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Activation of immune receptors, such as Toll-like (TLR), NOD-like (NLR) and Stimulator of Interferon Genes (STING) is critical for efficient innate and adaptive immunity. Gram-negative bacteria (G-NB) contain multiple TLR, NOD and STING agonists. Potential utility of G-NB for cancer immunotherapy is supported by observations of tumor regression in the setting of infection and Coley's Toxins. Coley reported that intravenous (i.v.) administration was likely most effective but produced uncontrollable toxicity. The discovery of TLRs and their agonists, particularly the potent TLR4 agonist lipopolysaccharide (LPS)-endotoxin, comprising ~75% of the outer membrane of G-NB, suggests that LPS may be both a critical active ingredient and responsible for dose-limiting i.v. toxicity of G-NB. This communication reports the production of killed, stabilized, intact bacteria products from non-pathogenic G-NB with ~96% reduction of LPS-endotoxin activity. One resulting product candidate, Decoy10, was resistant to standard methods of cell disruption and contained TLR2,4,8,9, NOD2 and STING agonist activity. Decoy10 also exhibited reduced i.v. toxicity in mice and rabbits, and a largely uncompromised ability to induce cytokine and chemokine secretion by human immune cells in vitro , all relative to unprocessed, parental bacterial cells. Decoy10 and a closely related product, Decoy20, produced single agent anti-tumor activity or combination-mediated durable regression of established subcutaneous, metastatic or orthotopic colorectal, hepatocellular (HCC), pancreatic, and non-Hodgkin's lymphoma (NHL) tumors in mice, with induction of both innate and adaptive immunological memory (syngeneic and human tumor xenograft models). Decoy bacteria combination-mediated regressions were observed with a low-dose, oral non-steroidal anti-inflammatory drug (NSAID), anti-PD-1 checkpoint therapy, low-dose cyclophosphamide (LDC), and/or a targeted antibody (rituximab). Efficient tumor eradication was associated with plasma expression of 15-23 cytokines and chemokines, broad induction of cytokine, chemokine, innate and adaptive immune pathway genes in tumors, cold to hot tumor inflammation signature transition, and required NK, CD4+ and CD8+ T cells, collectively demonstrating a role for both innate and adaptive immune activation in the anti-tumor immune response. [ABSTRACT FROM AUTHOR]

Lymphangioleiomyomatosis (LAM) is a devastating disease primarily found in women of reproductive age that leads to cystic destruction of the lungs. Recent work has shown that LAM causes immunosuppression and that checkpoint inhibitors can be used as LAM treatment. Toll-like receptor (TLR) agonists can also reactivate immunity, and the TLR9 agonist CpG oligodeoxynucleotide (CpG-ODN) has been effective in treating lung cancer in animal models. In this study, we investigated the use of TLR9 agonist CpG-ODN as LAM immunotherapy in combination with checkpoint inhibitor anti-PD1 and standard of care rapamycin, and determined the immune mechanisms underlying therapeutic efficacy. We used survival studies, flow cytometry, ELISA, and histology to assess immune response and survival after intranasal treatment with CpG-ODN in combination with rapamycin or anti-PD1 therapy in a mouse model of metastatic LAM. We found that local administration of CpG-ODN enhances survival in a mouse model of LAM. We found that a lower dose led to longer survival, likely because of fewer local side effects, but increased LAM nodule count and size compared with the higher dose. CpG-ODN treatment also reduced regulatory T cells and increased the number of T-helper type 17 cells as well as cytotoxic T cells. These effects appear to be mediated in part by plasmacytoid dendritic cells because depletion of plasmacytoid dendritic cells reduces survival and abrogates T-helper type 17 cell response. Finally, we found that CpG-ODN treatment is effective in early-stage and progressive disease and is additive with anti-PD1 therapy and rapamycin. In summary, we have found that TLR9 agonist CpG-ODN can be used as LAM immunotherapy and effectively synergizes with rapamycin and anti-PD1 therapy in LAM. [ABSTRACT FROM AUTHOR]

Activation of immune receptors, such as Toll-like (TLR), NOD-like (NLR) and Stimulator of Interferon Genes (STING) is critical for efficient innate and adaptive immunity. Gram-negative bacteria (G-NB) contain multiple TLR, NOD and STING agonists. Potential utility of G-NB for cancer immunotherapy is supported by observations of tumor regression in the setting of infection and Coley’s Toxins. Coley reported that intravenous (i.v.) administration was likely most effective but produced uncontrollable toxicity. The discovery of TLRs and their agonists, particularly the potent TLR4 agonist lipopolysaccharide (LPS)-endotoxin, comprising ~75% of the outer membrane of G-NB, suggests that LPS may be both a critical active ingredient and responsible for dose-limiting i.v. toxicity of G-NB. This communication reports the production of killed, stabilized, intact bacteria products from non-pathogenic G-NB with ~96% reduction of LPS-endotoxin activity. One resulting product candidate, Decoy10, was resistant to standard methods of cell disruption and contained TLR2,4,8,9, NOD2 and STING agonist activity. Decoy10 also exhibited reduced i.v. toxicity in mice and rabbits, and a largely uncompromised ability to induce cytokine and chemokine secretion by human immune cells in vitro, all relative to unprocessed, parental bacterial cells. Decoy10 and a closely related product, Decoy20, produced single agent anti-tumor activity or combination-mediated durable regression of established subcutaneous, metastatic or orthotopic colorectal, hepatocellular (HCC), pancreatic, and non-Hodgkin’s lymphoma (NHL) tumors in mice, with induction of both innate and adaptive immunological memory (syngeneic and human tumor xenograft models). Decoy bacteria combination-mediated regressions were observed with a low-dose, oral non-steroidal anti-inflammatory drug (NSAID), anti-PD-1 checkpoint therapy, low-dose cyclophosphamide (LDC), and/or a targeted antibody (rituximab). Efficient tumor eradication was associated with plasma expression of 15-23 cytokines and chemokines, broad induction of cytokine, chemokine, innate and adaptive immune pathway genes in tumors, cold to hot tumor inflammation signature transition, and required NK, CD4+ and CD8+ T cells, collectively demonstrating a role for both innate and adaptive immune activation in the anti-tumor immune response.

Influenza A viruses (IAVs) pose a significant global threat to human health. A tightly controlled host immune response is critical to avoid any detrimental effects of IAV infection. It is critical to investigate the association between the response of Toll-like receptors (TLRs) and influenza virus. Because TLRs may act as a double-edged sword, a balanced TLR response is critical for the overall benefit of the host. Consequently, a thorough understanding of the TLR response is essential for targeting TLRs as a novel therapeutic and prophylactic intervention. To date, a limited number of studies have assessed TLR and IAV interactions. Therefore, further research on TLR interactions in IAV infection should be conducted to determine their role in host–virus interactions in disease causation or clearance of the virus. Although influenza virus vaccines are available, they have limited efficacy, which should be enhanced to improve their efficacy. In this study, we discuss the current status of our understanding of the TLR response in IAV infection and the strategies adopted by IAVs to avoid TLR-mediated immune surveillance, which may help in devising new therapeutic or preventive strategies. Furthermore, recent advances in the use of TLR agonists as vaccine adjuvants to enhance influenza vaccine efficacy are discussed. [ABSTRACT FROM AUTHOR]

Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii, a designated potential agent of bioterrorism because of its route of transmission, resistance to disinfectants, and low infectious dose. The only vaccine licensed for human use is Q-VAX® (Seqirus, licensed in Australia), a formalin-inactivated whole-cell vaccine, which produces severe local and systemic reactogenic responses in previously sensitized individuals. Accordingly, the U.S. Food and Drug Administration and other regulatory bodies around the world, have been reluctant to approve Q-VAX for widespread use. To obviate these adverse reactions, we prepared recombinant protein subunit vaccine candidates containing purified CBU1910, CBU0307, CBU0545, CBU0612, CBU0891, and CBU1398 proteins and TLR triagonist adjuvants. TLR triagonist adjuvants combine different TLR agonists to enhance immune responses to vaccine antigens. We tested both the protective efficacy and reactogenicity of our vaccine candidates in Hartley guinea pigs using intratracheal infection with live C. burnetii. While all of our candidates showed varying degrees of protection during challenge, local reactogenic responses were significantly reduced for one of our vaccine candidates when compared with a formalin-inactivated whole-cell vaccine. Our findings show that subunit vaccines combined with novel TLR triagonist adjuvants can generate protective immunity to C. burnetii infection while reducing reactogenic responses.

