Potential Immune Adjuvant Research Articles

TLR5 agonist in combination with anti-PD-1 treatment enhances anti-tumor effect through M1/M2 macrophage polarization shift and CD8+ T cell priming

Immune checkpoint inhibitors have revolutionized anti-tumor therapy, notably improving treatment responses in various tumors. However, many patients remain non-responsive and do not experience benefits. Given that Toll-like receptors (TLRs) can counteract tumor immune tolerance by stimulating both innate and adaptive immune responses, TLR agonists are being explored as potential immune adjuvants for cancer treatment. In this study, we assessed the potential of enhancing the efficacy of immune checkpoint inhibitors by activating innate immunity with a TLR5 agonist. In a mouse tumor model, combination therapy with TLR5 agonist and anti-PD-1 significantly inhibited tumor growth. The TLR5 agonist shifted the balance from M2-like to M1-like macrophages and upregulated the expression of co-stimulatory molecules in macrophages. Furthermore, TLR5 agonist promoted the activation and tumor infiltration of CD8+ T cells. As a result, the TLR5 agonist augmented the anti-tumor efficacy of anti-PD-1, suggesting its potential in modulating the tumor microenvironment to enhance the anti-tumor response. Our findings point toward the possibility of optimizing immune checkpoint inhibitor therapy using TLR5 agonists.

A NF-κB-Based High-Throughput Screening for Immune Adjuvants and Inhibitors.

The nuclear factor-κB (NF-κB) family is crucial for regulating immune and inflammatory responses. The activation of the immune cell signaling pathway usually activates NF-κB, causing a protective immune response. NF-κB can also cause excessive inflammatory responses by activating a cascade reaction of pro-inflammatory mediators such as cytokines. In this study, we used an NF-κB luciferase reporter gene system. Out of more than 800 compounds screened, four NF-κB agonists were identified with strong activity at nontoxic concentrations. Subsequently, the adjuvant effect was verified on mouse bone marrow-derived dendritic cells (BMDCs) and macrophages RAW264.7. It was found that fostamatinib (R788) disodium increased the production of IL-6, IL-12p40, and TNF-α, indicating that R788 disodium could induce the maturation of antigen-presenting cells (APCs). In addition, three compounds were screened to significantly inhibit NF-κB at nontoxic doses, including dehydrocostus lactone (DHL)-a known NF-κB inhibitor. The results showed that DHL significantly reduced the release of LPS-induced inflammatory cytokines (including TNF-α, IL-6, and IL-12). Our findings indicate that the NF-κB-based high-throughput screening can be used to discover potential immune adjuvants and anti-inflammatory molecules.

Spiky titanium dioxide nanoparticles-loaded Plantaginis Semen polysaccharide as an adjuvant to enhance immune responses

The purpose of this study was to prepare spiky titanium dioxide nanoparticles-loaded Plantaginis Semen polysaccharide (SN-TiO2-PSP), and the structural characterization and immune response of infectious laryngotracheitis (ILT) vaccine in Hetian chickens were investigated. The structural characterization of SN-TiO2-PSP was analyzed by FT-IR, TEM, and TGA analysis. And the immune organs indexes, lymphocytes proliferation, specific antibody levels, and ratios of CD4+ and CD8+ T lymphocytes were studied. Structural characterization results showed that SN-TiO2-PSP has a typical polysaccharide absorption peak and good stability. The SN-TiO2-PSP's shape was similar to sea urchin, and its zeta potential and particle size were 27.56 mV and 976.11 nm, respectively. In vivo results showed that SN-TiO2-PSP could enhance the proliferation of peripheral lymphocytes, specific antibody levels, CD4+ and CD8+ T lymphocytes ratios, IL-4 and INF-γ levels in Hetian chickens vaccinated with ILT vaccine on D7, D14, D21, and D28. In addition, SN-TiO2-PSP not only enhanced the indexes of immune organs but also promoted the development of immune organs. Therefore, SN-TiO2-PSP has immune adjuvant activity and may become a new potential immune adjuvant.

Construction of Monomeric and Dimeric G-Quadruplex-Structured CpG Oligodeoxynucleotides for Enhanced Uptake and Activation in TLR9-Positive Macrophages.

