Non-invasive imaging of GFAP expression after neuronal damage in mice.

Up-regulation of glial fibrillary acidic protein (GFAP) expression is often used as a surrogate marker of neuronal damage. We have created a transgenic mouse line that carries the luciferase gene under the transcriptional control of the mouse GFAP promoter. Biophotonic imaging was used to non-invasively detect the increase in GFAP expression after kainic acid induced neuronal cell death. We demonstrate that after kainic acid treatment, strong biophotonic signals were detected from the brain area. This correlated with both endogenous GFAP and luciferase RNA levels as well as with hippocampal cell death observed histologically. The transgenic mouse line will provide a powerful tool to dynamically monitor neuronal cell death in the living animal and will aid in the discovery and development of drugs to treat damage due to stroke and other neurodegenerative diseases.

Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin.

The recognition of specific pathogen associated molecular patterns (PAMPs) by members of the Toll-like receptor (TLR) family is critical for the activation of the adaptive immune response. Thus, incorporation of PAMPs into vaccines should result in more potent, protective antigen-specific responses in the absence of adjuvants or complex formulations. Here we describe an influenza A vaccine that is refractory to the genetic instability of hemagglutinin and neuraminidase and includes a trigger of the innate immune response to enhance immunogenicity and efficacy. A recombinant protein comprising the TLR5 ligand flagellin fused to four tandem copies of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli and purified to homogeneity. This protein, STF2.4xM2e, retained TLR5 activity and displayed the protective epitope of M2e defined by a monoclonal antibody, 14C2. Mice immunized with STF2.4xM2e in aqueous buffer, without adjuvants or other formulation additives, developed potent M2e-specific antibody responses that were quantitatively and qualitatively superior to those observed with M2e peptide delivered in alum. The antibody response was dependent on the physical linkage of the antigen to flagellin and recognized the epitope defined by monoclonal antibody 14C2, which has been shown to protect mice from challenge with influenza A virus. Moreover, immunization with STF2.4xM2e at a dose of 0.3 microg per mouse protected mice from a lethal challenge with influenza A virus, and significantly reduced weight loss and clinical symptoms. These data demonstrate that the linkage of specific TLR ligand with influenza M2e yields a vaccine candidate that offers significant promise for widespread protection against multiple influenza A virus strains.

A West Nile virus recombinant protein vaccine that coactivates innate and adaptive immunity.

A chimeric protein West Nile virus (WNV) vaccine capable of delivering both innate and adaptive immune signals was designed by fusing a modified version of bacterial flagellin (STF2 Delta ) to the EIII domain of the WNV envelope protein. This fusion protein stimulated interleukin-8 production in a Toll-like receptor (TLR)-5-dependent fashion, confirming appropriate in vitro TLR5 bioactivity, and also retained critical WNV-E-specific conformation-dependent neutralizing epitopes as measured by enzyme-linked immunosorbent assay. When administered without adjuvant to C3H/HeN mice, the fusion protein elicited a strong WNV-E-specific immunoglobulin G antibody response that neutralized viral infectivity and conferred protection against a lethal WNV challenge. This potent EIII-specific immune response requires a direct linkage of EIII to STF2 Delta , given that a simple mixture of the 2 components failed to induce an antibody response or to provide protection against virus challenge. The presence of a functional TLR5 gene in vivo is also required--TLR5-deficient mice elicited only a minimal antigen-specific response. These results confirm that vaccines designed to coordinately regulate the innate and adaptive immune responses can induce protective immune responses without the need for potentially toxic adjuvants. They also support the further development of an effective WNV vaccine and novel monovalent and multivalent vaccines for related flaviviruses.

Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.

Micromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4.4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3' end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.