Simple Summary: Toll-like receptor (TLR) agonists induce strong immune responses, which are used as an effective adjuvant system for vaccine development. L-pampo™ is our innovative adjuvant system containing TLR2 and TLR3 agonists, and it provides robust humoral and cellular immune responses against infectious diseases. Cancer vaccines are a type of cancer immunotherapy that activate the host immune system to recognize and eliminate cancer cells. Here, we investigate the anti-tumor efficacy and the mechanism of action of vaccines formulated with L-pampo™. We demonstrate that L-pampo™ directly stimulates the maturation and function of dendritic cells, which are essential antigen-presenting cells. Additionally, the vaccine formulated with L-pampo™ induces the recruitment of dendritic cells into lymph nodes, where they prime antigen-specific T-cell responses and induce potent anti-tumor effects. Interestingly, combining vaccines formulated with L-pampo™ with immune checkpoint inhibitors induces a synergistic anti-tumor effect, indicating that L-pampo™ can be a powerful cancer vaccine adjuvant and a potent partner for combination immunotherapy. TLR agonists have emerged as an efficient cancer vaccine adjuvant system that induces robust immune responses. L-pampo™, a proprietary vaccine adjuvant of TLR2 and TLR3 agonists, promotes strong humoral and cellular immune responses against infectious diseases. In this study, we demonstrate that vaccines formulated with L-pampo™ affect the recruitment and activation of dendritic cells (DCs) in draining lymph nodes (dLNs) and leading to antigen-specific T-cell responses and anti-tumor efficacy. We analyzed DC maturation and T-cell proliferation using flow cytometry and ELISA. We determined the effect of L-pampo™ on DCs in dLNs and antigen-specific T-cell responses using flow cytometric analysis and the ELISPOT assay. We employed murine tumor models and analyzed the anti-tumor effect of L-pampo™. We found that L-pampo™ directly enhanced the maturation and cytokine production of DCs and, consequently, T-cell proliferation. OVA or OVA peptide formulated with L-pampo™ promoted DC migration into dLNs and increased activation markers and specific DC subsets within dLNs. In addition, vaccines admixed with L-pampo™ promoted antigen-specific T-cell responses and anti-tumor efficacy. Moreover, the combination of L-pampo™ with an immune checkpoint inhibitor synergistically improved the anti-tumor effect. This study suggests that L-pampo™ can be a potent cancer vaccine adjuvant and a suitable candidate for combination immunotherapy. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that play important roles in the early detection of pathogen-associated molecular patterns and shaping innate and adaptive immune responses, which may influence the consequences of infection. Similarly to other viral infections, human immunodeficiency virus type 1 (HIV-1) also modulates the host TLR response; therefore, a proper understanding of the response induced by human HIV-1 or co-infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), due to the common mode of transmission of these viruses, is essential for understanding HIV-1 pathogenesis during mono- or co-infection with HBV or HCV, as well as for HIV-1 cure strategies. In this review, we discuss the host TLR response during HIV-1 infection and the innate immune evasion mechanisms adopted by HIV-1 for infection establishment. We also examine changes in the host TLR response during HIV-1 co-infection with HBV or HCV; however, this type of study is extremely scarce. Moreover, we discuss studies investigating TLR agonists as latency-reverting agents and immune stimulators towards new strategies for curing HIV. This understanding will help develop a new strategy for curing HIV-1 mono-infection or co-infection with HBV or HCV. [ABSTRACT FROM AUTHOR]

Interferon-γ (IFN-γ) is a cytokine that plays an important role in immune regulation, especially in the activation and differentiation of immune cells. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that sense structural motifs related to pathogens and alert immune cells to the invasion. Both IFN-γ and TLR agonists have been used as immunoadjuvants to augment the efficacy of cancer immunotherapies and vaccines against infectious diseases or psychoactive compounds. In this study, we aimed to explore the potential of IFN-γ and TLR agonists being applied simultaneously to boost dendritic cell activation and the subsequent antigen presentation. In brief, murine dendritic cells were treated with IFN-γ and/or the TLR agonists, polyinosinic–polycytidylic acid (poly I:C), or resiquimod (R848). Next, the dendritic cells were stained for an activation marker, a cluster of differentiation 86 (CD86), and the percentage of CD86-positive cells was measured by flow cytometry. From the cytometric analysis, IFN-γ efficiently stimulated a considerable number of the dendritic cells, while the TLR agonists by themselves could merely activate a few compared to the control. The combination of IFN-γ with poly I:C or R848 triggered a higher amount of dendritic cell activation than IFN-γ alone. For instance, 10 ng/mL IFN-γ with 100 µg/mL poly I:C achieved 59.1% cell activation, which was significantly higher than the 33.4% CD86-positive cells obtained by 10 ng/mL IFN-γ. These results suggested that IFN-γ and TLR agonists could be applied as complementary systems to promote dendritic cell activation and antigen presentation. There might be a synergy between the two classes of molecules, but further investigation is warranted to ascertain the interaction of their promotive activities. [ABSTRACT FROM AUTHOR]

With growing interest and efforts to achieve a hepatitis B (HBV) cure, HBV therapeutics have increasingly entered the clinical testing phase. In designing an early phase clinical trial aimed at HBV cure, the heterogeneity in participants and the choice of a biomarker endpoint that signals a cure requires careful consideration. We describe the key elements to consider during the development of HBV clinical trials aimed at a functional cure, and how we have addressed them in the design of a phase II AIDS Clinical Trials Group (ACTG) study, A5394 (NCT05551273). The trial we present is for persons with both HIV and HBV, a unique population that has much to gain from an HBV cure. Our decisions on the design elements are specific to the study agent and the targeted population, but our deliberations may be informative in the emerging field of early phase HBV trials aimed at cure.