The G-quadruplex (GQ) structure has potential applications in nucleic acid drug delivery because of its superior stability. In this study, we added one G-tract (five guanines) to an unmethylated phosphodiester-linked cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), a potential immune adjuvant, to construct a GQ-structured CpG ODN with precise structural properties, increased biological stability, and efficient delivery to Toll-like receptor 9 (TLR9)-positive immune cells. A G-tract was added to phosphodiester-backboned CpG1668 at the 5'-end [1668(5'-G5)], 3'-end [1668(3'-G5)], or within the sequence [1668(mid-G5)]. Circular dichroism analysis showed that all CpG ODNs with a G-tract formed parallel GQ structures, irrespective of its position. Electrophoresis showed that 1668(5'-G5) formed a GQ dimer, whereas others remained GQ monomers. GQ-structured CpG ODNs induced greater tumor necrosis factor-α and interleukin-6 secretion from TLR9-positive mouse macrophage-like RAW264.7 cells than single-stranded CpG ODNs, with the highest for 1668(3'-G5). GQ structuration increased CpG ODN uptake by RAW264.7 cells, and 1668(3'-G5) decomposed more slowly in serum than 1668(5'-G5). Thus, GQ formation with one G-tract is a simple and efficient strategy for CpG ODN delivery to TLR9-positive cells, and addition of a G-tract to the 3'-end is effective in obtaining monomeric GQ-structured CpG ODN with high biological stability and immunostimulatory activity.

Surface Immunogenic Protein of Streptococcus Group B is an Agonist of Toll-Like Receptors 2 and 4 and a Potential Immune Adjuvant.

Vaccine-induced protection against pathogens, especially subunit-based vaccines, are related to antigen properties but mainly in their ability to stimulate the immune system by the use of an adjuvant. Modern vaccines are formulated with a high level of antigen purity, where an efficient adjuvant is necessary. In this context, the use of protein Toll-Like Receptor (TLR) agonists as vaccine adjuvants has been highlighted because of their optimal immunogenicity and minimal toxicity. The Surface Immunogenic Protein (SIP) from Group B Streptococcus (GBS) has gained importance as a new potential protein-based vaccine. Recently, we reported that recombinant SIP (rSIP) expressed by E. coli and purified by High Performance Liquid Chromatography (HPLC) alone induces a protective humoral immune response. In this study, we present the immunomodulatory properties of rSIP as a protein-based adjuvant, as an agonist of TLR. To this end, we showed that C57BL/6 bone marrow-derived dendritic cells pulsed by rSIP resulted in enhanced CD40, CD80, CD86, and Major Histocompatibility Complex (MHC) class II as well as increased secretion proinflammatory cytokines Interleukin (IL)-6, Interferon (IFN)-γ, Tumor Necrosis Factor (TNF)-α, and IL-10. Next, we investigated the in vivo effect of rSIP in the absence or presence of ovalbumin (OVA) on antigen-specific antibody secretion in C57BL/6 mice. Immunization with rSIP plus OVA showed that anti-OVA IgG2a and IgG1a increased significantly compared with OVA alone in C57BL/6 mice. Also, the immunization of rSIP plus OVA generates increased serum cytokines levels characterized by IL-12p70, IL-10, IL-4, and IFN-γ. Interestingly, we observed that rSIP stimulate Toll Like Receptor (TLR)2 and TLR4, individually expressed by Human embryonic kidney (HEK) 293-derived TLR reporter cells. These findings suggest that rSIP is a new potential protein TLR agonist adjuvant and may be employed in the development of new vaccines.

Modified chitosan-based nanoadjuvants enhance immunogenicity of protein antigens after mucosal vaccination

Nasal vaccination is considered to be an effective and convenient way of increasing immune responses both systemically and locally. Although various nanovaccine carriers have been introduced as potential immune adjuvants, further improvements are still needed before they can be taken to clinical usage. Chitosan-based nanovaccine carriers are one of the most widely studiedadjuvants, owing to the abilityof chitosan toopen tight junctions between nasal epithelial cells and enhance particle uptake as well as its inherent immune activating role. In present study, bovine serum albumin (BSA) loaded nanoparticles were prepared using novel aminated (aChi) and aminated plus thiolated chitosan (atChi) polymers, to further enhance mucoadhesiveness and adjuvanticity of the vaccine system by improving electrostatic interactions of polymers with negatively charged glycoproteins. Nanocarriers with optimum size and surface charge, high encapsulation efficiency of model antigen and good stability were developed. Negligible toxicity was observed in Calu-3 and A549 cell lines. In vivo studies, revealed high levels of systemic antibodies (IgG, IgG1 and IgG2a) throughout the study and presence of sIgA in vaginal washes showed that common mucosal system was successfully stimulated. Cytokine levels indicated a mixed Th1/Th2 immune response. A shift towards cellular immune responses was observed after nasal immunisation with antigen loaded nanoparticle formulations. These nanoparticles exhibit great potential for nasal application of vaccines.

