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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.

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.

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.

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.

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.

Background/Objectives: Freund’s adjuvants induce different immunomodulatory effects, but their underlying molecular mechanisms are unclear. In this study, we investigated whether the immune-stimulating effects of the complete Freund’s adjuvant (CFA) involve the mechanisms of trained immunity (TI). Methods: We examined bone marrow cells (BMCs) isolated from CFA-immunized A/J mice to address this question. Incomplete Freund’s adjuvant (IFA) and Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin (BCG) served as negative and positive controls, respectively. We evaluated cytokine profiles, metabolic, and epigenetic changes. Results: First, BMCs from all groups except saline showed varied levels of IL-1β, IL-6, and TNF-α. But expression of CCL5 and CXCL10 was significantly elevated only in the CFA and BCG groups. Transcriptionally, significant elevations were noted for TNF-α and IL-1β in the CFA and BCG groups, whereas CXCL10, IL-6, and IL-10 were upregulated in the CFA and BCG groups, respectively. Second, while BMCs from the BCG group expressed the markers of both the M1 and M2 macrophages, no clear trends were noted in the CFA and IFA groups. Third, cell lysates from the CFA group revealed metabolic reprogramming in the BMCs. Specifically, we observed an increased level of lactate, indicative of aerobic glycolysis, which is implicated in TI, and this was also detected in the IFA group. Fourth, epigenetic analysis revealed histone enrichment in the promoter region of TNF-α, in the CFA group, but to a lesser degree than the BCG group. However, no epigenetic changes were observed in the IFA group. Conclusions: Our data provide new insights into the mechanisms of Freund’s adjuvants and the immunomodulatory effects of CFA could involve the features of TI.

The carboxyl-terminal fragment of MSP-1 is a potential malaria vaccine candidate, but its limited immunogenicity in humans has slowed clinical progress, needing the optimization of formulation of adjuvant and construct. In this study, the N- and C-terminal fragments of the PyMSP-1 (PyMSP-1 N and PyMSP-1C) were immunized to mice with either incomplete Freund's adjuvant (IFA) plus CpG ODN 1826 or Aluminum salts (Alum) plus CpG, followed by a challenge with Plasmodium yoelii 17XNL to investigate vaccine efficacy. Humoral response and antigen-specific T-cell-derived IFN-γ cytokines were analyzed to compare both fragments. After challenge infection, all mice immunized by PyMSP-1C in IFA plus CpG ODN survived with low-grade parasitemia, while 50 % of mice immunized with PyMSP-1 N in Alum plus CpG ODN died with high levels of parasitemia. Co-immunized with both fragments prevented parasitemia entirely, with IFA plus CpG adjuvants proving more suitable than Alum plus CpG. Both fragments elicited a comparable humoral response when they were formulated with IFA plus CpG ODN but PyMSP-1 N formulated with Alum plus CpG ODN significantly decreased the antigen-specific IgG level. While both IgG1 and IgG2c levels were comparable in two fragments formulated by IFA plus CpG ODN, it was efficient to induce the level of IgG2c of PyMSP-1 N fragment (P < 0.0001). Likewise, IFN-γ from both CD8+ and CD4+ T-cells was significantly lower by PyMSP-1 N than PyMSP-1C formulated in IFA plus CpG ODN (P < 0.0001). In conclusion, the N-terminal fragment of PyMSP-1 protected mice although it showed lower humoral and cellular immune response compared to C-terminal of MSP-1 in IFA plus CpG. The antibody level of PyMSP-1 N was comparable to that of PyMSP-1C when it was formulated with IFA plus CpG.

Background Attentional deficits are among the most common pain-induced cognitive disorders. Pain disrupts attention and may excessively occupy attentional resources in pathological states, leading to daily function impairment and increased disability. However, the neural circuit mechanisms by which pain disrupts attention are incompletely understood. Methods We used a three-choice serial reaction time task (3CSRTT) to construct a sustained-attention task model in male C57BL/6J mice. Formalin or complete Freund's adjuvant was injected into a paw to establish an inflammatory pain model. We measured changes in 3CSRTT performance in the two inflammatory pain models, and investigated the neural circuit mechanisms of pain-induced attentional deficits. Results Acute inflammatory pain impaired 3CSRTT performance, while chronic inflammatory pain had no effect. Either inhibition of the ascending pain pathway by blockade of the conduction of nociceptive signals in the sciatic nerve using the local anesthetic lidocaine or chemogenetic inhibition of Ca 2+ /calmodulin-dependent protein kinase IIα (CaMKIIα) neurons in the lateral parabrachial nucleus (LPBN) attenuated the acute inflammatory pain-induced impairment of 3CSRTT performance, while chemogenetic activation of CaMKIIα neurons in the LPBN disrupted the 3CSRTT. Furthermore, the activity of CaMKIIα neurons in the LPBN was significantly lower on Day 2 after complete Freund's adjuvant injection than on the day of injection, which correlated with the recovery of 3CSRTT performance during chronic inflammatory pain. Conclusions Activation of excitatory neurons in the LPBN is a mechanism by which acute inflammatory pain disrupts sustained attention. This finding has implications for the treatment of pain and its cognitive comorbidities.