Early innate viral recognition by the host is critical for the rapid response and subsequent clearance of an infection. Innate immune cells patrol sites of infection to detect and respond to invading microorganisms including viruses. Surface Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) that can be activated by viruses even before the host cell becomes infected. However, the early activation of surface TLRs by viruses can lead to viral clearance by the host or promote pathogenesis. Thus, a plethora of research has attempted to identify specific viral ligands that bind to surface TLRs and mediate progression of viral infection. Herein, we will discuss the past two decades of research that have identified specific viral proteins recognized by cell surface-associated TLRs, how these viral proteins and host surface TLR interactions affect the host inflammatory response and outcome of infection, and address why controversy remains regarding host surface TLR recognition of viral proteins. [ABSTRACT FROM AUTHOR]

Emerging evidence indicates that the detection and clearance of cancer cells via phagocytosis induced by innate immune checkpoints play significant roles in tumor-mediated immune escape. The most well-described innate immune checkpoints are the "don't eat me" signals, including the CD47/signal regulatory protein α axis (SIRPα), PD-1/PD-L1 axis, CD24/SIGLEC-10 axis, and MHC-I/LILRB1 axis. Molecules have been developed to block these pathways and enhance the phagocytic activity against tumors. Several clinical studies have investigated the safety and efficacy of CD47 blockades, either alone or in combination with existing therapy in hematological malignancies, including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and lymphoma. However, only a minority of patients have significant responses to these treatments alone. Combining CD47 blockades with other treatment modalities are in clinical studies, with early results suggesting a synergistic therapeutic effect. Targeting macrophages with bispecific antibodies are being explored in blood cancer therapy. Furthermore, reprogramming of pro-tumor macrophages to anti-tumor macrophages, and CAR macrophages (CAR-M) demonstrate anti-tumor activities. In this review, we elucidated distinct types of macrophage-targeted strategies in hematological malignancies, from preclinical experiments to clinical trials, and outlined potential therapeutic approaches being developed. [ABSTRACT FROM AUTHOR]

Interferon-γ (IFN-γ) is a cytokine that plays an important role in immune regulation, especially in the activation and differentiation of immune cells. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that sense structural motifs related to pathogens and alert immune cells to the invasion. Both IFN-γ and TLR agonists have been used as immunoadjuvants to augment the efficacy of cancer immunotherapies and vaccines against infectious diseases or psychoactive compounds. In this study, we aimed to explore the potential of IFN-γ and TLR agonists being applied simultaneously to boost dendritic cell activation and the subsequent antigen presentation. In brief, murine dendritic cells were treated with IFN-γ and/or the TLR agonists, polyinosinic–polycytidylic acid (poly I:C), or resiquimod (R848). Next, the dendritic cells were stained for an activation marker, a cluster of differentiation 86 (CD86), and the percentage of CD86-positive cells was measured by flow cytometry. From the cytometric analysis, IFN-γ efficiently stimulated a considerable number of the dendritic cells, while the TLR agonists by themselves could merely activate a few compared to the control. The combination of IFN-γ with poly I:C or R848 triggered a higher amount of dendritic cell activation than IFN-γ alone. For instance, 10 ng/mL IFN-γ with 100 µg/mL poly I:C achieved 59.1% cell activation, which was significantly higher than the 33.4% CD86-positive cells obtained by 10 ng/mL IFN-γ. These results suggested that IFN-γ and TLR agonists could be applied as complementary systems to promote dendritic cell activation and antigen presentation. There might be a synergy between the two classes of molecules, but further investigation is warranted to ascertain the interaction of their promotive activities.

Toll-like receptors (TLRs) are a class of pattern recognition receptors that play a critical role in innate and adaptive immunity. Toll-like receptor agonists (TLRa) as vaccine adjuvant candidates have become one of the recent research hotspots in the cancer immunomodulatory field. Nevertheless, numerous current systemic deliveries of TLRa are inappropriate for clinical adoption due to their low efficiency and systemic adverse reactions. TLRa-loaded nanoparticles are capable of ameliorating the risk of immune-related toxicity and of strengthening tumor suppression and eradication. Herein, we first briefly depict the patterns of TLRa, followed by the mechanism of agonists at those targets. Second, we summarize the emerging applications of TLRa-loaded nanomedicines as state-of-the-art strategies to advance cancer immunotherapy. Additionally, we outline perspectives related to the development of nanomedicine-based TLRa combined with other therapeutic modalities for malignancies immunotherapy. [ABSTRACT FROM AUTHOR]

Purpose: The goal of this study was to characterize an acellular pertussis vaccine (Tdap) containing genetically modified pertussis toxin (gdPT) and TLR agonist adsorbed to AlOOH adjuvant. Methods: Several analytical tools including nanoDSF, FTIR, and LD were used to examine the conformation of novel gdPT and the composition of AlOOH adjuvant formulations adsorbed to pertussis vaccine. Results: DLS particle size results were 9.3 nm and 320 nm for gdPT. For pertussis toxoid (PT), the DLS particle size results were larger at ~440 nm. After adsorption to AlOOH, which was driven by the protein antigen, the size distribution ranged from 3.5 to 22 µm. Two thermal transitions were observed by DSC for gdPT at 70 °C and 102 °C. The main thermal transition was confirmed to be at 72 °C by nanoDSF. All three vaccine formulations showed one thermal transition: Tdap-AlOOH had a thermal transition of 74.6 °C, Tdap-E6020-AlOOH had a thermal transition at 74.2 °C, and Tdap-CpG-AlOOH had a thermal transition at 77.0 °C. Analysis of pertussis toxin (PTx) and gdPT was also performed by FTIR spectroscopy for the purpose of comparison. The second derivative of the FTIR spectra showed an additional feature for PTx at 1685 cm−1 compared to gdPT. The antigen’s amide I and II regions were largely unchanged after adsorption to AlOOH adjuvant as shown by FTIR, suggesting that there were no significant changes in the secondary structure. Conclusion: gdPT conformation was successfully characterized using an array of analytical methods. All three Tdap formulations have similar thermal stability as shown by nanoDSF, similar size distribution as shown by LD, and similar overall secondary structure as shown by FTIR. In-line particle sizing and IR can be used as in-process characterization tools to monitor consistency of adsorbed vaccine and to confirm product identity.

Early innate viral recognition by the host is critical for the rapid response and subsequent clearance of an infection. Innate immune cells patrol sites of infection to detect and respond to invading microorganisms including viruses. Surface Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) that can be activated by viruses even before the host cell becomes infected. However, the early activation of surface TLRs by viruses can lead to viral clearance by the host or promote pathogenesis. Thus, a plethora of research has attempted to identify specific viral ligands that bind to surface TLRs and mediate progression of viral infection. Herein, we will discuss the past two decades of research that have identified specific viral proteins recognized by cell surface-associated TLRs, how these viral proteins and host surface TLR interactions affect the host inflammatory response and outcome of infection, and address why controversy remains regarding host surface TLR recognition of viral proteins.

Conventional cancer vaccines based on soluble vaccines and traditional adjuvants have produced suboptimal therapeutic efficacy in clinical trials. Thus, there is an urgent need for vaccine technologies that can generate potent T cell responses with strong anti-tumor efficacy. We have previously reported the development of synthetic high-density protein (sHDL) nanodiscs for efficient lymph node (LN)-targeted co-delivery of antigen peptides and CpG oligonucleotides (a Toll-like receptor-9 agonist). Here, we performed a comparative study in mice and non-human primates (NHPs) to identify an ideal vaccine platform for induction of CD8+ T cell responses. In particular, we compared the efficacy of CpG class B, CpG class C, and polyICLC (a synthetic double-stranded RNA analog, a TLR-3 agonist), each formulated with antigen-carrying sHDL nanodiscs. Here, we report that sHDL-Ag admixed with polyICLC elicited robust Ag-specific CD8+ T cell responses in mice, and when used in combination with α-PD-1 immune checkpoint inhibitor, sHDL-Ag + polyICLC eliminated large established (~100 mm3) MC-38 tumors in mice. Moreover, sHDL-Gag + polyICLC induced robust Simian immunodeficiency virus Gag-specific, polyfunctional CD8+ T cell responses in rhesus macaques and could further amplify the efficacy of recombinant adenovirus-based vaccine. Notably, while both sHDL-Ag-CpG-B and sHDL-Ag-CpG-C generated strong Ag-specific CD8+ T cell responses in mice, their results were mixed in NHPs. Overall, sHDL combined with polyICLC offers a strong platform to induce CD8+ T cells for vaccine applications. [Display omitted] • Nano-vaccine elicits robust CD8+ T cell responses in mice and non-human primates. • Nano-vaccine combined with anti-PD-1 IgG therapy exert robust anti-tumor efficacy. • Nano-vaccine + polyICLC induce stronger T cell response than those with CpC-B or -C. [ABSTRACT FROM AUTHOR]

Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii, a designated potential agent of bioterrorism because of its route of transmission, resistance to disinfectants, and low infectious dose. The only vaccine licensed for human use is Q-VAX® (Seqirus, licensed in Australia), a formalin-inactivated whole-cell vaccine, which produces severe local and systemic reactogenic responses in previously sensitized individuals. Accordingly, the U.S. Food and Drug Administration and other regulatory bodies around the world, have been reluctant to approve Q-VAX for widespread use. To obviate these adverse reactions, we prepared recombinant protein subunit vaccine candidates containing purified CBU1910, CBU0307, CBU0545, CBU0612, CBU0891, and CBU1398 proteins and TLR triagonist adjuvants. TLR triagonist adjuvants combine different TLR agonists to enhance immune responses to vaccine antigens. We tested both the protective efficacy and reactogenicity of our vaccine candidates in Hartley guinea pigs using intratracheal infection with live C. burnetii. While all of our candidates showed varying degrees of protection during challenge, local reactogenic responses were significantly reduced for one of our vaccine candidates when compared with a formalin-inactivated whole-cell vaccine. Our findings show that subunit vaccines combined with novel TLR triagonist adjuvants can generate protective immunity to C. burnetii infection while reducing reactogenic responses.

Toll-like receptors (TLRs) are a class of pattern recognition receptors that play a critical role in innate and adaptive immunity. Toll-like receptor agonists (TLRa) as vaccine adjuvant candidates have become one of the recent research hotspots in the cancer immunomodulatory field. Nevertheless, numerous current systemic deliveries of TLRa are inappropriate for clinical adoption due to their low efficiency and systemic adverse reactions. TLRa-loaded nanoparticles are capable of ameliorating the risk of immune-related toxicity and of strengthening tumor suppression and eradication. Herein, we first briefly depict the patterns of TLRa, followed by the mechanism of agonists at those targets. Second, we summarize the emerging applications of TLRa-loaded nanomedicines as state-of-the-art strategies to advance cancer immunotherapy. Additionally, we outline perspectives related to the development of nanomedicine-based TLRa combined with other therapeutic modalities for malignancies immunotherapy.

Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii , a designated potential agent of bioterrorism because of its route of transmission, resistance to disinfectants, and low infectious dose. The only vaccine licensed for human use is Q-VAX® (Seqirus, licensed in Australia), a formalin-inactivated whole-cell vaccine, which produces severe local and systemic reactogenic responses in previously sensitized individuals. Accordingly, the U.S. Food and Drug Administration and other regulatory bodies around the world, have been reluctant to approve Q-VAX for widespread use. To obviate these adverse reactions, we prepared recombinant protein subunit vaccine candidates containing purified CBU1910, CBU0307, CBU0545, CBU0612, CBU0891, and CBU1398 proteins and TLR triagonist adjuvants. TLR triagonist adjuvants combine different TLR agonists to enhance immune responses to vaccine antigens. We tested both the protective efficacy and reactogenicity of our vaccine candidates in Hartley guinea pigs using intratracheal infection with live C. burnetii. While all of our candidates showed varying degrees of protection during challenge, local reactogenic responses were significantly reduced for one of our vaccine candidates when compared with a formalin-inactivated whole-cell vaccine. Our findings show that subunit vaccines combined with novel TLR triagonist adjuvants can generate protective immunity to C. burnetii infection while reducing reactogenic responses. [ABSTRACT FROM AUTHOR]

Toll-like receptor 7/8 agonists, such as imidazoquinolines (IMDQs), are promising for the de novo priming of antitumor immunity. However, their systemic administration is severely limited due to the off-target toxicity. Here, this work describes a sequential drug delivery strategy. The formulation is composed of two sequential modules: a tumor microenvironment remodeling nanocarrier (poly(l-glutamic acid)-graft-methoxy poly(ethylene glycol)/combretastatin A4, termed CA4-NPs) and an immunotherapy nanocarrier (apcitide peptide-decorated poly(l-glutamic acid)-graft-IMDQ-N 3 conjugate, termed apcitide-PLG-IMDQ-N 3 ). CA4-NPs, as a vascular disrupting agent, are utilized to remodel the tumor microenvironment for enhancing tumor coagulation and hypoxia. Subsequently, the apcitide-PLG-IMDQ-N 3 could identify and target tumor coagulation through the binding of surface apcitide peptide to the GPIIb-IIIa on activated platelets. Afterward, IMDQ is activated selectively through the conversion of "-N 3 " to "-NH 2 " in the presence of hypoxia. The biodistribution results confirm their high tumor uptake of activated IMDQ (22.66%ID/g). By augmenting the priming and immunologic memory of tumor-specific CD8 + T cells, 4T1 and CT26 tumors with a size of ≈500 mm 3 are eradicated without recurrence in mouse models., (© 2024 Wiley‐VCH GmbH.)

Abstract Objectives Loss of tumor‐inherent type I interferon (IFN) signalling has been closely linked to accelerated metastatic progression via decreased immunogenicity and antitumor immunity. Previous studies in murine models of triple‐negative breast cancer (TNBC) demonstrate that systemic IFN inducers are effective antimetastatic agents, via sustained antitumor CD8+ T‐cell responses. Repeated systemic dosing with recombinant IFNs or IFN inducers is associated with significant toxicities; hence, the use of alternate intratumoral agents is an active area of investigation. It is critical to investigate the impact of intratumoral agents on subsequent metastatic spread to predict clinical impact. Methods In this study, the local and systemic impact of the intratumoral Toll‐like receptor (TLR) 7/8 agonist 3M‐052 alone or in combination with anti‐PD1 was evaluated in metastatic TNBC models. The IFN‐α receptor (IFNAR1) blocking antibody, MAR1‐5A3, along with immune‐deficient mice and ex vivo assays are utilised to examine the key targets of this agent that are critical for an antimetastatic response. Results Single intratumoral administration of 3M‐052 reduced mammary tumor growth, induced a T‐cell‐inflamed tumor microenvironment (TME) and reduced metastatic spread to lung. Metastasis suppression was reliant on IFN signalling and an antitumor immune response, in contrast to primary tumor growth inhibition, which was retained in NSG and CD8+ T‐cell‐depleted mice. 3M‐052 action was demonstrated via dendritic cell activation and production of type I IFN and other pro‐inflammatory cytokines to initiate a T‐cell‐inflamed TME and promote tumor cell antigen presentation. Conclusion This work supports neoadjuvant TLR agonist‐based immunotherapeutics as realistic options for immune activation in the TME and long‐term metastatic protection in TNBC.

