Theiler's Murine Encephalomyelitis Virus as a Vaccine Candidate for Immunotherapy

The induction of sterilizing T-cell responses to tumors is a major goal in the development of T-cell vaccines for treating cancer. Although specific components of anti-viral CD8+ immunity are well characterized, we still lack the ability to mimic viral CD8+ T-cell responses in therapeutic settings for treating cancers. Infection with the picornavirus Theiler's murine encephalomyelitis virus (TMEV) induces a strong sterilizing CD8+ T-cell response. In the absence of sterilizing immunity, the virus causes a persistent infection. We capitalized on the ability of TMEV to induce strong cellular immunity even under conditions of immune deficiency by modifying the virus to evaluate its potential as a T-cell vaccine. The introduction of defined CD8+ T-cell epitopes into the leader sequence of the TMEV genome generates an attenuated vaccine strain that can efficiently drive CD8+ T-cell responses to the targeted antigen. This virus activates T-cells in a manner that is capable of inducing targeted tissue damage and glucose dysregulation in an adoptive T-cell transfer model of diabetes mellitus. As a therapeutic vaccine for the treatment of established melanoma, epitope-modified TMEV can induce strong cytotoxic T-cell responses and promote infiltration of the T-cells into established tumors, ultimately leading to a delay in tumor growth and improved survival of vaccinated animals. We propose that epitope-modified TMEV is an excellent candidate for further development as a human T-cell vaccine for use in immunotherapy

Evidence of homing of black rockfish Sebastes inermis using biotelemetry

The black rockfish Sebastes inermis is one of the most important fishery species along the coast from southern Hokkaido to Kyushu, Japan and is often found in rocks and Zostera areas. We conducted biotelemetry using coded ultrasonic transmitters to clarify the movement of the black rockfish that inhabited the seawall of the Kansai International Airport. We released 25 black rockfish at two points. One was the airport seawall and the other was side shallows off the Sensyu district. Seventeen black rockfish returned to their capture site after release. We used the V-test to determine whether the direction of movement was random or orientated. The black rockfish moved at random along the seawall within some hours after release (P> 0.05). Four hours after release, they moved to their home site intentionally (P< 0.0025)

Role of olfaction and vision in homing behaviour of black rockfish Sebastes inermis

How fish find their original habitat and natal home remains an unsolved riddle of animal behaviour. Despite extensive efforts to study the homing behaviour of diadromous fish, relatively little attention has been paid to that of non-diadromous marine fish. Among these, most rockfish of the genus Sebastes exhibit homing ability and/or a strong fidelity to their habitats. However, how these rockfish detect the homeward direction has not been clarified. The goal of the present research was to investigate the sensory mechanisms involved in the homing behaviour of the black rockfish Sebastes inermis, using acoustic telemetry. Vision-blocked or olfactory-ablated rockfish were released in natural waters and their homing behaviours compared with those of intact or control individuals. Blind rockfish showed homing from both inside and outside their habitat. The time taken by blind fish to reach their home habitat was not significantly different from that of the control fish. In contrast, most olfactory-ablated fish did not successfully reach their original habitat. Our results indicate that black rockfish predominantly use the olfactory sense in their homing behaviour

Induction of diabetes with TMEV-L/OVA using RIP-OVA mice given OT-1 T-cell transfer.

<p>Representative pancreatic islets from RIP-OVA mice given TMEV-wt (A) or TMEV-L/OVA (B) without OT-1 transfer. (C) Pancreatic islet infiltration observed in OT-1 transferred RIP-OVA receiving TMEV-L/OVA vaccine compared to TMEV-wt vaccine (D). (E) Blood glucose levels observed in RIP-OVA mice given TMEV-wt or TMEV-L/OVA vaccines. Increased blood glucose was observed on day 6 (p = 0.007), 7 (p<0.001), 8 (p<0.001) and 9 (p<0.001) in mice receiving both OT-1 transfer and TMEV-L/OVA vaccine compared to transfer with TMEV-wt.</p

Generation of epitope specific CD8+ T-cell responses with TMEV-L/OVA.

<p>(A) FACS analysis of brain infiltrating lymphocytes (BIL) from mice infected with TMEV-L/OVA for 6 days. The proportion of OVA₂₅₇ specific T-cells increases in the absence of viral specific CD8+ T-cells (p<0.001). (B) In vitro cytotoxic activity of BIL as measured by a 4 hour chromium release assay using VP2₁₂₁ and OVA₂₅₇ peptide pulsed targets. (C) OVA₂₅₇ specific in vivo killing of labeled target cells in 6 day TMEV-L/OVA infected mice was increased compared to TMEV-wt (p = <0.001).</p

The generation of epitope modified TMEV vaccine.

<p>(A) The genome of TMEV contains an Xho I restriction site within the leader sequence which can be used for insertion of MHC class I peptide epitopes. (B) Sequence of LCMV and ovalbumin linked epitopes inserted into the Xho I restriction site of the pDAFL₃ vector. (C) Productive infection and plaques from wild-type and modified TMEV virus. (D) Plaque assay (left) of virus recovered from the brains of mice infected with virus for 6 days (*p = 0.014). Absence of detectable viral transcripts in the brain 21 days after inoculation with TMEV-L/OVA vaccine in B6 and FVB mice (right).</p

MHC class I epitope specific protection and targeting of B16-OVA melanoma.

<p>(A) Vaccination with TMEV-wt or TMEV-L/OVA virus did not delay tumor outgrowth using the parental B16 tumor model. (B), Tumor outgrowth in mice challenged with B16-OVA tumor and vaccinated with TMEV-wt (top) or with TMEV-L/OVA (bottom). Tumor sizes were significantly different in these treatment groups on days 10 through 19 (* designates p<0.05). (C) G418 resistance and growth of tumor cells recovered from mice vaccinated with TMEV-wt or with TMEV-L/OVA (left). Quantitation of cresyl violet stained tumor cells recovered from vaccinated mice. Data expressed as the percent of well area containing stained tumor cells from TMEV-wt and TMEV-L/OVA vaccine treated mice (middle) (p<0.05). RNA isolated from recovered tumors was analyzed by qRT-PCR for the presence of ovalbumin specific transcripts (p = 0.009).</p

Delayed tumor outgrowth using TMEV-L/OVA vaccine in a therapeutic model of established tumor burden.

<p>FACS analysis of 6 day BIL (A) and TIL (B) from mice given TMEV vaccines on day 9 after B16-OVA implantation. Lymphocytes were assessed for the presence of OVA₂₅₇, VP2₁₂₁ and E7₄₉ specific CD8 T-cell responses. Percentages are the percent of tetramer specific CD8 cells. Numbers represent the absolute numbers of cells per 100,000 events. (C) Four hour chromium release assay using day 15 TIL from mice treated with TMEV-L/OVA or TMEV-wt 9 days after B16-OVA challenge. (D) (left) Observed tumor growth in mice treated with TMEV-wt or TMEV-L/OVA. (p<0.05 at day 21 and 24). Tumor outgrowth and survival of individual animals treated with TMEV vaccines. Significant differences in survival were observed between the treatment groups at the conclusion of the 30 day observation period (p<0.05).</p