Magnitude and Breadth of Antibody Cross-reactivity Induced by Recombinant Influenza Hemagglutinin Trimer Vaccine Is Enhanced by Combination Adjuvants

Abstract The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Of all the formulations tested, a combination of CpG, MPLA and AddaVAX (termed “IVAX-1”) yielded the greatest breadth and magnitude of Ab responses, particularly against the HA1 region (which includes the variable head domain) of HA. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNg gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.

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A Novel Toll-Like Receptor 9 Agonist, MGN1703, Enhances HIV-1 Transcription and NK Cell-Mediated Inhibition of HIV-1-Infected Autologous CD4+T Cells

Journal of Virology ◽

10.1128/jvi.00222-16 ◽

2016 ◽

Vol 90 (9) ◽

pp. 4441-4453 ◽

Cited By ~ 48

Author(s):

Rasmus Offersen ◽

Sara Konstantin Nissen ◽

Thomas A. Rasmussen ◽

Lars Østergaard ◽

Paul W. Denton ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Mononuclear Cells ◽

Nk Cell ◽

Killer Cell ◽

Clinical Testing ◽

Toll Like Receptor ◽

Peripheral Blood Mononuclear ◽

Tlr Agonists ◽

Hiv 1

ABSTRACTToll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a “shock-and-kill” approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703ex vivoexhibited increased secretion of interferon alpha (IFN-α) (P= 0.005) and CXCL10 (P= 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56dimCD16+NK cells (P= 0.001) as well as higher proportions of CD107a-positive (P= 0.002) and IFN-γ-producing (P= 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4+and CD8+T cells. Furthermore, CD4+T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA (P= 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4+T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (P= 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4+T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials.IMPORTANCEWe demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the CpG-oligodeoxynucleotide (CpG-ODN) family are due to its novel “dumbbell-shape” structure made of covalently closed, natural DNA. In our study, we found that incubation of peripheral blood mononuclear cells with MGN1703 results in natural killer cell activation and increased natural killer cell function, which significantly inhibited the spread of HIV in a culture of autologous CD4+T cells. Furthermore, we discovered that MGN1703-mediated activation can enhance HIV-1 transcription in CD4+T cells, suggesting that this molecule may serve a dual purpose in HIV-1 eradication therapy: enhanced immune function and latency reversal. These findings provide a strong preclinical basis for the inclusion of MGN1703 in an HIV eradication clinical trial.

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Engineering Therapeutic Strategies in Cancer Immunotherapy via Exogenous Delivery of Toll-like Receptor Agonists

Pharmaceutics ◽

10.3390/pharmaceutics13091374 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1374

Author(s):

Sehwan Jeong ◽

Yunyoung Choi ◽

Kyobum Kim

Keyword(s):

T Cells ◽

Immune Responses ◽

Cancer Immunotherapy ◽

Cell Activation ◽

The Body ◽

Toll Like Receptor ◽

Tlr Agonists ◽

Cytokines And Chemokines ◽

Adaptive Immune Cells ◽

Delivery Strategies

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.

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Differential indirect activation of human invariant natural killer T cells by Toll-like receptor agonists

Immunotherapy ◽

10.2217/imt.09.30 ◽

2009 ◽

Vol 1 (4) ◽

pp. 557-570

Author(s):

María Moreno ◽

Berber M Mol ◽

Silvia von Mensdorff-Pouilly ◽

René HM Verheijen ◽

Esther C de Jong ◽

...

Keyword(s):

T Cells ◽

Natural Killer ◽

Mononuclear Cells ◽

Toll Like Receptor ◽

Inkt Cells ◽

Peripheral Blood Mononuclear ◽

Tlr Agonists ◽

Killer T Cells ◽

Invariant Natural Killer ◽

Dc Maturation

Aim: Invariant natural killer (iNK) T cells are activated by bacterial glycosphingolipids presented by CD1d on dendritic cells (DCs). Here, it was investigated whether Toll-like receptor (TLR) ligands derived from various microorganisms can either directly or indirectly (through DC activation) activate iNKT cells. Materials & Methods: TLR expression by iNKT cells was examined and the ability of various TLR ligands to activate iNKT cells was evaluated. Results: Although human iNKT cells express all TLRs, apart from TLR8, they did not respond directly to TLR ligands. However, iNKT cells became strongly activated when total peripheral blood mononuclear cells were stimulated with TLR2/6, 7/8 and 9 ligands, but not or to a lesser extent with TLR3, 4 and 5 ligands. TLR-stimulated monocyte-derived DCs promoted iNKT cell phenotypic activation and, in turn, these activated iNKT cells further enhanced DC maturation. Conclusion: TLR agonists may act as strong adjuvants for immunotherapy by promoting combined and reciprocal activation of iNKT cells and DCs.