Objectives: Loss of tumor‐inherent type I interferon (IFN) signalling has been closely linked to accelerated metastatic progression via decreased immunogenicity and antitumor immunity. Previous studies in murine models of triple‐negative breast cancer (TNBC) demonstrate that systemic IFN inducers are effective antimetastatic agents, via sustained antitumor CD8+ T‐cell responses. Repeated systemic dosing with recombinant IFNs or IFN inducers is associated with significant toxicities; hence, the use of alternate intratumoral agents is an active area of investigation. It is critical to investigate the impact of intratumoral agents on subsequent metastatic spread to predict clinical impact. Methods: In this study, the local and systemic impact of the intratumoral Toll‐like receptor (TLR) 7/8 agonist 3M‐052 alone or in combination with anti‐PD1 was evaluated in metastatic TNBC models. The IFN‐α receptor (IFNAR1) blocking antibody, MAR1‐5A3, along with immune‐deficient mice and ex vivo assays are utilised to examine the key targets of this agent that are critical for an antimetastatic response. Results: Single intratumoral administration of 3M‐052 reduced mammary tumor growth, induced a T‐cell‐inflamed tumor microenvironment (TME) and reduced metastatic spread to lung. Metastasis suppression was reliant on IFN signalling and an antitumor immune response, in contrast to primary tumor growth inhibition, which was retained in NSG and CD8+ T‐cell‐depleted mice. 3M‐052 action was demonstrated via dendritic cell activation and production of type I IFN and other pro‐inflammatory cytokines to initiate a T‐cell‐inflamed TME and promote tumor cell antigen presentation. Conclusion: This work supports neoadjuvant TLR agonist‐based immunotherapeutics as realistic options for immune activation in the TME and long‐term metastatic protection in TNBC. [ABSTRACT FROM AUTHOR]

Human trichinellosis is acquired by eating raw or undercooked meats carrying muscle larvae of Trichinella spp. Toll-like receptors (TLRs) are essential components of the innate immune system. However, little is known about the potential application of TLR agonists for immunotherapy against Trichinella spiralis (T. spiralis) infection. Here, we evaluated the effects of four TLR agonists (i.e., TLR3, TLR4, TLR8, and TLR9 agonists) on T. spiralis infection in mice. The reduction rate of worm burden showed that TLR3 agonist poly(I:C) significantly reduced T. spiralis infection rather than TLR4, TLR8, and TLR9 agonists (p < 0.05). Moreover, TLR3 showed a continuous high-level of expression during 6–35 days post infection (dpi). The levels of interferon-gamma (IFN-γ), interleukin (IL)-2, and IL-6 increased significantly in mice serum compared with control group after treatment with TLR3 agonist at 0, 3, 6, 9, 12, 15, 18, 21, 28, and 35 dpi (p < 0.05). A significant decreasing trend was also detected in levels of IL-10 and IL-4 after treatment with TLR3 agonist compared with control group at 0, 3, 6, 9, 12, 15, 18, 21, 28, and 35 dpi (p < 0.05). Overall, this study suggested that TLR3-targeted therapies might be effective on worm burden reduction by regulation of the cytokine levels in the mice infected with T. spiralis. [ABSTRACT FROM AUTHOR]

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to coronavirus disease (COVID-19), a global health pandemic causing millions of deaths worldwide. However, the immunopathogenesis of COVID-19, particularly the interaction between SARS-CoV-2 and host innate immunity, remains unclear. The innate immune system acts as the first line of host defense, which is critical for the initial detection of invading pathogens and the activation and shaping of adaptive immunity. Toll-like receptors (TLRs) are key sensors of innate immunity that recognize pathogen-associated molecular patterns and activate downstream signaling for pro-inflammatory cytokine and chemokine production. However, TLRs may also act as a double-edged sword, and dysregulated TLR responses may enhance immune-mediated pathology, instead of providing protection. Therefore, a proper understanding of the interaction between TLRs and SARS-CoV-2 is of great importance for devising therapeutic and preventive strategies. The use of TLR agonists as vaccine adjuvants for human disease is a promising approach that could be applied in the investigation of COVID-19 vaccines. In this review, we discuss the recent progress in our understanding of host innate immune responses in SARS-CoV-2 infection, with particular focus on TLR response. In addition, we discuss the use of TLR agonists as vaccine adjuvants in enhancing the efficacy of COVID-19 vaccine.

As a currently spotlighted method for cancer treatment, cancer immunotherapy has made a lot of progress in recent years. Among tremendous cancer immunotherapy boosters available nowadays, Toll-like receptor (TLR) agonists were specifically selected, because of their effective activation of innate and adaptive immune cells, such as dendritic cells (DCs), T cells, and macrophages. TLR agonists can activate signaling pathways of DCs to express CD80 and CD86 molecules, and secrete various cytokines and chemokines. The maturation of DCs stimulates naïve T cells to differentiate into functional cells, and induces B cell activation. Although TLR agonists have anti-tumor ability by activating the immune system of the host, their drawbacks, which include poor efficiency and remarkably short retention time in the body, must be overcome. In this review, we classify and summarize the recently reported delivery strategies using (1) exogenous TLR agonists to maintain the biological and physiological signaling activities of cargo agonists, (2) usage of multiple TLR agonists for synergistic immune responses, and (3) co-delivery using the combination with other immunomodulators or stimulants. In contrast to naked TLR agonists, these exogenous TLR delivery strategies successfully facilitated immune responses and subsequently mediated anti-tumor efficacy.

The development of COVID-19 vaccines is critical in controlling global health issues under the COVID-19 pandemic. The subunit vaccines are the safest and most widely used vaccine platform and highly effective against a multitude of infectious diseases. An adjuvant is essential for subunit vaccines to enhance the magnitude and durability of immune responses. In this study, we determined whether a combination of toll-like receptor (TLR)1/2 and TLR3 agonists (L-pampo) can be a potent adjuvant for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine. We measured a neutralizing antibody (nAb) and an angiotensin-converting enzyme 2 (ACE2) receptor-blocking antibody against SARS-CoV-2 receptor-binding domain (RBD). We also detected interferon-gamma (IFN-γ) production by using ELISPOT and ELISA assays. By employing a ferret model, we detected nAbs and IFN-γ producing cells and measured viral load in nasal wash after the challenge of SARS-CoV-2. We found that SARS-CoV-2 antigens with L-pampo stimulated robust humoral and cellular immune responses. The efficacy of L-pampo was higher than the other adjuvants. Furthermore, in the ferret model, SARS-CoV-2 antigens with L-pampo elicited nAb response and antigen-specific cellular immune response against SARS-CoV-2, resulting in substantially decreased viral load in their nasal wash. Our study suggests that SARS-CoV-2 antigens formulated with TLR agonists, L-pampo, can be a potent subunit vaccine to promote sufficient protective immunity against SARS-CoV-2.

Adjuvants have been used in vaccines for over a century, however, the search for safe and effective vaccine adjuvants continues. In recent decades toll-like-receptor (TLR) agonists have been investigated as potential vaccine adjuvants. In this regard, the majority of the currently investigated TLR agonists are non-protein microbial components such as lipopolysaccharides, oligonucleotides, and lipopeptides. On the other hand, a growing number of studies reveal that TLR signaling and immune responses can be activated by numerous bacterial proteins. However, their potential roles as adjuvants have been somewhat overlooked. Herein, we discuss several such bacterial proteins which exhibit adjuvant properties, including the activation of TLR signaling, antigen presenting cell maturation, pro-inflammatory cytokine production and adaptive immune response. The protein nature of these TLR agonists presents several unique features not shared by non-protein TLR agonists. These properties include the amenability for modifying the structure and function as necessary for optimal immunogenicity and minimal toxicity. Protein adjuvants can be genetically fused to protein antigens which ensure the co-delivery of adjuvant-antigen not only into the same cell but also in the same endocytic cargo, leading to more effective activation of innate and adaptive immune response.

