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Background Cancer vaccines require adjuvants to induce effective immune responses; however, there is no consensus on optimal adjuvants. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC or TLR4 agonist lipopolysaccharide (LPS), combined with CD4 T cell activation, would support strong and durable CD8 + T cell responses, whereas addition of an incomplete Freund’s adjuvant (IFA) would reduce magnitude and persistence of immune responses. Patients and methods Participants with resected stage IIB-IV melanoma received a vaccine comprised of 12 melanoma peptides restricted by Class I MHC (12MP), plus a tetanus helper peptide (Tet). Participants were randomly assigned 2:1 to cohort 1 (LPS dose-escalation) or cohort 2 (polyICLC). Each cohort included 3 subgroups (a-c), receiving 12MP + Tet + TLR agonist without IFA (0), or with IFA in vaccine one (V1), or all six vaccines (V6). Toxicities were recorded (CTCAE v4). T cell responses were measured with IFNγ ELIspot assay ex vivo or after one in vitro stimulation (IVS). Results Fifty-three eligible patients were enrolled, of which fifty-one were treated. Treatment-related dose-limiting toxicities (DLTs) were observed in 0/33 patients in cohort 1 and in 2/18 patients in cohort 2 (11%). CD8 T cell responses to 12MP were detected ex vivo in cohort 1 (42%) and in cohort 2 (56%) and in 18, 50, and 72% for subgroups V0, V1, and V6, respectively. T cell responses to melanoma peptides were more durable and of highest magnitude for IFA V6. Conclusions LPS and polyICLC are safe and effective vaccine adjuvants when combined with IFA. Contrary to the central hypothesis, IFA enhanced T cell responses to peptide vaccines when added to TLR agonists. Future studies will aim to understand mechanisms underlying the favorable effects with IFA. Trial registration The clinical trial Mel58 was performed with IRB (#15781) and FDA approval and is registered with Clinicaltrials.gov on April 25, 2012 (NCT01585350). Patients provided written informed consent to participate. Enrollment started on June 24, 2012.

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8–37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8–37–cholestanol, but not unconjugated CGRP8–37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8–37–cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund’s adjuvant more effectively than unconjugated CGRP8–37. Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

BackgroundExperimental cancer vaccines are traditionally administered by injection in subcutaneous tissue or muscle, commonly with adjuvants that create chronic inflammatory depots. Injection of melanoma-derived peptides induces T cell responses; however, the depots that form following injection may inhibit optimization of the immune response. In skin, epidermal Langerhans cells (LC) are a dominant source of professional antigen presenting cells. We hypothesized that: (1) applying melanoma-derived peptides topically, in proximity to LC, could be immunogenic and safe, with low vaccine-site toxicity and (2) topical toll-like receptor 7 (TLR7) agonist would increase immunogenicity of the peptide vaccine.MethodsTwelve melanoma peptides plus a tetanus helper peptide were combined with granulocyte macrophage colony stimulating factor (GM-CSF) and were administered topically on days 1, 8, and 15, to 28 patients randomized to one of four adjuvant preparations: (1) incomplete Freund’s adjuvant (IFA); (2) IFA plus a TLR7 agonist (imiquimod) administered on days 0, 7, 14; (3) dimethyl sulfoxide (DMSO) or (4) DMSO+ imiquimod administered on day 0, 7, 14. Every 3 weeks thereafter (x 6), the peptides were combined with GM-CSF and were injected into the dermis and subcutis in an emulsion with IFA. Toxicities were recorded and immune responses assayed by ELIspot.ResultsCD8+ T cell responses to transdermal vaccination in DMSO occurred in 83% of participants in group 3 and 86% in group 4, and responses to vaccination in IFA were observed in 29% of participants in group 1 and 14% in group 2. Overall, 61% of participants had CD4+ T cell immune responses to the tetanus peptide, with large, durable responses in groups 3 and 4. Five of seven participants in group 4 had a severe rash, one that was dose limiting. Ten-year overall survival was 67% and disease-free survival was 44%.ConclusionsThese data provide proof of principle for immunogenicity in humans of transdermal immunization using peptides in DMSO. Further study is warranted into the pharmacokinetics and immunobiology of TLR agonists as vaccine adjuvants during transcutaneous application. Overall survival is high, supporting further investigation of this immunization approach.

