Kamal U. Saikh

Protective Immunization against Inhalational Anthrax: A Comparison of Minimally Invasive Delivery Platforms

A new anthrax vaccine under clinical investigation is based on recombinant Bacillus anthracis protective antigen (rPA). Here, we investigated microneedle-based cutaneous and nasal mucosal delivery of rPA in mice and rabbits. In mice, intradermal (id) delivery achieved up to 90% seroconversion after a single dose, compared with 20% after intramuscular (im) injection. Intranasal (inl) delivery of a liquid formulation required 3 doses to achieve responses that were comparable with those achieved via the id or im routes. In rabbits, id delivery provided complete protection against aerosol challenge with anthrax spores; in addition, novel powder formulations administered inl provided complete protection, whereas a liquid formulation provided only partial protection. These results demonstrate, for the first time, that cutaneous or nasal mucosal administration of rPA provides complete protection against inhalational anthrax in rabbits. The novel vaccine/device combinations described here have the potential to improve the efficacy of rPA and other biodefense vaccines.

Human Monocytes Infected with Yersinia pestis Express Cell Surface TLR9 and Differentiate into Dendritic Cells

TLR9 recognizes DNA sequences containing hypomethylated CpG motifs and is a component of the innate immune system highly conserved during eukaryotic evolution. Previous reports suggested that the expression of TLR9 is restricted to plasmacytoid dendritic cells and B lymphocytes. Our results indicate that low levels of TLR9 are present on the cell surface of freshly isolated human monocytes, and expression is greatly increased by infection with Yersinia pestis. Enhanced cell surface TLR9 coincided with elevated levels of cytoplasmic TLR9 and recruitment of MyD88. Infected monocytes differentiated into mature dendritic cells, expressed IFN-α, and stimulated proliferative and cytotoxic T cell responses specific to Y. pestis. Furthermore, uninfected B cells and monocytes both increased cell surface TLR9, CD86, and HLA-DR in response to treatment with CpG-containing oligonucleotides, whereas cell surface TLR9 was down-modulated on infected dendritic cells by the addition of agonist oligonucleotide. Our results suggest that increased expression of TLR9 on the surface of infected cells may serve a role as an activation signal to other cells of the immune system.

Toll-Like Receptor and Cytokine Expression Patterns of CD56+ T Cells Are Similar to Natural Killer Cells in Response to Infection with Venezuelan Equine Encephalitis Virus Replicons

Using the natural killer (NK) cell-surface marker CD56 to study NK T cells in peripheral blood, we found that their frequency in mononuclear cells among healthy individuals was 1%-20% (average, 7.3%) and sporadically increased 4-5-fold within individuals over the course of 8 months. Infection of mononuclear cells in vitro with Venezuelan equine encephalitis virus replicon particles (VRPs) resulted in a significant increase in CD56(+) T cells and in the expression of interferon-alpha, tumor necrosis factor (TNF)-alpha, and interferon-gamma by CD56(+) but not CD56(-) T cells. NK and CD56(+) T cells expressed higher levels of Toll-like receptor (TLR)-3 and TLR4 after infection with VRPs, whereas only NK cells expressed inducible TNF-alpha and TLR2. Most of these effects were duplicated by activating mononuclear cells with double-stranded RNA. These expression patterns indicate that T cells coexpressing NK markers respond like NK cells to viral infection or double-stranded RNA, potentially fulfilling innate and adaptive immune functions.

Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules

Ligands binding to Toll-like receptor (TLR), interleukin 1 receptor (IL-1R), or IFN-γR1 are known to trigger MyD88-mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which bind to MHC class II molecules on APCs and cross link T cell receptors, activate MyD88- mediated pro-inflammatory cytokine responses. We also reported that MyD88−/− mice were resistant to SE- induced toxic shock and had reduced levels of serum cytokines. In this study, we investigated whether MHC class II- SE interaction by itself is sufficient to activate MyD88 in MHC class II+ cells and induce downstream pro-inflammatory signaling and production of cytokines such as TNF-α and IL-1β. Here we report that human monocytes treated with SEA, SEB, or anti-MHC class II monoclonal antibodies up regulated MyD88 expression, induced activation of NF-kB, and increased expression of IL-1R1 accessory protein, TNF-α and IL-1β. MyD88 immunoprecipitated from cell extracts after SEB stimulation showed a greater proportion of MyD88 phosphorylation compared to unstimulated cells indicating that MyD88 was a component of intracellular signaling. MyD88 downstream proteins such as IRAK4 and TRAF6 were also up regulated in monocytes after SEB stimulation. In addition to monocytes, primary B cells up regulated MyD88 in response to SEA or SEB stimulation. Importantly, in contrast to primary B cells, MHC class II deficient T2 cells had no change of MyD88 after SEA or SEB stimulation, whereas MHC class II-independent activation of MyD88 was elicited by CpG or LPS. Collectively, these results demonstrate that MHC class II utilizes a MyD88-mediated signaling mechanism when in contact with ligands such as SEs to induce pro-inflammatory cytokines.