Ra-SBRT is Potential Immune Adjuvant for Innate Immune Cell Populations in Advance Stage NSCLC Patients

Bystander toxicity and tissue fibrosis are the major complications with conventional radiation therapy for cancer patients. In this context, we here propose RapidArc - Stereotactic Body Radiation Therapy (Ra-SBRT) as a non-invasive and immune adjuvant approach for the successful eradication of advance stage NSCLC. Ra-SBRT is highly focused and capable of destroying tumors with high grade metastatic lesions and spared normal tissues. Follow up of stage 4th NSCLC patient revealed that Ra-SBRT is potentially immunogenic which was evident by increased number of iNOS+ Tumor Associated macrophages (M1-TAM), Siglac-8+ eosinophils, basophils and subsequent prolongation of disease free survival of 4th stage NSCLC patients by 3 years. This study demonstrated M1 retuning potential of Ra-SBRT which is a pre-requisite of effective management of inoperable and highly metastatic tumors of lung with least or no bystander impact.

Polysaccharide from Phellinus Igniarius activates TLR4-mediated signaling pathways in macrophages and shows immune adjuvant activity in mice

Polysaccharide from Phellinus igniarius (PPI) is known for its immune-regulating effect with low toxicity. Toll like receptor 4 (TLR4) is important in both innate and adaptive immune responses and considered to be a promising target for new immune adjuvants. In this study, PPI was investigated for its effect on activating TLR4 in RAW264.7 and peritoneal macrophages. The adjuvant potential of PPI was evaluated in OVA-immunized mice. The results showed PPI treatment significantly increased the secretion and the mRNA expression of both MyD88 dependent and TRIF dependent cytokines. IRAK-1, a key molecule on the downstream of MyD88, was polyubiquitinated while IRF-3, another key molecule on the downstream of TRIF, was phosphorylated obviously after the treatment of PPI. The phosphorylation of molecules involved in both NF-κB pathway and MAPK pathway were significantly up-regulated after PPI treatment. In addition, the effects of PPI on the macrophages almost completely disappeared after treating the cells with the TLR4 antagonist TAK-242. Further in vivo results showed PPI significantly increased the serum OVA-specific antibody and the OVA-specific spleen cell proliferation. Taken together, PPI can specifically stimulate TLR4 and activate both MyD88 and TRIF pathways. PPI has immune adjuvant activity and may become a new potential immune adjuvant.

Nanoparticle-induced inflammation can increase tumor malignancy.

Many different types of nanoparticles have been shown to possess immunomodulatory properties, depending on their physicochemical parameters. This can potentially be harnessed as a possible antitumor therapy. However, in the current work we show that inflammation elicited by nanomaterials can have grave effects in pathophysiological conditions, where non-specific inflammation was found to increase cancer cell mobility and tumor malignancy. These data show that immunomodulatory properties of nanomaterials must be carefully controlled to avoid any undesired side-effects.

Rearrangement reactions in the fluorination of d-glucopyranoside at the C-4 position by DAST

Attempts to synthesize 4-deoxy-4-fluoro-KRN-7000 (2), a potential immune adjuvant, by fluorination of 4-hydroxy of α-d-glucopyranoside using DAST, were not successful. Instead, the unusual 5-deoxy-5-fluoro β-l-altrofurnoside and the corresponding 4-deoxy-4-fluoro α-d-glucopyranoside with retained configuration were obtained. In the presence of polar solvents, the reaction afforded the epoxide product, suggesting the bicyclic oxiranium ion intermediate to be involved in this reaction. Compound 2 was eventually achieved by treating 4-methanesufonated glucopyranoside with fluoride ion and found to be a weak agonist for CD1d and NKT cell activation.

