Immunodominant fragments of myelin basic protein initiate T cell-dependent pain.

BACKGROUND: The myelin sheath provides electrical insulation of mechanosensory Aß-afferent fibers. Myelin-degrading matrix metalloproteinases (MMPs) damage the myelin sheath. The resulting electrical instability of Aß-fibers is believed to activate the nociceptive circuitry in Aß-fibers and initiate pain from innocuous tactile stimulation (mechanical allodynia). The precise molecular mechanisms, responsible for the development of this neuropathic pain state after nerve injury (for example, chronic constriction injury, CCI), are not well understood. METHODS AND RESULTS: Using mass spectrometry of the whole sciatic nerve proteome followed by bioinformatics analyses, we determined that the pathways, which are classified as the Infectious Disease and T-helper cell signaling, are readily activated in the nerves post-CCI. Inhibition of MMP-9/MMP-2 suppressed CCI-induced mechanical allodynia and concomitant TNF-α and IL-17A expression in nerves. MMP-9 proteolysis of myelin basic protein (MBP) generated the MBP84-104 and MBP68-86 digest peptides, which are prominent immunogenic epitopes. In agreement, the endogenous MBP69-86 epitope co-localized with MHCII and MMP-9 in Schwann cells and along the nodes of Ranvier. Administration of either the MBP84-104 or MBP68-86 peptides into the naïve nerve rapidly produced robust mechanical allodynia with a concomitant increase in T cells and MHCII-reactive cell populations at the injection site. As shown by the genome-wide expression profiling, a single intraneural MBP84-104 injection stimulated the inflammatory, immune cell trafficking, and antigen presentation pathways in the injected naïve nerves and the associated spinal cords. Both MBP84-104-induced mechanical allodynia and characteristic pathway activation were remarkably less prominent in the T cell-deficient athymic nude rats. CONCLUSIONS: These data implicate MBP as a novel mediator of pain. Furthermore, the action of MMPs expressed within 1 day post-injury is critical to the generation of tactile allodynia, neuroinflammation, and the immunodominant MBP digest peptides in nerve. These MBP peptides initiate mechanical allodynia in both a T cell-dependent and -independent manner. In the course of Wallerian degeneration, the repeated exposure of the cryptic MBP epitopes, which are normally sheltered from immunosurveillance, may induce the MBP-specific T cell clones and a self-sustaining immune reaction, which may together contribute to the transition of acute pain into a chronic neuropathic pain state.

Immunoselective algorithm to devise multi-epitope subunit vaccine fighting against human cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus (HCMV) generally causes asymptomatic infection, but sometimes it may cause severe complications among immunocompromised individuals. It may also promote various malignancies like prostate cancer and breast cancer. However, even after having this severe illness, there is no effective cure yet. This situation urges the need for effective chemotherapeutics or vaccination to tackle this severe complication. METHODS: A combinatorial screening algorithm was applied to design a subunit vaccine consisting of B-cell epitopes, CTL- and HTL epitopes along with a suitable adjuvant (TLR-4 agonist) and linkers. The conservancy of CTL, HTL, and B-cell epitopes was also determined. Further, physicochemical characterization, antigenicity, and allergenicity were determined to check the safety and immunogenic behavior of the designed vaccine candidate. Later on, the 3D structure of the vaccine protein was determined, followed by molecular docking and molecular dynamics simulation with TLR-4 to check their binding free energy and complex stability. RESULT: A subunit vaccine of 964 amino acid residues was developed, having good immunogenicity and non-allergenicity behavior. The designed subunit vaccine has HTL epitopes with their ability to induce the release of IFN-γ cytokine. The sorted HTL and CTL epitopes were found to be conserved among two available strains of HCMV. It has also shown an excellent binding affinity with the TLR-4 receptor along with the formation of the stable complex as determined by a molecular dynamics simulation study. CONCLUSION: The designed subunit vaccine may have the ability to induce an immunogenic response and memory cell formation to protect against the HCMV mediated disease conditions.

Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes.

A melanoma vaccine composed of HLA-A2-restricted peptide YLEPGPVTA (gp100(280)), with or without a modified T-helper epitope from tetanus toxoid AQYIKANSKFIGITEL, has been evaluated in a Phase I trial to assess safety and immunological response. The vaccines were administered s.c. in either of two adjuvants, Montanide ISA-51 or QS-21, to 22 patients with high-risk resected melanoma (stage IIB-IV). Local and systemic toxicities were mild and transient. We detected CTL responses to the gp100(280) peptide in peripheral blood in 14% of patients. Helper T-cell responses to the tetanus helper peptide were detected in 79% of patients and had a Th1 cytokine profile. One patient with a CTL response to gp100 had a recurrence in a lymph node 2 years later; her nodes contained CD8+ cells reactive to gp100(280) (0.24%), which proliferated in response to peptide. The overall survival of patients is 75% (95% confidence interval, 57-94%) at 4.7 years follow-up, which compares favorably with expected survival. Four of 14 patients who completed at least six vaccines subsequently developed metastases, all of which were solitary and surgically resectable. They remain alive and clinically free of disease at last follow-up. Data from this trial demonstrate immunogenicity of the gp100(280) peptide and suggest that immune responses may persist long-term in some patients. The frequency and magnitude of the CTL response may be improved with more aggressive vaccination regimens. Although this Phase I study was not intended to evaluate clinical benefit, the excellent survival of patients on this protocol suggests the possibility of a benefit that should be assessed in future studies.