Are DNA-based vaccines useful for protection against secreted bacterial toxins? Tetanus toxin test case.

Polypeptide and DNA vaccine alternatives to the conventional tetanus toxoid were compared. Mouse immunizations with plasmid DNA that encoded the tetanus toxin C fragment polypeptide induced consistently lower antibody responses than direct immunization with the C fragment polypeptide or toxoid, yet provided some degree of protection from a lethal toxin challenge. Cytotoxic T-cell responses dominated DNA immunizations, while specific T-cell proliferation resulted from all vaccines tested. Immune responses to the DNA vaccine exhibited a T-helper type-1 propensity, while polypeptides elicited T-helper type-2 responses. The lower antibody response to the plasmid vaccine was not due to insufficient quantity of C fragment in vivo but was likely the result of a mode of antigen presentation that was less efficient for supporting antibody production. Collectively, these results suggest that polypeptide or toxoid vaccines are preferable to plasmid-based vaccination for control of diseases caused by tetanus toxin.

A small molecule that mimics the BB-loop in the Toll interleukin-1 (IL-1) receptor domain of MyD88 attenuates staphylococcal enterotoxin B-induced pro-inflammatory cytokine production and toxicity in mice.

Toxic shock syndrome (TSS) is a clinical consequence of the profound amplification of host pro-inflammatory cytokine signaling that results from staphylococcal enterotoxin (SE) exposure. We recently reported that MyD88−/− mice were resistant to SEA or SEB toxic shock and displayed reduced levels of pro-inflammatory cytokines in their serum. Here we report that SEB stimulation of total mononuclear cells up-regulated MyD88 in monocytes and T cells. Further, MyD88 gene silencing in primary human cells using siRNA prevented SEB or SEB plus lipopolysaccharide (LPS) induction of interleukin-1β (IL-1β) transcriptional activation, suggesting that MyD88-mediated signaling is an essential component of SEB toxicity. We synthesized small molecules that mimic the conserved BB-loop in the Toll/IL-1 receptor (TIR) domain of MyD88. In primary human cells, these mimetics attenuated SEB-induced pro-inflammatory cytokine production. SEB stimulation of primary cells with mimetic affected newly synthesized MyD88 and downstream signaling components. Furthermore, LPS-induced MyD88 signaling was likewise inhibited in a cell-based reporter assay. More importantly, administration of mimetic reduced cytokine responses and increased survivability in a murine SEB challenge model. Collectively, these results suggest that MyD88 BB-loop mimetics interfere with SEB-induced pro-inflammatory signaling and toxicity, thus offering a potential approach in the therapy of toxic shock.

Staphylococcal enterotoxin A induction of pro-inflammatory cytokines and lethality in mice is primarily dependent on MyD88.

Staphylococcal enterotoxin (SE) ‐induced toxic shock is triggered by inflammatory cytokine signal amplification after SE binding to major histocompatibility complex class II molecules on antigen‐presenting cells and T‐cell receptors. Identifying host cellular elements contributing to this pro‐inflammatory signal amplification is critical for developing a strategy for therapeutic intervention. Myeloid differentiation primary‐response protein 88 (MyD88) is an intracellular signalling adaptor protein primarily known for mediating pro‐inflammatory cytokine responses. We investigated the role of MyD88 in staphylococcal enterotoxin A (SEA) ‐treated cell cultures and mouse models of toxic shock. Our results demonstrated that elevated levels of tumour necrosis factor‐α, interferon‐γ, interleukin‐1α/β (IL‐1α/β), IL‐2 and IL‐6 production correlated with up‐regulation of MyD88 after treatment of spleen cells and mice with SEA alone or in combination with lipopolysaccharide (LPS). The SEA‐induced lethality was also observed in (LPS‐independent) d‐galactosamine‐sensitized mice. While LPS potentiated SEA‐induced cytokine responses, d‐galactosamine treatment had no additive effect. Most importantly, our results demonstrated that MyD88−/− mice were resistant to SEA‐induced toxic shock and had reduced pro‐inflammatory cytokine responses. These results suggest that SEA‐induced lethality is primarily dependent on MyD88. Our findings offer an important insight on potential therapeutic treatment of SEA‐induced toxic shock targeting MyD88.

