The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are ...active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Childhood infection with Epstein–Barr virus (EBV) is often asymptomatic and may result in mild flu-like symptoms, but exposure during adolescence and young adulthood can lead to acute ...infectious mononucleosis (AIM) with a pathology characterized by swollen lymph nodes, sore throat, and severe fatigue lasting weeks or months. A vaccine targeting the envelope glycoprotein gp350 adjuvanted with aluminum hydroxide complexed with the TLR4 agonist monophosphoryl lipid A (MPLA) achieved a 78% reduction in AIM incidence in a small phase II trial of college-age individuals, but development of this vaccine was halted by the manufacturer. Here, we report the evaluation in mice and rabbits of an EBV-gp350 vaccine combined with an adjuvant composed of the synthetic TLR4 agonist glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE). In mice, GLA/SE-adjuvanted gp350 generated high IgG titers (both IgG1 and IgG2a/c subtypes), elevated EBV-neutralizing antibody titers, and robust poly-functional anti-gp350 CD4+ T cell responses. In addition, GLA/SE routinely demonstrated superior performance over aluminum hydroxide in all immunological readouts, including induction of durable neutralizing antibody titers out to at least 1 year post-vaccination. Both components of the GLA/SE adjuvant were found to be required to get optimal responses in both arms of the immune response: specifically, SE for neutralizing antibodies and GLA for induction of T cell responses. Furthermore, this vaccine also elicited high neutralizing antibody titers in a second species, rabbit. These promising results suggest that clinical development of a vaccine comprised of EBV-gp350 plus GLA/SE has the potential to prevent AIM in post-adolescents.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Several prophylactic vaccines targeting herpes simplex virus 2 (HSV-2) have failed in the clinic to demonstrate sustained depression of viral shedding or protection from recurrences. Although these ...vaccines have generated high titers of neutralizing antibodies (NAbs), their induction of robust CD8 T cells has largely been unreported, even though evidence for the importance of HSV-2 antigen-specific CD8 T cells is mounting in animal models and in translational studies involving subjects with active HSV-2-specific immune responses. We developed a subunit vaccine composed of the NAb targets gD and gB and the novel T cell antigen and tegument protein UL40, and we compared this vaccine to a whole-inactivated-virus vaccine (formaldehyde-inactivated HSV-2 FI-HSV-2). We evaluated different formulations in combination with several Th1-inducing Toll-like receptor (TLR) agonists
In mice, the TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide formulated in a squalene-based oil-in-water emulsion promoted most robust, functional HSV-2 antigen-specific CD8 T cell responses and high titers of neutralizing antibodies, demonstrating its superiority to vaccines adjuvanted by monophosphoryl lipid A (MPL)-alum. We further established that FI-HSV-2 alone or in combination with adjuvants as well as adjuvanted subunit vaccines were successful in the induction of NAbs and T cell responses in guinea pigs. These immunological responses were coincident with a suppression of vaginal HSV-2 shedding, low lesion scores, and a reduction in latent HSV-2 DNA in dorsal root ganglia to undetectable levels. These data support the further preclinical and clinical development of prophylactic HSV-2 vaccines that contain appropriate antigen and adjuvant components responsible for programming elevated CD8 T cell responses.
Millions of people worldwide are infected with herpes simplex virus 2 (HSV-2), and to date, an efficacious prophylactic vaccine has not met the rigors of clinical trials. Attempts to develop a vaccine have focused primarily on glycoproteins necessary for HSV-2 entry as target antigens and to which the dominant neutralizing antibody response is directed during natural infection. Individuals with asymptomatic infection have exhibited T cell responses against specific HSV-2 antigens not observed in symptomatic individuals. We describe for the first time the immunogenicity profile in animal models of UL40, a novel HSV-2 T cell antigen that has been correlated with asymptomatic HSV-2 disease. Additionally, vaccine candidates adjuvanted by a robust formulation of the CpG oligonucleotide delivered in emulsion were superior to unadjuvanted or MPL-alum-adjuvanted formulations at eliciting a robust cell-mediated immune response and blocking the establishment of a latent viral reservoir in the guinea pig challenge model of HSV-2 infection.
Toll‐like receptors (TLRs) are a family of molecules that function as sensors for the detection of foreign pathogens through the recognition of nonvariable microbial motifs. Although numerous studies ...have focused on singular TLRs, less attention has been focused on how simultaneous signaling of multiple TLRs may result in counter‐regulation of the effects of each. Here, we examine the counter‐regulation that occurs during simultaneous stimulation of TLR7 and TLR9 on human plasmacytoid dendritic cels (PDCs) and B cells. Interestingly, we observed that the capacity for potent IFN‐α‐induction by TLR9 ligands like CpG‐C and CpG‐A is markedly reduced by concurrent small molecule TLR7 stimulation. However, this inhibition is specific to particular CpG motif‐containing immunostimulatory sequence (ISS) functions such as IFN‐α induction and BDCA‐2 down‐regulation. Other ISS activities such as PDC expression of CD80/CD86, secretion of IL‐6, and B cell proliferation are not altered by the presence of TLR7 ligands (TLR7Ls). In concordance with the ability of TLR7Ls to decrease IFN‐α secretion induced by ISS, we also find that the expression of interferon regulatory factor‐7 (IRF‐7), a transcriptional factor critical for IFN‐α expression, is reduced. Furthermore, down‐regulation of TLR9 mRNA expression is accelerated after TLR7 stimulation. These data indicate that TLR7 and TLR9 costimulation do not combine synergistically for IFN‐α induction and demonstrate that, instead, a negative feedback mechanism has evolved, possibly to prevent levels of IFN‐α secretion potentially detrimental to the host.