The development of vaccines and other immunotherapies has been complicated by heterogeneous antigen display and the use of incompletely defined immune adjuvants with complex mechanisms of action. We have observed strong antibody responses in mice without the coadministration of any additional adjuvant by noncovalently assembling a T and B cell epitope peptide into nanofibers using a short C-terminal peptide extension. Self-assembling peptides have been explored recently as scaffolds for tissue engineering and regenerative medicine, but our results indicate that these materials may also be useful as chemically defined adjuvants. In physiological conditions, these peptides self-assembled into long, unbranched fibrils that displayed the epitope on their surfaces. IgG1, IgG2a, and IgG3 were raised against epitope-bearing fibrils in levels similar to the epitope peptide delivered in complete Freund's adjuvant (CFA), and IgM production was even greater for the self-assembled epitope. This response was dependent on self-assembly, and the self-assembling sequence was not immunogenic by itself, even when delivered in CFA. Undetectable levels of interferon-gamma, IL-2, and IL-4 in cultures of peptide-challenged splenocytes from immunized mice suggested that the antibody responses did not involve significant T cell help.

We conducted a randomized clinical trial in 45 patients with resected AJCC stage IIB-IV melanoma to characterize cellular and molecular events at sites of immunization with incomplete Freund’s adjuvant (IFA) alone, or a melanoma vaccine in IFA. At a primary vaccine site, all patients received a multi-peptide melanoma vaccine in IFA. At a replicate vaccine site, which was biopsied, group 1 received IFA only; group 2 received vaccine in IFA. Lymphocytes isolated from replicate vaccine site microenvironments (VSME) were compared to time-matched peripheral blood mononuclear cells (PBMC) in ELISpot and flow cytometry assays. Compared to PBMC, the VSME had fewer naïve and greater proportions of effector memory CD8 + T cells (T CD8 ). The vast majority of T CD8 within the VSME were activated (CD69 + ), with a concentration of antigen-specific (tetramer pos ) cells in the VSME, particularly in vaccine sites with peptide (group 2). CXCR3 + lymphocytes were concentrated in the VSME of all patients, suggesting IFA-induced chemokine recruitment. T CD8 expression of retention integrins αEβ7 and α1β1 was elevated in VSME, with the highest levels observed in antigen-specific cells in VSME containing peptide (group 2). T CD8 retained in the VSME of both groups were strikingly dysfunctional, with minimal IFN-γ production in response to peptide stimulation and few tetramer pos cells producing IFN-γ. These data suggest that vaccine-induced selective retention and dysfunction of antigen-specific T CD8 within VSME may represent a significant mechanism underlying transient immune responses and low clinical response rates to peptide vaccines administered in IFA.

The developmental programs that generate a broad repertoire of regulatory T cells (T reg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature T reg cells were generated through two distinct developmental programs involving CD25 + T reg cell progenitors (CD25 + T reg P cells) and Foxp3 lo T reg cell progenitors (Foxp3 lo T reg P cells). CD25 + T reg P cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3 lo T reg P cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3 lo T reg P cells. The development of both CD25 + T reg P cells and Foxp3 lo T reg P cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25 + T reg P cells and Foxp3 lo T reg P cells arose by coopting negative-selection programs and positive-selection programs, respectively. T reg cells derived from CD25 + T reg P cells, but not those derived from Foxp3 lo T reg P cells, prevented experimental autoimmune encephalitis. Our findings indicate that T reg cells arise through two distinct developmental programs that are both required for a comprehensive T reg cell repertoire capable of establishing immunotolerance. Farrar and colleagues show that thymic T reg cells are generated through two distinct developmental programs that are both required for a comprehensive T reg cell repertoire.

This research explored the potential of a synthesised pyrazoline derivative 5‐ethoxy 5‐hydroxy 3‐methyls 4, 5‐dihydro 1Hpyrazol 1 yl (pyridine 4 yl) methanone [5‐E‐5‐H‐PD], against arthritis using a Complete Freund's Adjuvant (CFA)‐induced arthritis in a rat model. Sprague–Dawley rats were used to induce arthritis via subplantar injection of CFA (0.1 mL) into their right hind paw. Animals were divided into 6 groups ( n = 4): normal, arthritis, standard drug (methotrexate 1 mg/kg intraperitoneally), and 3 treatment groups receiving 5‐E‐5‐H‐PD, 10, 20 and 40 mg/kg orally for 21 days. Clinical signs (paw volume and arthritis score), pro‐inflammatory cytokines, and histopathological alterations were evaluated. The 5‐E‐5‐H‐PD groups showed a reduction in paw edema in a dose‐dependent manner. On day 21, paw volume in the 40 mg/kg dose animals decreased significantly to 2.31 ± 0.12 mm compared to 4.82 ± 0.14 mm in the disease animals ( p < 0.001). Arthritis scores reduced from 3.8 ± 0.2 (control) to 1.5 ± 0.3 in the high‐dose treatment group. Serum IL‐10, TNF‐α, and NF‐κB levels were significantly reduced to 66.75 ± 3.0 pg/mL, 34.50 ± 1.8 pg/mL and 9.50 ± 0.6 pg/mL respectively, compared to the arthritis induced rats 129.8 ± 2.0 pg/mL, 77.75 ± 1.5 pg/mL and 28.50 ± 1.3 pg/mL respectively, compared to the arthritis induced rats (112.3 ± 5.5, 96.8 ± 4.3, 123.1 ± 6.2 pg/mL, p < 0.001). Histopathology analysis confirmed reduced synovial hyperplasia and inflammatory infiltration in treated joints. The pyrazoline derivative, 5‐E‐5‐H‐PD, demonstrated significant anti‐arthritic effects in the CFA‐induced rat model by reducing inflammation, cytokine expression and joint destruction. These findings support further investigation into pyrazoline‐based compounds as promising therapeutic agents for RA.

