Bax deficiency extends the survival of Ku70 knockout mice that develop lung and heart diseases.

Ku70 (Lupus Ku autoantigen p70) is essential in nonhomologous end joining DNA double-strand break repair, and ku70(-/-) mice age prematurely because of increased genomic instability and DNA damage responses. Previously, we found that Ku70 also inhibits Bax, a key mediator of apoptosis. We hypothesized that Bax-mediated apoptosis would be enhanced in the absence of Ku70 and contribute to premature death observed in ku70(-/-) mice. Here, we show that ku70(-/-) bax(+/-) and ku70(-/-) bax(-/-) mice have better survival, especially in females, than ku70(-/-) mice, even though Bax deficiency did not decrease the incidence of lymphoma observed in a Ku70-null background. Moreover, we found that ku70(-/-) mice develop lung diseases, like emphysema and pulmonary arterial (PA) occlusion, by 3 months of age. These lung abnormalities can trigger secondary health problems such as heart failure that may account for the poor survival of ku70(-/-) mice. Importantly, Bax deficiency appeared to delay the development of emphysema. This study suggests that enhanced Bax activity exacerbates the negative impact of Ku70 deletion. Furthermore, the underlying mechanisms of emphysema and pulmonary hypertension due to PA occlusion are not well understood, and therefore ku70(-/-) and Bax-deficient ku70(-/-) mice may be useful models to study these diseases.

Intracellular delivery of a novel multiepitope peptide vaccine by an amphipathic peptide carrier enhances cytotoxic T-cell responses in HLA-A*201 mice.

Cytotoxic T lymphocytes (CTL) are key players in the neutralization of viruses and killing of tumor cells. However, for generating an optimal CTL response by vaccination, the antigen has to be delivered directly into the cytoplasm for presentation by the conventional MHC class I pathway. To mimic the presentation of multiple epitopes by a tumor or virus infected cell, we have designed a multiepitope peptide vaccine incorporating thee CTL epitopes in tandem with double arginine spacers to facilitate efficient cleavage of the individual epitopes. To deliver the multiepitope peptide vaccine into the cytoplasm of mature dendritic cells for presentation by the MHC class I pathway we made use of an amphipathic peptide carrier. Direct injection of a non-covalent complex of the multiepitope peptide vaccine and amphipathic peptide carrier in an aqueous formulation into HLA-A*0201 (HHD) transgenic mice enhanced the cytotoxic T-cell responses by two to sixfold compared with multiepitope peptide vaccination alone. This novel antigen delivery strategy may find general application in the development of more effective vaccines for the treatment of cancer and infectious disease.

Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein.

The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) of B. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 microg of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.