSA infection.
Project description:Staphylococcus aureus is the leading cause of skin and skin structure infection (SSSI), a primary portal of entry for invasive infection. Our prior studies discovered a role for protective innate memory against recurrent methicillin-resistant S. aureus (MRSA) SSSI. In the present study, the dynamics and mechanisms of this response were explored in recurrent SSSI in WT mice. Priming by prior infection reduced skin lesion severity and MRSA burden, and protected against dissemination at day 7 but not day 2. Cytokine and cellular signatures in SSSI differed at day 2 versus 7, and were distinct in skin versus blood or spleen. Cytokines associated with protection in skin included increased IL-17, IL-6, monokine inducible by IFN-γ (MIG), and RANTES, while increased IP-10 correlated with protection from dissemination. Cellular signatures of protection included increased Th17, M1 macrophage, and dendritic cell populations in abscesses, and total macrophages in lymph nodes. Priming potentiated S. aureus-specific phagocytic killing by bone marrow-derived macrophages in vitro, and their adoptive transfer into naïve skin afforded protective efficacy in vivo. Present findings indicate that protective immunity in recurrent S. aureus infection is locally targeted, and involves specific memory conferred by macrophages. These insights provide targets for vaccine and immunotherapeutic development against MRSA.
Project description:The investigational vaccine, NDV-3, contains the N-terminal portion of the Candida albicans agglutinin-like sequence 3 protein (Als3p) formulated with an aluminum hydroxide adjuvant in phosphate-buffered saline. Preclinical studies demonstrated that the Als3p vaccine antigen protects mice from oropharyngeal, vaginal and intravenous challenge with C. albicans and other selected species of Candida as well as both intravenous challenge and skin and soft tissue infection with Staphylococcus aureus. The objectives of this first-in-human Phase I clinical trial were to evaluate the safety, tolerability and immunogenicity of NDV-3 at two different antigen levels compared to a saline placebo. Forty healthy, adult subjects were randomized to receive one dose of NDV-3 containing either 30 or 300 ?g of Als3p, or placebo. NDV-3 at both dose levels was safe and generally well-tolerated. Anti-Als3p total IgG and IgA1 levels for both doses reached peak levels by day 14 post vaccination, with 100% seroconversion of all vaccinated subjects. On average, NDV-3 stimulated peripheral blood mononuclear cell (PBMC) production of both IFN-? and IL-17A, which peaked at day 7 for subjects receiving the 300 ?g dose and at day 28 for those receiving the 30 ?g dose. Six months after receiving the first dose of NDV-3, nineteen subjects received a second dose of NDV-3 identical to their first dose to evaluate memory B- and T-cell immune responses. The second dose resulted in a significant boost of IgG and IgA1 titers in >70% of subjects, with the biggest impact in those receiving the 30 ?g dose. A memory T-cell response was also noted for IFN-? in almost all subjects and for IL-17A in the majority of subjects. These data support the continued investigation of NDV-3 as a vaccine candidate against Candida and S. aureus infections.
Project description:Staphylococcus aureus is an opportunistic pathogen that colonizes the skin of patients with atopic dermatitis (AD) and aggravates their disease. Neutrophils and the cytokines IL-17A and IL-17F, which drive the expression of the neutrophil-attracting chemokines, are important for the clearance of S aureus infection. The cytokine IL-22 is often coproduced by IL-17-secreting cells. The levels of IL-22 are elevated in AD skin lesions.We sought to determine the role of IL-22 in the clearance of S aureus infection of mouse skin subjected to tape stripping, a surrogate for scratching, a cardinal feature of AD.S aureus was applied to the tape-stripped skin of wild-type and Il22-/- mice. Bacterial burden was evaluated by enumerating colony-forming units. Quantitative PCR and ELISA were performed to quantify Il22 mRNA and IL-22 protein in mouse and human skin. Flow cytometry was used to enumerate neutrophils in the skin.Scratching the skin of healthy adults and tape stripping of mouse skin induced local expression of Il22 mRNA and IL-22 protein. Induction of Il22 expression by tape stripping was dependent on IL-23 and ?? T cells. Clearance of S aureus from tape-stripped skin was significantly impaired in Il22-/- mice. Neutrophil infiltration and upregulation of expression of genes encoding the antimicrobial peptides antigen-6/urokinase-type plasminogen activator receptor related protein-1 and ?-DEFENSIN 14 and the chemokine (C-X-C motif) ligand following tape stripping were significantly impaired in Il22-/- mice.These findings show that IL-22 is important for limiting the growth of S aureus on mechanically injured skin and caution that IL-23 and IL-22 blockade in patients with AD may enhance susceptibility to staphylococcal skin infection.