Interleukin-15 Increases Vaccine Efficacy through a Mechanism Linked to Dendritic Cell Maturation and Enhanced Antibody Titers

ABSTRACT Interleukin-15 (IL-15) is generally considered to sustain T-cell memory and to be a growth factor for natural killer cells. Previous data from our laboratory demonstrated that IL-15 is also an important factor for developing human dendritic cells. For this study, we investigated the effects of IL-15 on antibody responses in mice to a recombinant staphylococcal enterotoxin B (SEB) vaccine (STEBVax) in a preclinical model of toxic shock syndrome induced by SEB. We observed that mouse spleen cells treated with IL-15 in ex vivo culture gained a dendritic cell-like phenotype. Administration of IL-15 to mice also resulted in an increased number of mature CD11c+ dendritic cells in mouse spleens. A significant, IL-15 dose-dependent increase in antigen-specific antibody was observed after coadministration with the vaccine and an aluminum-based adjuvant (alhydrogel). Furthermore, the coadministration of IL-15 with STEBVax and alhydrogel also protected mice from lethal toxic shock above the levels that obtained without IL-15. Thus, the vaccine response enhanced by IL-15 appears to be mediated by mature dendritic cells and results in prevalent seroconversion to Th2-dependent antibodies. This suggests a potential use of IL-15 as an adjuvant for antibody-dependent responses to vaccines.

Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

Staphylococcal enterotoxin B (SEB) exposure triggers an exaggerated pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. MyD88 mediates pro-inflammatory cytokine signaling induced by SEB exposure and MyD88−/− mice are resistant to SEB intoxication, suggesting that MyD88 may be a potential target for therapeutic intervention. We targeted the BB loop region of the Toll/IL-1 receptor (TIR) domain of MyD88 to develop small-molecule therapeutics. Here, we report that a synthetic compound (EM-163), mimic to dimeric form of BB-loop of MyD88 attenuated tumor necrosis factor (TNF)- α, interferon (IFN)-γ, interleukin (IL)-1β, IL-2 and IL-6 production in human primary cells, whether administered pre- or post-SEB exposure. Results from a direct binding assay, and from MyD88 co-transfection/co-immunoprecipitation experiments, suggest that EM-163 inhibits TIR-TIR domain interaction. Additional results indicate that EM-163 prevents MyD88 from mediating downstream signaling. In an NF-kB-driven reporter assay of lipopolysaccharide-stimulated MyD88 signaling, EM-163 demonstrated a dose-dependent inhibition of reporter activity as well as TNF-α and IL-1β production. Importantly, administration of EM-163 pre- or post exposure to a lethal dose of SEB abrogated pro-inflammatory cytokine responses and protected mice from toxic shock-induced death. Taken together, our results suggest that EM-163 exhibits a potential for therapeutic use against SEB intoxication.

MyD88-dependent pro-inflammatory cytokine response contributes to lethal toxicity of staphylococcal enterotoxin B in mice

An elevated pro-inflammatory cytokine response is the primary cause of death by toxic shock after exposure to staphylococcal enterotoxin B (SEB). Identifying an intracellular signal mediator that predominantly controls the pro-inflammatory response is important for developing a therapeutic strategy. We examined the role of the signaling adaptor MyD88 in cell culture and in a mouse model of toxic shock. Our results indicated that elevated tumor necrosis factor-α, interferon-γ, interleukin (IL)-1α/β and IL-6 production from mouse spleen cells treated with SEB alone or in combination with lipopolysaccharide (LPS) was regulated by MyD88. Elevated levels of MyD88 protein in spleen cells, as well as in CD11c+ or Mac3+ cells, and activation of nuclear factor-κB in spleen cells were observed in mice treated with SEB. An SEB-dose dependent lethality was observed in LPS-potentiated and in D-galactosamine-sensitized mice. D-Galactosamine treatment of spleen cells had no effect in cytokine induction but rather increased the sensitivity to toxic shock in mice. Our results demonstrated an impaired pro-inflammatory cytokine production by spleen cells of MyD88–/– mice in response to SEB or SEB plus LPS. Most importantly, MyD88–/– mice were resistant to SEB-induced death. These results demonstrate that MyD88-dependent pro-inflammatory signaling is responsible for SEB intoxication. In addition, our studies also demonstrated that LPS potentiation, in comparison to D-galactosamine sensitization, contributes to a stronger SEB–induced lethality. This is due to the pro-inflammatory cytokine response elicited by MyD88 after exposure to SEB and LPS. These findings offer an important insight upon SEB intoxication and subsequent therapy targeting MyD88.