Systems vaccinology approaches have been used successfully to define early signatures of the vaccine-induced immune response. However, the possibility that transcriptomics can also identify a correlate or surrogate for vaccine inflammation has not been fully explored. We have compared four licensed vaccines with known safety profiles, as well as three agonists of Toll-like receptors (TLRs) with known inflammatory potential, to elucidate the transcriptomic profile of an acceptable response to vaccination versus that of an inflammatory reaction. In mice, we looked at the transcriptomic changes in muscle at the injection site, the lymph node that drained the muscle, and the peripheral blood mononuclear cells (PBMCs)isolated from the circulating blood from 4 hr after injection and over the next week. A detailed examination and comparative analysis of these transcriptomes revealed a set of novel biomarkers that are reflective of inflammation after vaccination. These biomarkers are readily measurable in the peripheral blood, providing useful surrogates of inflammation, and provide a way to select candidates with acceptable safety profiles.

The presence of inhibitory immune cells and difficulty in generating activated effector T cells remain obstacles to development of effective cancer vaccines. We designed a vaccine regimen combining human telomerase reverse transcriptase (hTERT) peptides with concomitant therapies targeting regulatory T cells (Tregs) and cyclooxygenase-2 (COX2)-mediated immunosuppression. This Phase 1 trial combined an hTERT-derived 7-peptide library, selected to ensure presentation by both HLA class-I and class-II in 90% of patients, with oral low-dose cyclophosphamide (to modulate Tregs) and the COX2 inhibitor celecoxib. Adjuvants were Montanide and topical TLR-7 agonist, to optimise antigen presentation. The primary objective was determination of the safety and tolerability of this combination therapy, with anti-cancer activity, immune response and detection of antigen-specific T cells as additional endpoints. Twenty-nine patients with advanced solid tumours were treated. All were multiply-pretreated, and the majority had either colorectal or prostate cancer. The most common adverse events were injection-site reactions, fatigue and nausea. Median progression-free survival was 9 weeks, with no complete or partial responses, but 24% remained progression-free for ≥6 months. Immunophenotyping showed post-vaccination expansion of CD4 + and CD8 + T cells with effector phenotypes. The in vitro re-challenge of T cells with hTERT peptides, TCR sequencing, and TCR similarity index analysis demonstrated the expansion following vaccination of oligoclonal T cells with specificity for hTERT. However, a population of exhausted PD-1 + cytotoxic T cells was also expanded in vaccinated patients. This vaccine combination regimen was safe and associated with antigen-specific immunological responses. Clinical activity could be improved in future by combination with anti-PD1 checkpoint inhibition to address the emergence of an exhausted T cell population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zareian, Eremin, Pandha, Baird, Kwatra, Funingana, Verma, Choy, Hargreaves, Moghimi, Shepherd, Lobo, Eremin, Farzaneh, Kordasti and Spicer.)

It is critical to emphasize the importance of vaccination as it protects us against harmful pathogens. Despite significant progress in vaccine development, there is an ongoing need to develop vaccines that are not only safe but also highly effective in protecting against severe infections. Subunit vaccines are generally safe, but they frequently fail to elicit strong immune responses. As a result, there is a need to improve vaccine effectiveness by combining them with adjuvants, which have the potential to boost the immune system many folds. The process of developing these adjuvants requires searching for molecules capable of activating the immune system, combining these promising compounds with an antigen, and then testing this combination using animal models before approving it for clinical use. Liposomal adjuvants work as delivery adjuvants and its activity depends on certain parameters such as surface charge, vesicle size, surface modification and route of administration. Self-assembly property of peptide adjuvants and discovery of hybrid peptides have widened the scope of peptides in vaccine formulations. Since most pathogenic molecules are not peptide based, phage display technique allows for screening peptide mimics for such pathogens that have potential as adjuvants. This chapter discusses about peptide and liposome-based adjuvants focusing on their properties imparting adjuvanticity along with the methods of formulating them. Methods of adjuvant characterization important for an adjuvant to be approved for clinical trials are also discussed. These include assays for cytotoxicity, T-lymphocyte proliferation, dendritic cell maturation, cytokine and antibody production, toll-like receptor dependent signaling and adjuvant half-life., (Copyright © 2024. Published by Elsevier Inc.)

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that mediate first line of host defence to pathogens. TLR agonists are potent immunostimulatory agents that help to prime a robust adaptive immune response. In the present study, adjuvant potential of Poly I:C and lipopolysaccharide (LPS) were evaluated with live Newcastle disease virus (NDV) vaccine. Cornish chickens were immunized with live Newcastle disease virus (NDV) vaccine (R2B-mesogenic strain) adjuvanted either with Poly I:C (TLR3 agonist) or LPS-TLR4 agonist and both. Humoral Immune response to ND vaccine was evaluated through haemagglutination inhibition (HI) test and ELISA, while the cellular immune response (CMI) was quantified by lymphocyte transformation test (LTT). IL-1β cytokine mRNA levels in spleen tissue were also quantified by real time PCR. The results suggest that TLR3 and TLR4 agonists are an efficient immune-stimulators separately, as LPS co-administered group has shown significantly higher serum titre on second week post-immunization and Poly I:C group on third week post-immunization both by HI and ELISA (P < 0.01), however, the combined administration of both LPS and Poly I:C did not give any complementary effect on serum titre. There were no significant differences in stimulation indices (SI) and IL-1β cytokine levels between groups at different intervals post-immunization. Hence, TLR agonists LPS followed by Poly I:C could be used as adjuvant to enhance the immune response to NDV vaccine in chicken. [ABSTRACT FROM AUTHOR]

• GLA-SE combined with EBOV VLP confers lasting protection against ma-EBOV. • GLA-SE inclusion elicits Th1-skewed antibodies. • GLA-SE enhances EBOV-specific polyfunctional T cells. The identification of adjuvants that promote lasting antigen-specific immunity and augment vaccine efficacy are integral to the development of new protein-based vaccines. The Ebola virus-like particle (VLP) vaccine expressing Ebola virus glycoprotein (GP) and matrix protein (VP40) was used in this study to evaluate the ability of TLR4 agonist glucopyranosyl lipid adjuvant (GLA) formulated in a stable emulsion (SE) to enhance immunogenicity and promote durable protection against mouse-adapted Ebola virus (ma-EBOV). Antibody responses and Ebola-specific T cell responses were evaluated post vaccination. Survival analysis after lethal ma-EBOV challenge was performed 4 weeks and 22 weeks following final vaccination. GLA-SE enhanced EBOV-specific immunity and resulted in long-term protection against challenge with ma-EBOV infection in a mouse model. Specifically, GLA-SE elicited Th1-skewed antibodies and promoted the generation of EBOV GP-specific polyfunctional T cells. These results provide further support for the utility of TLR4 activating GLA-SE-adjuvanted vaccines. [ABSTRACT FROM AUTHOR]