Immunocontraceptive vaccines targeting gonadotropin-releasing hormone (GnRH) are in high demand for controlling population growth and managing the temperament of both wild and domesticated animals. Achieving adequate efficacy, especially with a single-dose immunization, requires potent vaccines and adjuvants. However, commercial vaccines face challenges: some, like Gonacon®, have regulatory issues with veterinary authorities, while others, like Bopriva®, lack potency and require multiple booster doses. Thus, there is a critical need for highly effective vaccines with robust adjuvants suitable for easy, less invasive single-dose administration. Recent studies have shown that peptide vaccines adjuvanted with 15-mer polyleucine (L15) or poly(methyl acrylate) (PMA) demonstrate potent immunogenicity, comparable to complete Freund's adjuvant (CFA), after a single dose against Streptococcus pyogenes. Our research aims to evaluate the performance of these promising vaccine adjuvant as single-dose contraceptive vaccines in mice, compared to well-established commercial and experimental adjuvants, such as AddaVax®, Incomplete Freunds Adjuvant (IFA), and CFA. To develop a vaccine with sufficient HLA coverage for cattle immunocontraception, we evaluated a peptide vaccine incorporating three cattle-compatible helper T cell epitopes. We evaluated the immunogenicity of constructs in mice to that of a construct with the universal mouse-compatible PADRE (P) helper T cell epitope. Various vaccines were prepared to investigate: (A) the impact of incorporating cattle-compatible helper T cells on immunogenicity, and (B) the effectiveness of different adjuvant systems compared to CFA. The vaccines were administered subcutaneously to C57BL/6 mice, and serological assays revealed that the L15/Quil A-based vaccine system was highly immunogenic, with performance comparable to CFA without the need for reactogenic mycobacterial components. Our vaccines significantly reduced serum progesterone levels in mice, making the L15/Quil A system a strong candidate for single-dose anti-fertility application, followed by PMA, and AddaVax® adjuvanted GnRH.

•MVA85A boost did not improve the immune response induced by 6611 strain + IFA adjuvant.•Priming with 6611 + ISA201 and boosting with MVA85A elicited the highest immune response.•The protection provided by the 6611 + ISA201 vaccine is enhanced by the MVA85A boost. Vaccination is the best strategy to control Paratuberculosis (PTB), which is a significant disease in cattle and sheep. Previously we showed the humoral and cellular immune response induced by a novel vaccine candidate against PTB based on the Argentinian Mycobacterium avium subspecies paratuberculosis (Map) 6611 strain. To improve 6611 immunogenicity and efficacy, we evaluated this vaccine candidate in mice with two different adjuvants and a heterologous boost with a recombinant modified vaccinia Ankara virus (MVA) expressing the antigen 85A (MVA85A). We observed that boosting with MVA85A did not improve total IgG or specific isotypes in serum induced by one or two doses of 6611 formulated with incomplete Freund’s adjuvant (IFA). However, when 6611 was formulated with ISA201 adjuvant, MVA85A boost enhanced the production of IFNγ, Th1/Th17 cytokines (IL-2, TNF, IL-17A) and IL-6, IL-4 and IL-10. Also, this group showed the highest levels of IgG2b and IgG3 isotypes, both important for better protection against Map infection in the murine model. Finally, the heterologous scheme elicited the highest levels of protection after Map challenge (lowest CFU count and liver lesion score). In conclusion, our results encourage further evaluation of 6611 strain + ISA201 prime and MVA85A boost in bovines.