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Effector CD8 T cells possess suppressor function after 4-1BB and Toll-like receptor triggering

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0837611100 ◽

2003 ◽

Vol 100 (9) ◽

pp. 5348-5353 ◽

Cited By ~ 47

Author(s):

L. Myers ◽

C. Takahashi ◽

R. S. Mittler ◽

R. J. Rossi ◽

A. T. Vella

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Toll Like Receptor ◽

Suppressor Function

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Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle

Scientific Reports ◽

10.1038/s41598-021-81474-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Josephine F. Reijneveld ◽

Mira Holzheimer ◽

David C. Young ◽

Kattya Lopez ◽

Sara Suliman ◽

...

Keyword(s):

T Cells ◽

Fatty Acid ◽

Immune System ◽

T Cell ◽

Cross Reactivity ◽

Lipid Binding ◽

Human Immune System ◽

Human T Cell ◽

Human T Cells ◽

Pure Compounds

AbstractThe cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

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Induction of Tertiary Phase Epileptiform Discharges after Postasphyxial Infusion of a Toll-Like Receptor 7 Agonist in Preterm Fetal Sheep

International Journal of Molecular Sciences ◽

10.3390/ijms22126593 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6593

Author(s):

Kenta H.T. Cho ◽

Mhoyra Fraser ◽

Bing Xu ◽

Justin M. Dean ◽

Alistair J. Gunn ◽

...

Keyword(s):

Moderate Hypertension ◽

Epileptiform Activity ◽

Ischemic Brain Injury ◽

Toll Like Receptor ◽

Fetal Sheep ◽

Ischemic Brain ◽

Tlr Agonists ◽

Epileptiform Discharges ◽

Subcortical Regions ◽

Total Burden

Background: Toll-like receptor (TLR) agonists are key immunomodulatory factors that can markedly ameliorate or exacerbate hypoxic–ischemic brain injury. We recently demonstrated that central infusion of the TLR7 agonist Gardiquimod (GDQ) following asphyxia was highly neuroprotective after 3 days but not 7 days of recovery. We hypothesize that this apparent transient neuroprotection is associated with modulation of seizure-genic processes and hemodynamic control. Methods: Fetuses received sham asphyxia or asphyxia induced by umbilical cord occlusion (20.9 ± 0.5 min) and were monitored continuously for 7 days. GDQ 3.34 mg or vehicle were infused intracerebroventricularly from 1 to 4 h after asphyxia. Results: GDQ infusion was associated with sustained moderate hypertension that resolved after 72 h recovery. Electrophysiologically, GDQ infusion was associated with reduced number and burden of postasphyxial seizures in the first 18 h of recovery (p < 0.05). Subsequently, GDQ was associated with induction of slow rhythmic epileptiform discharges (EDs) from 72 to 96 h of recovery (p < 0.05 vs asphyxia + vehicle). The total burden of EDs was associated with reduced numbers of neurons in the caudate nucleus (r2 = 0.61, p < 0.05) and CA1/2 hippocampal region (r2 = 0.66, p < 0.05). Conclusion: These data demonstrate that TLR7 activation by GDQ modulated blood pressure and suppressed seizures in the early phase of postasphyxial recovery, with subsequent prolonged induction of epileptiform activity. Speculatively, this may reflect delayed loss of early protection or contribute to differential neuronal survival in subcortical regions.

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184 Two types of anti-TIGIT antibodies with distinct binding epitope and functional activities

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0184 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A198-A198

Author(s):

Tingting Zhong ◽

Xinghua Pang ◽

Zhaoliang Huang ◽

Na Chen ◽

Xiaoping Jin ◽

...