Self-amplifying RNA (saRNA) is well suited as a vaccine platform against chlamydia, as it is relatively affordable and scalable, has been shown to induce immunity against multivalent antigens, and can result in protein expression for up to 60 days. Cationic adjuvant formulations (CAFs) have been previously investigated as an adjuvant for protein subunit vaccines; here we optimize the CAFs for delivery of saRNA in vivo and observe the immunogenicity profile in the context of both cellular and humoral immunity against the major outer membrane protein (MOMP) of Chlamydia trachomatis. We tested both liposomal and emulsion based CAFs with solid and fluid phase lipids, with or without the TLR agonists R848 and 3M-052, for in vitro transfection efficiency and cytotoxicity. We then optimized the RNA/delivery system ratio for in vivo delivery using saRNA coding for firefly luciferase (fLuc) as a reporter protein in vivo. We observed that while the fluid phase liposome formulations showed the highest in vitro transfection efficiency, the fluid and solid phase liposomes had equivalent luciferase expression in vivo. Incorporation of R848 or 3M-052 into the formulation was not observed to affect the delivery efficiency of saRNA either in vitro or in vivo. MOMP-encoding saRNA complexed with CAFs resulted in both MOMP-specific cellular and humoral immunity, and while there was a slight enhancement of IFN-γ+ T-cell responses when R848 was incorporated into the formulation, the self-adjuvanting effects of RNA appeared to dominate the immune response. These studies establish that CAFs are efficient delivery vehicles for saRNA both for in vitro transfections and in vivo immunogenicity and generate cellular and humoral responses that are proportionate to protein expression. Unlabelled Image • Cationic adjuvant formulations (CAFs) were optimized for saRNA delivery in vivo. • Fluid phase liposomes yielded the highest protein expression in vivo. • Incorporating TLR-agonists did not inhibit the saRNA delivery. • MOMP-encoding saRNA resulted in both cellular and humoral immunity. [ABSTRACT FROM AUTHOR]

Adjuvants have been used in vaccines for over a century, however, the search for safe and effective vaccine adjuvants continues. In recent decades toll-like-receptor (TLR) agonists have been investigated as potential vaccine adjuvants. In this regard, the majority of the currently investigated TLR agonists are non-protein microbial components such as lipopolysaccharides, oligonucleotides, and lipopeptides. On the other hand, a growing number of studies reveal that TLR signaling and immune responses can be activated by numerous bacterial proteins. However, their potential roles as adjuvants have been somewhat overlooked. Herein, we discuss several such bacterial proteins which exhibit adjuvant properties, including the activation of TLR signaling, antigen presenting cell maturation, pro-inflammatory cytokine production and adaptive immune response. The protein nature of these TLR agonists presents several unique features not shared by non-protein TLR agonists. These properties include the amenability for modifying the structure and function as necessary for optimal immunogenicity and minimal toxicity. Protein adjuvants can be genetically fused to protein antigens which ensure the co-delivery of adjuvant-antigen not only into the same cell but also in the same endocytic cargo, leading to more effective activation of innate and adaptive immune response. [ABSTRACT FROM AUTHOR]

• CDNs induce high titer vaccine antibody responses. • STING pathway stimulation leads to innate immune activation with low NO and ROS production. • Low NO production is associated with higher DC secretion of and concentration of serum BAFF. • In vivo inhibition of NO production by a TLR based adjuvant leads to improved antibody responses. • Relative to TLR agonists, CDNs result in high titer antibody responses in aged animals. One of the most concerning public health issues, related to vaccination and disease prevention, is the inability to induce durable immune responses following a single-dose immunization. In this regard, the nature of the inflammatory environment induced by vaccine adjuvants can negatively impact the resulting immune response. To address these concerns, new strategies to vaccine design are needed in order to improve the outcomes of immune responses, particularly in immunologically disadvantaged populations. Comparisons of the scope of innate immune activation induced by TLR agonists versus cyclic dinucleotides (CDNs) was performed. Their effects on the activation characteristics (e.g., metabolism, cytokine secretion) of bone marrow derived dendritic cells (BMDCs) were studied. In addition, the differential effects on in vivo induction of antibody responses were measured. As compared to TLR ligands, the stimulation of BMDCs with CDNs induced distinctly different metabolic outcomes. Marked differences were observed in the production of nitric oxide (NO) and the cytokine BAFF. These distinct differences were correlated with improved (i.e., more rapid and persistent) vaccine antibody responses in both aged and young mice. Our results illustrate that the innate immune pathway targeted by adjuvants can critically impact the outcome of the immune response post-vaccination. Specifically, CDN stimulation of APCs induced an activation phenotype that was characterized by decreased innate effector molecule production (e.g., NO) and increased BAFF. This was attributed to the induction of an innate inflammatory environment that enabled the host to make the most of the existing B lymphocyte potential. The use of adjuvants that differentially engage mechanisms of innate immune activation would be particularly advantageous for the generation of robust, single dose vaccines. The results of this study demonstrated that CDNs induced differential innate activation and enhanced vaccine induced antibody responses in both young and aged mice. [ABSTRACT FROM AUTHOR]

Abstract Pattern recognition receptors, including the Toll-like receptors (TLRs), are important in the induction and activation of two critical arms of the host defence to pathogens and microorganisms: the rapid innate immune response (as characterised by the production of Th1 promoting cytokines and type 1 interferons) and the adaptive immune response. Through this activation, ligands and agonists of TLRs can enhance immunotherapeutic efficacy. Resiquimod is a small (water-soluble) agonist of the endosome-located Toll-like receptors 7 and 8 (TLR7/8). However due to its molecular attributes it rapidly distributes throughout the body after injection. To circumvent this, these TLR agonists can be incorporated within delivery systems, such as liposomes, to promote the co-delivery of both antigen and agonists to antigen presenting cells. In this present study, resiquimod has been chemically conjugated to a lipid to form a lipid-TLR7/8 agonist conjugate which can be incorporated within immunogenic cationic liposomes composed of dimethyldioctadecylammonium bromide (DDA) and the immunostimulatory glycolipid trehalose 6,6′ – dibehenate (TDB). This DDA:TDB-TLR7/8 formulation offers similar vesicle characteristics to DDA:TDB (size and charge) and offers high retention of both resiquimod and the electrostatically adsorbed TB subunit antigen Ag85B-ESAT6-Rv2660c (H56). Following immunisation through the intramuscular (i.m.) route, these cationic DDA:TDB-TLR7/8 liposomes form a vaccine depot at the injection site. However, immunisation studies have shown that this biodistribution does not translate into notably increased antibody nor Th1 responses at the spleen and draining popliteal lymph node compared to DDA:TDB liposomes. This work demonstrates that the conjugation of TLR7/8 agonists to cationic liposomes can promote co-delivery but the immune responses stimulated do not merit the added complexity considerations of the formulation. Graphical abstract Unlabelled Image Highlights • Resiquimod is a small water-soluble agonist of Toll-like receptors 7 and 8. • A lipid conjugate of resiquimod was synthesised using a two-step process. • The lipid-resiquimod conjugate was incorporated into a liposomal adjuvant. • Co-delivery of antigen, TLR and liposomal adjuvant was achieved. • This co-delivery did not notably enhance the already strong adjuvant action of the cationic liposomes. [ABSTRACT FROM AUTHOR]

Improving the immunogenicity of subunit vaccines, in particular skewing of the immune response towards Th1 type immunity, is crucial for the development of effective vaccines against intracellular infections and for the development of anti-cancer vaccines. Small molecule TLR7/8 agonist hold high potential for this purpose, but suffer from an undesirable pharmacokinetic profile, resulting in systemic inflammatory responses. An effective solution to this problem is covalent ligation to a larger carrier. Here, a degradable nanogel carrier containing a covalently linked imidazoquinoline (IMDQ) TLR7/8 agonist is explored as adjuvant for vaccination against the respiratory syncytial virus (RSV). In vitro and in vivo experiments in mice provide a solid rational base for preferring nanogels over soluble polymers as IMDQ carrier in terms of cellular uptake and lymph node accumulation. [ABSTRACT FROM AUTHOR]