Project description:Linezolid resistance mediated by the cfr gene in MRSA represents a global concern. We investigated relevant phenotype differences between cfr-positive and -negative MRSA that contribute to pathogenesis, and the efficacy of linezolid-based combination therapies in murine models of bacteremia and skin and skin structure infection (SSSI). As a group, cfr-positive MRSA exhibited significantly reduced susceptibilities to the host defense peptides tPMPs, human neutrophil peptide-1 (hNP-1), and cathelicidin LL-37 (P < 0.01). In addition, increased binding to fibronectin (FN) and endothelial cells paralleled robust biofilm formation in cfr-positive vs. -negative MRSA. In vitro phenotypes of cfr-positive MRSA translated into poor outcomes of linezolid monotherapy in vivo in murine bacteremia and SSSI models. Importantly, rifampicin showed synergistic activity as a combinatorial partner with linezolid, and the EC50 of linezolid decreased 6-fold in the presence of rifampicin. Furthermore, this combination therapy displayed efficacy against cfr-positive MRSA at clinically relevant doses. Altogether, these data suggest that the use of linezolid in combination with rifampicin poses a viable therapeutic alternative for bacteremia and SSSI caused by cfr-positive multidrug resistant MRSA.
Project description:Atopic dermatitis (AD) is a chronic inflammatory immune-mediated skin disease characterized by skin colonization by Staphylococcus aureus. Interleukin (IL)-22, in cooperation with IL-17, triggers antimicrobial peptide elaboration and enhances certain immunological responses. In AD, IL-22 is related to epidermal hyperplasia, keratinocyte apoptosis, and inhibition of antimicrobial peptide (AMP) production. We aimed to evaluate the impact of staphylococcal enterotoxins on the Tc22/Th22 induction in the peripheral blood of AD patients and on CD4+/CD8+ T cells expressing IL-22 in AD skin. Our study showed inhibition of the staphylococcal enterotoxins A and B (SEA and SEB) response by Th22 (CD4+IL-22+IL-17A-IFN-?-) cells in AD patients. In contrast, Tc22 (CD8+IL-22+IL-17A-IFN-?-) cells were less susceptible to the inhibitory effects of staphylococcal enterotoxins and exhibited an enhanced response to the bacterial stimuli. In AD skin, we detected increased IL-22 transcript expression and T lymphocytes expressing IL-22. Together, our results provide two major findings in response to staphylococcal enterotoxins in adults with AD: dysfunctional CD4+ IL-22 secreting T cells and increased Tc22 cells. Our hypothesis reinforces the relevance of CD8 T cells modulated by staphylococcal enterotoxins as a potential source of IL-22 in adults with AD, which is relevant for the maintenance of immunological imbalance.