Full text
Available for:
FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Summary
Immunostimulatory sequences (ISS) that contain CpG motifs have been demonstrated to exert antipathogen and antitumour immunity in animal models through several mechanisms, including the ...activation of natural killer (NK) cells to secrete interferon‐γ (IFN‐γ) and to exert lytic activity. Since NK cells lack the ISS receptor TLR9, the exact pathway by which NK cells are activated by ISS is unclear. We determined that ISS‐induced IFN‐γ from NK cells is primarily dependent upon IFN‐α release from plasmacytoid dendritic cells (PDCs), which directly activates the NK cell. However, further analysis indicated that other PDC‐released soluble factor(s) may contribute to IFN‐γ induction. Indeed, tumour necrosis factor‐α (TNF‐α) was identified as a significant contributor to ISS‐mediated activation of NK cells and was observed to act in an additive fashion with IFN‐α in the induction of IFN‐γ from NK cells and to up‐regulate CD69 expression on NK cells. This activity of TNF‐α, however, was dependent upon the presence of PDC‐derived factors such as type I interferon. These results illustrate an important function for type I interferon in innate immunity, which is not only to activate effectors like NK cells directly, but also to prime them for enhanced activation by other factors such as TNF‐α.
Full text
Available for:
BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
We have investigated the adjuvant activity of both CpG motif-containing ImmunoStimulatory Sequences (ISS) and the saponin/phospholipid-based Iscomatrix (IMX) on HBV-specific humoral and ...cell-mediated immunity in C57BL/6 mice. Mice were immunized at weeks 0 and 2 with several formulations that incorporated HBV surface antigen (HBsAg), HBV core antigen (HBcAg), ISS, and IMX. ISS was added to antigen in soluble form or conjugated with HBsAg or HBcAg. Bleeds were performed 2 weeks post 1st immunization (2wp1) and 2wp2 and sera analyzed for anti-HBV IgG1 and IgG2a levels. Mice were also splenectomized at 2wp2 and HBV-specific cell-mediated immunity was examined. HBsAg + HBcAg + 1018 ISS + IMX resulted in high levels of HBsAg-specific and HBcAg-specific IFN-g induction that exceeded that induced by either adjuvant alone. Conjugates of HBV surface or core protein to ISS ODNs also provided for higher levels of antigen-specific IFN-g production and CTL activation. These results indicate that HBV therapeutic formulations that incorporate ISS and/or IMX may stimulate for enhanced levels of anti-HBsAg antibody and augmented anti-HBcAg CMI (cell-mediated immunity) and are promising candidates to test in human clinical trials.
Abstract only
HBV infects approximately 350 million people worldwide, and carriers are at increased risk for cirrhosis and hepatocellular carcinoma. Current HBV therapies do not eradicate HBV and ...have limited long‐term efficacy. We have developed a novel immunotherapeutic vaccine for treatment of chronic HBV infection. The vaccine, composed of HBsAg (HBV surface antigen), HBcAg (HBV core antigen) and a proprietary adjuvant, was designed to induce virus‐specific cellular immune responses such as are typically correlated with HBV clearance. Mice immunized with the vaccine exhibited robust cellular and humoral immune responses to both HBcAg and HBsAg. Cell‐mediated responses, as determined by ELISPOT and intracellular cytokine staining (ICS), demonstrated that CD8 T cell responses were dominated by HBcAg‐specific immunity, while the majority of the CD4 response was HBsAg‐directed. The vaccine also elicited strong anti‐HBsAg and anti‐HBcAg immunoglobulin responses in both IgG1 and IgG2a isotypes. In rhesus macaques, 4 monthly immunizations induced high frequencies of antigen‐specific IFN‐g/IL‐2‐secreting T cells in response to HBsAg and HBcAg overlapping peptide pools as measured by ELISPOT and ICS. Vaccinated rhesus also exhibited >10
5
mIU/mL anti‐HBsAg Ig, after only two immunizations, and substantial levels of anti‐HBcAg Ig as well. These data suggest this dual‐antigen immunotherapeutic vaccine may be valuable in the treatment of chronic HBV infection.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The in vivo persistence of adoptively transferred T cells is predictive of antitumor response. Identifying functional properties of infused T cells that lead to in vivo persistence and tumor ...eradication has remained elusive. We profiled CD19-specific chimeric antigen receptor (CAR) T cells as the infusion products used to treat large B cell lymphomas using high-throughput single-cell technologies based on time-lapse imaging microscopy in nanowell grids (TIMING), which integrates killing, cytokine secretion, and transcriptional profiling. Our results show that the directional migration of CD19-specific CAR T cells is correlated with multifunctionality. We showed that CD2 on T cells is associated with directional migration and that the interaction between CD2 on T cells and CD58 on lymphoma cells accelerates killing and serial killing. Consistent with this, we observed that elevated CD58 expression on pretreatment tumor samples in patients with relapsed or refractory large B cell lymphomas treated with CD19-specific CAR T cell therapy was associated with complete clinical response and survival. These results highlight the importance of studying dynamic T cell-tumor cell interactions in identifying optimal antitumor responses.
PDF