Peptide-based vaccines for cancer have many advantages however, for optimization these immunogens should incorporate peptide epitopes that induce CD8, as well as CD4 responses, antibody and long term immunity. Cell penetrating peptides (CPP) with a capacity of cytosolic delivery have been used to deliver antigenic peptides and proteins to antigen presenting cells to induce cytotoxic T cell, helper T cell and humoral responses in mice. For this study, a tripartite CPP including a mucin 1 (MUC1) variable number of tandem repeat (VNTR) containing multiple T cell epitopes and tetanus toxoid universal T helper epitope peptide (tetCD4) was synthesised (AntpMAPMUC1tet) and immune responses investigated in mice. Mice vaccinated with AntpMAPMUC1tet + CpG show enhanced antigen-specific interferon-gamma (IFN-γ) and IL-4 T cell responses compared with AntpMAPMUC1tet vaccination alone and induced a Th1 response, characterised by a higher ratio of IgG2a antibody/IgG1 antibodies. Furthermore, vaccination generated long term MUC1-specific antibody and T cell responses and delayed growth of MUC1+ve tumours in mice. This data demonstrates the efficient delivery of branched multiple antigen peptides incorporating CPP and that the addition of CpG augments immune responses. [ABSTRACT FROM AUTHOR]

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is characterized by severe immunosuppression in young chicks of 3 to 6 week age group. Although vaccines are available to prevent IBD, outbreaks of disease are still noticed in the field among vaccinated flocks. Further, the birds surviving IBD become susceptible to secondary infections caused by various viral and bacterial agents. This study assessed the immunoprophylactic potential of Cytosine-guanosinedeoxynucleotide (CpG) oligodeoxynucleotides (ODN) and Tinospora cordifolia stem aqueous extract in the specific pathogen free (SPF) chicks, experimentally infected with very virulent IBDV (vvIBDV). Both of these agents (CpG ODN and herbal extract) showed significant increase in the IFN-γ, IL-2, IL-4, and IL-1 levels in the peripheral blood mononuclear cells (PBMCs) (p < 0.05) of chickens in the treatment groups following IBD infection. Further we found significant reduction in mortality rate in vvIBDV infected chicks treated with either, or in combination, compared with the birds of control group. Additionally, the adjuvant or immune enhancing potential of these two immunomodulatory agents with the commercially available IBDV vaccine was determined in chicks. The augmentation of vaccine response in terms of an enhanced antibody titer after vaccination, along with either or a combination of the two agents was noticed. The findings provide a way forward to counter the menace of IBDV in the poultry sector through use of these herbal or synthetic immunomodulatory supplements.

Immunomics tools and databases play an important role in the designing of prophylactic or therapeutic vaccines against pathogenic bacteria and viruses. Therefore, we aimed to illustrate the different immunological databases and web servers used to design a chimeric vaccine candidate against human cervix papilloma. Initially, cellular immunity inducing major histocompatibility complex class I and II epitopes from L2 protein of papilloma 58 strain were predicted using the IEDB, NetMHC, and Tepi tools. Then, the overlapped segments from the above analysis were used to calculate efficiency on interferon-gamma and humoral immunity production. In addition, the allergenicity, antigenicity, cross-reactivity with human proteomes, and epitope conservancy of elite segments were determined. The chimeric vaccine candidate (SGD58) was constructed with two different overlapped peptide segments (23⁻36) and (29⁻42), adjuvants (flagellin and RS09), two Th epitopes, and amino acid linkers. The results of homology modeling demonstrated that SGD58 have 88.6% of favored regions based on Ramachandran plot. Protein⁻protein docking with Swarm Dock reveals SGD58 with receptor complex have −54.74 kcal/mol of binding energy with more than 20 interacting residues. Docked complex are stable in 100ns of molecular dynamic simulation. Further, coding sequences of SGD58 also show elevated gene expression in E. coli. In conclusion, SGD58 may prompt vaccine against cervix papilloma. This study provides insight of vaccine design against different pathogenic microbes as well.

The human papillomavirus (HPV) 58 is considered to be the second most predominant genotype in cervical cancer incidents in China. HPV type-restriction, non-targeted delivery, and the highcost of existing vaccines necessitate continuing research on the HPV vaccine. We aimed to explore the papillomaviral proteome in order to identify potential candidates for a chimeric vaccine against cervix papilloma using computational immunology and structural vaccinology approaches. Two overlapped epitope segments (23–36) and (29–42) from the N-terminal region of the HPV58 minor capsid protein L2 are selected as capable of inducing both cellular and humoral immunity. In total, 318 amino acid lengths of the vaccine construct SGD58 contain adjuvants (Flagellin and RS09), two Th epitopes, and linkers. SGD58 is a stable protein that is soluble, antigenic, and non-allergenic. Homology modeling and the structural refinement of the best models of SGD58 and TLR5 found 96.8% and 93.9% favored regions in Rampage, respectively. The docking results demonstrated a HADDOCK score of −62.5 ± 7.6, the binding energy (−30 kcal/mol) and 44 interacting amino acid residues between SGD58-TLR5 complex. The docked complex are stable in 100 ns of simulation. The coding sequences of SGD58 also show elevated gene expression in Escherichia coli with 1.0 codon adaptation index and 59.92% glycine-cysteine content. We conclude that SGD58 may prompt the creation a vaccine against cervix papilloma.

Introduction: Gold nanoparticles are versatile carriers for delivery of biomacromolecules. Here, we have developed spiky gold nanoparticles (SGNPs) that can efficiently deliver immunostimulatory agents. Objectives: Our goal was to develop a platform technology for co-delivery of multiple adjuvant molecules for synergistic stimulation and maturation of innate immune cells. Methods: SGNPs were synthesized by a seed-mediated, surfactant-free synthesis method and incorporated with polyinosinic-polycytidylic acid (pIC) and DNA oligonucleotide containing unmethylated CpG motif (CpG) by an electrostatic layer-by-layer approach. Adjuvant-loaded SGNP nano-complexes were examined for their biophysical and biochemical properties and studied for immune activation using bone marrow-derived dendritic cells (BMDCs). Results: We have synthesized SGNPs with branched nano-spikes layered with pIC and/or CpG. Adjuvant-loaded SGNP nano-complexes promoted cellular uptake of the adjuvants. Importantly, we achieved spatio-temporal control over co-delivery of pIC and CpG via SGNPs, which produced synergistic enhancement in cytokine release (IL-6, TNF-α) and upregulation of co-stimulatory markers (CD40, CD80, CD86) in BMDCs, compared with pIC, CpG, or their admixtures. Conclusion: SGNPs serve as a versatile delivery platform that allows flexible and on-demand cargo fabrication for strong activation of innate immune cells. [ABSTRACT FROM AUTHOR]

TLR3 belong to the Toll-like receptors family, it is mainly expressed on immune cells where it senses pathogen-associated molecular patterns and initiates innate immune response. TLR3 agonist poly(I:C) was developed to mimic pathogens infection and boost immune system activation to promote anti-cancer therapy. Accordingly, TLR agonists were included in the National Cancer Institute list of immunotherapeutic agents with the highest potential to cure cancer. Besides well known effects on immune cells, poly(I:C) was also shown, in experimental models, to directly induce apoptosis in cancer cells expressing TLR3. This review presents the current knowledge on the mechanism of poly(I:C)-induced apoptosis in cancer cells. Experimental evidences on positive or negative regulators of TLR3-mediated apoptosis induced by poly(I:C) are reported and strategies are proposed to successfully promote this event in cancer cells. Cancer cells apoptosis is an additional arm offered by poly(I:C), besides activation of immune system, for the treatment of various type of cancer. A further dissection of TLR3 signaling would contribute to greater resolution of the critical steps that impede full exploitation of the poly(I:C)-induced apoptosis. Experimental evidences about negative regulator of poly(I:C)-induced apoptotic program should be considered in combinations with TLR3 agonists in clinical trials. [ABSTRACT FROM PUBLISHER]