Project description:Abstract Background Omadacycline (OMC) was FDA approved to treat acute bacterial skin and skin structure infection (ABSSSI) and community-acquired bacterial pneumonia (CABP) for indicated organisms in 2018. Phase 2 OMC clinical trials for uncomplicated urinary tract infection (uUTI; NCT03425396) and acute pyelonephritis (NCT03757234) are ongoing. OMC is active against bacterial isolates expressing common tetracycline, penicillin, fluoroquinolone, and macrolide resistance mechanisms. Methods Isolates (14,000) were collected in 2018 from 31 medical centers located in the United States and 38 medical centers in Europe, including 3,458 staphylococci, 1,551 streptococci, 746 enterococci, 574 Haemophilus spp., and 5,690 Enterobacterales isolates. One isolate per patient infection episode was tested. Identifications were confirmed by MALDI-TOF MS and susceptibility testing was performed using CLSI broth microdilution methods. Results OMC (MIC50/90, 0.12/0.25 mg/L) was highly active against S. aureus isolates from skin and skin structure infection (SSSI; 99.3% susceptible [S]) including MRSA (97.7%S) and MSSA (99.9%S) (table). Similarly, OMC demonstrated potent activity against S. aureus isolates from respiratory tract infection (RTI; MIC50/90, 0.12/0.25 mg/L) including MSSA (98.2%S). All S. lugdunensis isolates from SSSI were S (100.0%) to OMC. All Streptococcus anginosus group (100.0%) and 97.6% of S. pyogenes isolates from SSSI were S to OMC as were 98.0% of S. pneumoniae from RTI. No streptococci were resistant (R) to OMC. OMC (MIC50/90 0.12/0.25 mg/L) had potent activity against E. faecalis isolates from SSSI (99.0%S). OMC S against E. cloacae and K. pneumoniae isolates from SSSI was 92.1% S and 89.4% S, respectively. Similarly, 86.2% of K. pneumoniae isolates from RTI were S to OMC. Susceptibility of H. influenzae isolates from RTI to OMC was 99.8%S (no isolates were R). ?90.0% of E. coli (MIC50/90, 1/2 mg/L) and K. pneumoniae (MIC50/90, 2/4 mg/L) UTI isolates were inhibited by ?4 mg/L of OMC. Conclusion OMC was highly active against bacterial pathogens associated with ABSSSI, CABP, and UTI including staphylococci (97.7%-100.0%S), streptococci (97.6%-100.0%S), E. faecalis (99.0%S). E. cloacae (92.1%S), K. pneumoniae (86.2%-89.4%S), and E. coli. Disclosures All authors: No reported disclosures.
Project description:T cell cytokines contribute to immunity against Staphylococcus aureus, but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL-17 response was mediated by ?? T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1?, IL-1?, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4 However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, V?6+ T cells were a predominant ?? T cell subset that produced IL-17A as well as IL-22, TNF, and IFN?, indicating a broad and substantial role for clonal V?6+V?4+ T cells in immunity against S. aureus skin infections.
Project description:Staphylococcus aureus (S. aureus) is a commensal bacterium in the human body; however, the bacterium frequently generates serious inflammation and infectious diseases. Some strains of S. aureus, such as methicillin-resistant Staphylococcus aureus (MRSA), are still a serious problem in public health facilities. Thus, an effective protection strategy is eagerly expected for the prevention and cure of MRSA infection. Here, we report that a specific fraction of an S. aureus lipoprotein (SA-LP) established a protective response against MRSA infection. The fractionated S. aureus lipoprotein SA-LP-F2, which is contained in 30-50 kDa of crude S. aureus lipoprotein (SA-LP-C), effectively activated dendritic cells (DCs) and the SA-LP-F2-pulsed DCs generated IFN-?+CD4+ T (Th1) and IL-17A+CD4+ T (Th17) cells by in vitro antigen presentation. The SA-LP-F2 immunization upregulated the Th1 and Th17 populations so that MRSA colonization on the skin was suppressed during the challenge phase with MRSA. By following the effector T cell upregulation, the neutrophil function, which was a substantial effector cell against MRSA, was also enhanced in the SA-LP-F2-immunized mice. Finally, we found that the protective effect of SA-LP-F2 immunization was maintained for at least 90 days because the immunized mice continued to show a protective response during the MRSA challenge period. In the MRSA challenge, reactivated Th1 and Th17 populations were maintained in the SA-LP-F2-immunized mice as compared to naive mice. In addition, the neutrophil population was also upregulated in the mice. The memory CD4+ T cell (central memory T; TCM and effector memory T; TEM) population was established by SA-LP-F2 immunization and was maintained at higher levels than usual. Taken together, our findings may provide a breakthrough in the establishment of an immunization strategy against MRSA infection.