Contribution of Antibody-Mediated Effector Functions to the Mechanism of Efficacy of Vaccines for Opioid Use Disorders.

Vaccines and mAbs offer promising strategies to treat substance use disorders (SUDs) and prevent overdose. Despite vaccines and mAbs against SUDs demonstrating proof of efficacy, selectivity, and safety in animal models, it is unknown whether the mechanism of action of these immunotherapeutics relies exclusively on the formation of Ab/drug complexes, or also involves Ab-mediated effector functions. Hence, this study tested whether the efficacy of active and passive immunization against drugs of abuse requires phagocytosis, the intact Fc portion of the anti-drug Ab, FcγRs, or the neonatal FcR (FcRn). The efficacy of a lead vaccine against oxycodone was not diminished in mice after depletion of macrophages or granulocytes. Anti-oxycodone F(ab')2 fragments resulted in lower serum levels of F(ab')2 compared with intact mAbs, and F(ab')2s were not as effective as the parent mAbs in reducing distribution of oxycodone to the brain. The efficacy of vaccines and mAbs against oxycodone was preserved in either FcγIII or FcγI-IV ablated mice, suggesting that FcγRs are not required for Ab efficacy. Finally, both active and passive immunization against oxycodone in FcRn-/- mice yielded reduced efficacy compared with wild-type control mice. These data identified a role for FcRn, but not for phagocytosis or Fc-dependent effector functions, in mediating the efficacy of vaccines and mAbs against SUD. This study supports rational design of vaccines and mAbs engineered for maximal neutralization activity and optimal FcRn binding.

Mechanisms of interleukin 4 mediated increase in efficacy of vaccines against opioid use disorders.

Opioid use disorders (OUD) affect over 27 million people worldwide. Anti-opioid vaccines offer a promising strategy to treat OUD and prevent overdose. Using immunomodulation of cytokine signaling to increase vaccine efficacy, this study found that blocking IL-4 improved the efficacy of vaccines targeting oxycodone and fentanyl in male and female mice. Genetic deletion of the IL-4 receptor, STAT6, or antibody-based depletion of IL-13, did not increase vaccine efficacy against opioids, suggesting the involvement of type I IL-4 receptors. Enhancement of vaccine efficacy with blockade of IL-4 was associated with improved germinal center formation in secondary lymphoid organs and selective transcriptome signatures in the activated CD4+ T cell population subset. These data suggest that IL-4 is both a pharmacological target and a potential biomarker of vaccine efficacy against OUD.

Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2Db class I molecule.

Brain atrophy is a common feature of numerous neurologic diseases in which the role of neuroinflammation remains ill-defined. In this study, we evaluated the contribution of major histocompatibility complex class I molecules to brain atrophy in Theiler's murine encephalomyelitis virus (TMEV)-infected transgenic FVB mice that express the Db class I molecule. FVB/Db and wild-type FVB mice were evaluated for changes in neuroinflammation, virus clearance, neuropathology, and development of brain atrophy via T2-weighted MRI and subsequent 3-dimensional volumetric analysis. Significant brain atrophy and hippocampal neuronal loss were observed in TMEV-infected FVB/Db mice, but not in wild-type FVB mice. Brain atrophy was observed at 1 mo postinfection and persisted through the 4-mo observation period. Of importance, virus-infected FVB/Db mice elicited a strong CD8 T-cell response toward the immunodominant Db-restricted TMEV-derived peptide, VP2121-130, and cleared TMEV from the CNS. In addition, immunofluorescence revealed CD8 T cells near virus-infected neurons; therefore, we hypothesize that class I restricted CD8 T-cell responses promote development of brain atrophy. This model provides an opportunity to analyze the contribution of immune cells to brain atrophy in a system where persistent virus infection and demyelination are not factors in long-term neuropathology.-Huseby Kelcher, A. M., Atanga, P. A., Gamez, J. D., Cumba Garcia, L. M., Teclaw, S. J., Pavelko, K. D., Macura, S. I., Johnson. A. J. Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2Db class I molecule.

Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique.

Effective recovery of activated brain infiltrating lymphocytes is critical for investigations involving murine neurological disease models. To optimize lymphocyte recovery, we compared two isolation methods using brains harvested from seven-day Theiler's murine encephalomyelitis virus (TMEV) and TMEV-OVA infected mice. Brains were processed using either a manual dounce based approach or enzymatic digestion using type IV collagenase. The resulting cell suspensions from these two techniques were transferred to a percoll gradient, centrifuged, and lymphocytes were recovered. Flow cytometric analysis of CD45hi cells showed greater percentage of CD44hiCD62lo activated lymphocytes and CD19+ B cells using the dounce method. In addition, we achieved a 3-fold greater recovery of activated virus-specific CD8 T cells specific for the immunodominant Db:VP2121-130 and engineered Kb:OVA257-264 epitopes through manual dounce homogenization approach as compared to collagenase digest. A greater percentage of viable cells was also achieved through dounce homogenization. Therefore, we conclude that manual homogenization is a superior approach to isolate activated T cells from the mouse brain.

The Effect of Vector Silencing during Picornavirus Vaccination against Experimental Melanoma and Glioma.

Virus vector-based vaccination against tumor-specific antigens remains a promising therapeutic approach to overcome the immune suppressive tumor microenvironment. However, the extent that the desired CD8 T cell response against the targeted tumor antigen is impacted by the CD8 T cell response against the virus vector is unclear. To address this question, we used picornavirus vaccination with Theiler's murine encephalomyelitis virus (TMEV) as our vector against tumor-expressed ovalbumin (OVA257-264) antigen in both the B16-OVA murine melanoma and GL261-quad cassette murine glioma models. Prior to vaccination, we employed vector silencing to inhibit the CD8 T cell response against the immunodominant TMEV antigen, VP2121-130. We then monitored the resulting effect on the CD8 T cell response against the targeted tumor-specific antigen, ovalbumin. We demonstrate that employing vector silencing in the context of B16-OVA melanoma does not reduce tumor burden or improve survival, while TMEV-OVA vaccination without vector silencing controls tumor burden. Meanwhile, employing vector silencing during picornavirus vaccination against the GL261-quad cassette glioma resulted in a lower frequency of tumor antigen-specific CD8 T cells. The results of this study are relevant to antigen-specific immunotherapy, in that the virus vector-specific CD8 T cell response is not competing with tumor antigen-specific CD8 T cells. Furthermore, vector silencing may have the adverse consequence of reducing the tumor antigen-specific CD8 T cell response, as demonstrated by our findings in the GL261-quad cassette model.