Antitumor effects of Newcastle disease virus hemagglutinin-neuraminidase used as a molecular adjuvant

Gene therapy is a potentially powerful tool used in cancer therapy. The strength of immune responses induced by some strategies is usually low, therefore, the development of agents capable of enhancing these responses is highlighted. The authors investigated the potential of an approach based on the hemagglutinin-neuraminidase(HN) of Newcastle disease virus(NDV) as a potential immune adjuvant. It was found that recombinant adenovirus(Ad) infected SGC7901 cells expressing HN exhibited both hemagglutinin(HA) and neuraminidase(NA) activities. It was demonstrated that administration of HN induced higher levels of the effector cytokines TNF-α, IFN-α and IFN-γ and increased natural killer(NK) cell activity. Based on the therapeutic tumor model, the results show that the administration of HN with Apoptin led to improved survival and tumor suppression. In conclusion, this study indicates that HN stimulates innate immune responses to make the activity of NK cells increased, which highlights the potential adjuvant activity of HN in cancer gene therapy.

Synthesis and Immunological Characterization of Toll-Like Receptor 7 Agonistic Conjugates

Activation of toll-like receptors (TLRs) on cells of the innate immune system initiates, amplifies, and directs the antigen-specific acquired immune response. Ligands that stimulate TLRs, therefore, represent potential immune adjuvants. In this study, a potent TLR7 agonist was conjugated to phospholipids, poly(ethylene glycol) (PEG), or phospholipid-PEG via a versatile benzoic acid functional group. Compared to the unmodified TLR7 agonist, each conjugate displayed a distinctive immunological profile in vitro and in vivo. In mouse macrophages and human peripheral blood mononuclear cells, the phospholipid TLR7 agonist conjugate was at least 100-fold more potent than the free TLR7 ligands, while the potency of PEG−phospholipid conjugate was similar to that of the unmodified TLR7 agonist. When administered systemically in mice, the phospholipid and phospholipid−PEG TLR7 conjugates induced prolonged increases in the levels of proinflammatory cytokines in serum, compared to the unmodified TLR7 activator. When the conjugates were used as adjuvants during vaccination, only the phospholipid TLR7 agonist conjugates induced both Th1 and Th2 antigen-specific immune responses. These data show that the immunostimulatory activity of a TLR7 ligand can be amplified and focused by conjugation, thus broadening the potential therapeutic application of these agents.

The PorB porin from commensal Neisseria lactamica induces Th1 and Th2 immune responses to ovalbumin in mice and is a potential immune adjuvant

Porins from pathogenic Neisseriae are among several bacterial products with immune adjuvant activity. Neisseria meningitidis (Nme) PorB, has been shown to induce immune cells activation in a TLR2-dependent manner and acts as a vaccine immune adjuvant. The PorB porin from Neisseria lactamica (Nlac), a common nasopharyngeal commensal, shares significant structural and functional similarities with Nme PorB. In this work we ask whether the immune adjuvant ability of porins from pathogenic Neisserial strains is a characteristic shared with porins from non-pathogenic Neisserial species or whether it is unique for bacterial products derived from microorganisms capable of inducing inflammation and disease. We evaluated the potential immune adjuvant effect of Nlac PorB in mice using ovalbumin (OVA) as a prototype antigen. Immunization with Nlac PorB/OVA induced high OVA-specific IgG and IgM titers compared to OVA alone, similar to other adjuvants such as Nme PorB and alum. High titers of IgG1 and IgG2b were detected as well as production of IL-4, IL-10, IL-12 and INF-gamma in response to Nlac PorB, consistent with induction of both a Th1-type and a Th2-type immune response. OVA-specific proliferation was also determined in splenocytes from Nlac PorB/OVA-immunized mice. In addition, B cell activation in vitro and cytokine production in response to Nlac PorB was found to be mediated by TLR2, in a similar manner to Nme PorB.

Co-injection of interleukin 8 with the glycoprotein gene from viral haemorrhagic septicemia virus (VHSV) modulates the cytokine response in rainbow trout ( Oncorhynchus mykiss)

Since previous results showed that interleukin 8 (IL-8) was induced in rainbow trout ( Oncorhynchus mykiss) in response to viral hemorrhagic septicemia virus (VHSV) infection, we have cloned IL-8 in an expression vector (pIL8+) and studied its possible adjuvant effect on the early response to a VHSV immunization model, focusing on the early response of several cytokines induced by a vector coding for the glycoprotein of VHSV (pMCV1.4-G) in the spleen and head kidney. First, we demonstrated that the pIL8+ successfully transcribed IL-8, by induction of IL-8 transcription in the muscle and blood, and by a massive infiltration of neutrophils at the muscle inoculation site. We have studied the effect of pIL8+ co-administration on the expression of two pro-inflammatory cytokines, such as IL-1β and tumour necrosis factor α (TNF-α); cytokines that have mainly an inhibitory role, IL-11 and transforming growth factor β (TGF-β); and a Th1 type cytokine, IL-18. We demonstrated that the co-administration of pIL8+ with pMCV1.4-G modulates the cytokine response that is induced, mainly by having its effect increasing pro-inflammatory cytokines (IL-1β and TNF-α1), with a greater impact on the spleen, and to a lesser extent in the head kidney. All these data suggest that IL-8 is able to modulate the early cytokine immune response that is produced in response to a DNA vaccine, and therefore, might be a potential immune adjuvant in fish viral vaccination. More work should be done to determine if this modulation has a beneficial effect on protection as seen in other mammal viral models.