Overcoming immunogenicity associated with the use of biopharmaceuticals.

The safety and efficacy of biopharmaceuticals can be severely impaired by their immunogenicity. A risk-based strategy should be used to assess immunogenicity on a case-by-case basis using standardized methods to correlate anti-drug antibody levels with clinical outcome. In silico and in vitro techniques allow putative T-cell epitopes to be identified and eliminated in candidate molecules while maintaining structure and function. Putative T-cell epitopes can be studied in the context of the HLA allotypes representative of the target population in vitro and in transgenic mice that express human HLA genes. Mice immune tolerant to human proteins allow the study of the effect of factors such as aggregation on the loss of immune tolerance. However, significant challenges remain in order to be able predict the immunogenicity of a therapeutic protein in a particular individual.

Phase I active immunotherapy with combination of two chimeric, human epidermal growth factor receptor 2, B-cell epitopes fused to a promiscuous T-cell epitope in patients with metastatic and/or recurrent solid tumors.

PURPOSE To evaluate the maximum-tolerated dose (MTD), safety profile, and immunogenicity of two chimeric, B-cell epitopes derived from the human epidermal growth factor receptor (HER2) extracellular domain in a combination vaccine with a promiscuous T-cell epitope (ie, MVF) and nor-muramyl-dipeptide as adjuvant emulsified in SEPPIC ISA 720. PATIENTS AND METHODS Eligible patients with metastatic and/or recurrent solid tumors received three inoculations on days 1, 22, and 43 at doses of total peptide that ranged from 0.5 to 3.0 mg. Immunogenicity was evaluated by enzyme-linked immunosorbent assay, flow cytometry, and HER2 signaling assays. Results Twenty-four patients received three inoculations at the intended dose levels, which elicited antibodies able to recognize native HER2 receptor and inhibited both the proliferation of HER2-expressing cell lines and phosphorylation of the HER2 protein. The MTD was determined to be the highest dose level of 3.0 mg of the combination vaccine. There was a significant increase from dose level 1 (0.5 mg) to dose level 4 (3.0 mg) in HER2-specific antibodies. Four patients (one each with adrenal, colon, ovarian, and squamous cell carcinoma of unknown primary) were judged to have stable disease; two patients (one each with endometrial and ovarian cancer) had partial responses; and 11 patients had progressive disease. Patients with stable disease received 6-month boosts, and one patient received a 20-month boost. CONCLUSION The combination vaccines were safe and effective in eliciting antibody responses in a subset of patients (62.5%) and were associated with no serious adverse events, autoimmune disease, or cardiotoxicity. There was preliminary evidence of clinical activity in several patients.

Antigen-specific CD8+ T cells mediate a peptide-induced fatal syndrome.

Peptide immunotherapy both activates and suppresses the T cell response against known peptide Ags. Although pretreatment with VP2(121-130) peptide inhibits the development of antiviral CTL specific for the immunodominant D(b):VP2(121-130) epitope expressed during acute Theiler's murine encephalomyelitis virus infection, i.v. injection of this same peptide or MHC tetramers containing the peptide during an ongoing antiviral CTL response results in a peptide-induced fatal syndrome (PIFS) within 48 h. Susceptibility to PIFS is dependent on peptide-specific CD8(+) T cells, varies among inbred strains of mice, and is not mediated by traditionally defined mechanisms of shock. Analyses using bone marrow chimeras and mutant mice demonstrate that susceptibility to PIFS is determined by the genotype of bone marrow-derived cells and requires the expression of perforin. Animals responding to peptide treatment with PIFS develop classical stress responses in the brain. These findings raise important considerations for the development of peptide therapies for active diseases to modify immune responses involving expanded populations of T cells. In summary, treatment with peptides or MHC-tetramers during a peptide-specific immune response can result in a fatal shock-like syndrome. Susceptibility to the syndrome is genetically determined, is mediated by CD8(+) T cells, and requires expression of perforin. These findings raise concerns about the use of peptides and MHC tetramers in therapeutic schemes.