Keyword(s):

T Cells ◽

Mixed Culture ◽

Cell Activity ◽

Cross Reactivity ◽

Binding Activity ◽

Jurkat Cells ◽

List Type ◽

Dependent Manner ◽

Vitro Assay

BackgroundTIGIT is an inhibitory receptor mainly expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and Treg cells. TIGIT competes with CD226 for binding with CD155. In cancers, CD155 has been reported to up-regulate on tumor cells, and TIGIT was found to increase on TILs.1 Activation of TIGIT/CD155 pathway would mediate immunosuppression in tumor; while blockade of TIGIT promotes anti-tumor immune response.MethodsAK126 and AK113 are two humanized anti-human TIGIT monoclonal antibodies developed by Akesobio. Binding activity of AK126 and AK113 to human TIGIT, and competitive binding activity with CD155 and CD112, were performed by using ELISA, Fortebio, and FACS assays. Cross-reactivity with cynomolgus monkey TIGIT and epitope binning were also tested by ELISA assay. In-vitro assay to investigate the activity to promote IL-2 secretion was performed in mixed-culture of Jurkat-TIGIT cells and THP-1 cells.ResultsAK126 and AK113 could specifically bind to human TIGIT with comparative affinity and effectively blocked the binding of human CD155 and CD112 to human TIGIT. X-ray crystal structure of TIGIT and PVR revealed the C’-C’’ loop and FG loop regions of TIGIT are the main PVR interaction regions.2 The only amino acid residue differences in these regions between human and monkey TIGIT are 70C and 73D. AK126 binds to both human and monkey TIGIT, AK113 binds only to monkey TIGIT. This suggests that these residues are required for AK113 binding to human TIGIT, but not required for AK126. Interestingly, results from cell-based assays indicated that AK126 and AK113 showed significantly different activity to induce IL-2 secretion in mixed-culture of Jurkat-TIGIT cells and THP-1 cells (figure 1A and B), in which AK126 had a comparable capacity of activity to 22G2, a leading TIGIT mAb developed by another company, to induce IL-2 secretion, while, AK113 showed a significantly higher capacity than 22G2 and AK126.Abstract 184 Figure 1Anti-TIGIT Antibodies Rescues IL-2 Production in Vitro T-Cell Activity Assay in a dose dependent manner. Jurkat-TIGIT cells (Jurkat cells engineered to over-express human TIGIT) were co-cultured with THP-1 cells, and stimulated with plate-bound anti-CD3 mAb in the presence of TIGIT ligand CD155 (A) or CD112 (B) with anti-TIGIT antibodies. After incubated for 48h at 37° C and 5.0% CO2, IL-2 levels were assessed in culture supernatants by ELISA. Data shown as mean with SEM for n = 2.ConclusionsWe discovered two distinct types of TIGIT antibodies with differences in both epitope binding and functional activity. The mechanism of action and clinical significance of these antibodies require further investigation.ReferencesSolomon BL, Garrido-Laguna I. TIGIT: a novel immunotherapy target moving from bench to bedside. Cancer Immunol Immunother 2018;67:1659–1667.Stengel KF, Harden-Bowles K, Yu X, et al. Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering. Proc Natl Acad Sci USA 2012;109:5399–5404.

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CD4+CD25+Foxp3+ regulatory T cells regulate immune balance in unexplained recurrent spontaneous abortion via the Toll-like receptor 4/nuclear factor-κB pathway

Journal of International Medical Research ◽

10.1177/0300060520980940 ◽

2020 ◽

Vol 48 (12) ◽

pp. 030006052098094

Author(s):

Shuang Qin ◽

Li Li ◽

Jia Liu ◽

Jinrui Zhang ◽

Qing Xiao ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Inflammatory Response ◽

Mouse Model ◽

Nuclear Factor Κb ◽

Nuclear Factor ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Unexplained Recurrent Spontaneous Abortion ◽

Tlr4 Antagonist

Objective The present study aimed to evaluate the effects of cluster of differentiation (CD)4+CD25+ forkhead box p3 (Foxp3)+ regulatory T cells (Tregs) on unexplained recurrent spontaneous abortion (URSA) and the associated mechanisms. Methods The proportion of CD4+CD25+Foxp3+ Tregs and inflammatory cytokine concentrations in the peripheral blood of women with URSA were measured by flow cytometry and enzyme-linked immunosorbent assay, respectively. CBA/JxDBA/2J mating was used to establish an abortion-prone mouse model and the model mice were treated with the Toll-like receptor 4 (TLR4) antagonist E5564 and the TLR4 agonist lipopolysaccharide. Results The proportion of CD4+CD25+Foxp3+ Tregs was decreased and the inflammatory response was increased in women with URSA. In the abortion-prone mouse model, E5564 significantly increased the proportion of CD4+CD25+Foxp3+ Tregs, decreased the inflammatory response, and increased Foxp3 mRNA and protein expression. Lipopolysaccharide had adverse effects on the abortion-prone model. Conclusions These data suggest that CD4+CD25+Foxp3+ Tregs regulate immune homeostasis in URSA via the TLR4/nuclear factor-κB pathway, and that the TLR4 antagonist E5564 may be a novel and potential drug for treating URSA.

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