Immunostimulatory neem leaf preparation acts as an adjuvant to enhance the efficacy of poorly immunogenic B16 melanoma surface antigen vaccine

Immunogenecity of the poorly immunogenic B16 melanoma cell surface antigen (B16MelSAg) was enhanced by combining B16MelSAg with NLP in C57BL/6 mice, as evidenced by ELISA and flow cytometry. NLP was as effective as Freund's complete and incomplete adjuvant to generate antibodies recognizing the B16MelSAg. The NLP generated antibody was a gamma globulin with a subtype of IgG1. Splenic lymphocytes from B16MelSAg + NLP treated mice proliferated more rapidly in vitro when stimulated by specific (B16MelSAg) and nonspecific (ConA) stimulators, in comparison to the proliferation detected in B16MelSAg and NLP treated groups. Vaccination of mice with B16MelSAg + NLP more efficiently prevented the growth of B16 melanoma tumor than mice immunized with B16MelSAg or NLP alone. In another experiment, the immune sera (B16MelSAg + NLP) was mixed with B16Mel tumors and injected subcutaneously into syngenic C57BL/6 mice. Tumor burden was less in mice receiving a tumor along with B16MelSAg + NLP generated immune sera than other groups. The B16MelSAg + NLP generated immune sera induced antibody dependent cellular cytotoxicity specifically towards B16Mel tumor cells in vitro. We concluded that NLP might be a potential immune adjuvant for inducing active immunity towards tumor antigens.

Decreased stimulation of CD4+ T cell proliferation and IL-2 production by highly enriched populations of HIV-infected dendritic cells.

APC infection and dysfunction may contribute to the immunopathogenesis of HIV disease. In this study, we examined immunologic function of highly enriched populations of HIV-infected monocyte-derived dendritic cells (DC). Compared with uninfected DC, HIV-infected DC markedly down-regulated surface expression of CD4. HIV p24(+) DC were then enriched by negative selection of CD4(+)HIV p24(-) DC and assessed for cytokine secretion and immunologic function. Although enriched populations of HIV-infected DC secreted increased IL-12p70 and decreased IL-10, these cells were poor stimulators of allogeneic CD4(+) T cell proliferation and IL-2 production. Interestingly, HIV-infected DC secreted HIV gp120 and the addition of soluble (s) CD4 (a known ligand for HIV gp120) to DC-CD4(+) T cell cocultures restored T cell proliferation in a dose-dependent manner. By contrast, addition of antiretroviral drugs did not affect CD4(+) T cell proliferation. Furthermore, recombinant HIV gp120 inhibited proliferation in uninfected cocultures of allogeneic DC and CD4(+) T cells, an effect that was also reversed by addition of sCD4. In summary, we show that HIV gp120 produced by DC infected by HIV in vitro impairs normal CD4(+) T cell function and that sCD4 completely reverses HIV gp120-mediated immunosuppression. We hypothesize that HIV-infected DC may contribute to impaired CD4(+) T cell-mediated immune responses in vivo and that agents that block this particular immunosuppression may be potential immune adjuvants in HIV-infected individuals.

Cloning and sequencing of the cDNA for ovine granulocyte-macrophage colony-stimulating factor (GM-CSF)

Colony-stimulating factors (CSFs) are not only regulators of haemopoiesis but can also enhance the function of mature myeloid cells, and are therefore potential immune adjuvants. By use of the polymerase chain reaction (PCR) with primers based on the bovine granulocyte-macrophage CSF (GM-CSF) sequence, we have amplified the cDNA for ovine GM-CSF, produced from crude mRNA extracted from alveolar macrophages. The PCR product was cloned into pUC119, and electroporated into Escherichia coli. The complete nucleotide sequence of two clones, and the partial sequence of eight others, was determined. At the nucleotide and amino acid levels, the ovine and bovine GM-CSF sequences are 91% and 81% homologous, respectively.