The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen.

Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity.

Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice.

Background: Recombinant Salmonella vaccine vectors may potentially be used to induce specific CD4+ T cell responses against foreign viral antigens. Such immune responses are required features of vaccines against pathogens such as human immunodeficiency virus type 1 (HIV-1). The aim of this study was to investigate the induction of systemic HIV-1-specific CD4+ T helper (Th) responses in mice after oral immunization with a live attenuated Salmonella vaccine vector that expressed HIV-1 subtype C Gag.

A prime-boost immunisation regimen using recombinant BCG and Pr55(gag) virus-like particle vaccines based on HIV type 1 subtype C successfully elicits Gag-specific responses in baboons.

Mycobacterium bovis BCG is considered an attractive live bacterial vaccine vector. In this study, we investigated the immune response of baboons to a primary vaccination with recombinant BCG (rBCG) constructs expressing the gag gene from a South African HIV-1 subtype C isolate, and a boost with HIV-1 subtype C Pr55(gag) virus-like particles (Gag VLPs). Using an interferon enzyme-linked immunospot assay, we show that although these rBCG induced only a weak or an undetectable HIV-1 Gag-specific response on their own, they efficiently primed for a Gag VLP boost, which strengthened and broadened the immune responses.

An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice.

Background: The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP) model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli beta-galactosidase alpha-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated.

Creation and characterisation of a high-copy-number version of the pAL5000 mycobacterial replicon.

The majority of mycobacterial plasmid vectors are derived from the pAL5000 replicon and maintained at approximately five copies per cell. We have devised a method that directly selects for high-copy-number plasmids. This involves enriching for high copy number plasmids by repeatedly isolating and retransforming plasmids from a mutant library.

Genetic alteration of Mycobacterium smegmatis to improve mycobacterium-mediated transfer of plasmid DNA into mammalian cells and DNA immunization.

Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M.

Evaluation of recombinant BCG expressing rotavirus VP6 as an anti-rotavirus vaccine.

Recombinant BCG expressing rotavirus VP6 was explored as an anti-rotavirus vaccine in a mouse model. Three promoters and five ribosome-binding sites were used in episomal and integrative E. coli-mycobacterium shuttle vectors to express VP6 in BCG.

Design and preclinical evaluation of a multigene human immunodeficiency virus type 1 subtype C DNA vaccine for clinical trial.

In this study, the design and preclinical development of a multigene human immunodeficiency virus type 1 (HIV-1) subtype C DNA vaccine are described, developed as part of the South African AIDS Vaccine Initiative (SAAVI). Genetic variation remains a major obstacle in the development of an HIV-1 vaccine and recent strategies have focused on constructing vaccines based on the subtypes dominant in the developing world, where the epidemic is most severe. The vaccine, SAAVI DNA-C, contains an equimolar mixture of two plasmids, pTHr.

The immune response of the Chacma baboon to Bacille Calmette Guerin: development of a primate model for BCG-based vaccine research.

Mycobacterium bovis Bacilli Calmette-Guerin (BCG) is used increasingly as an efficient vector for expression of recombinant proteins to induce a strong cell-mediated immunity. Here, we tested the immune response of Chacma baboons to the Tokyo and Pasteur strains of BCG in order to obtain base-line information on the response of this primate to BCG. While a humoral immune response to BCG was detected only in some vaccinated baboons, a cellular immune response characterized by a PPD-specific delayed hypersensitivity response and BCG-specific IFN-gamma production from PBMC was a consistent finding.

O(6)-alkylguanine-DNA alkyltransferase DNA repair in mycobacteria: pathogenic and non-pathogenic species differ.

Setting: DNA repair genes assist the organism in maintaining DNA integrity in the face of environmental (mutagenic) stress. The genome sequences of M. tuberculosis and M.

Molecular evolution of Mycobacterium tuberculosis: phylogenetic reconstruction of clonal expansion.

Setting: M. tuberculosis isolates were collected from patients attending health clinics in a high incidence urban community and in a low incidence rural setting in South Africa.

Objective: To reconstruct the evolutionary history of a group of closely related M.

Novel method for rapid measurement of growth of mycobacteria in detergent-free media.

We describe a novel, rapid, and inexpensive method for the measurement of growth of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium smegmatis in the presence or absence of detergent. The method, which employs hot NaOH treatment of mycobacterial cells to release total cellular protein, compares favorably with other methods for monitoring mycobacterial growth but is particularly useful for heavily clumped cultures grown in defined minimal medium.

Computer assisted identification and classification of streptomycete promoters.

Short sequences that were over represented in a database of Streptomyces promoter region sequences were identified. These sequences and others that were selected on the basis of the characteristics of known promoters, were tested to determine if they were found predominantly at particular distances from the transcription start site. In several cases obvious clusters were recorded.

Influence of ascorbic acid esters on acetaminophen-induced hepatotoxicity in mice.

Groups of male Swiss-Webster mice were gavaged with acetaminophen (APAP), APAP + ascorbyl stearate (AS), or APAP + ascorbyl palmitate (AP) at a dose of 600 mg/kg for each chemical. APAP alone caused a significant increase in liver weight/body weight ratio and hepatic glutathione (GSH) depletion. Co-administration of the ascorbate esters AP or AS with APAP prevented an increase in liver weight/body weight ratios and hepatic glutathione depletion.

Effect of CGS-8216 on high-dose flurazepam convulsive threshold.

Flurazepam, a clinically proven and widely accepted sedative hypnotic, has been shown by a number of investigators to produce convulsions at toxic doses. In the present study, CGS-8216, a benzodiazepine receptor antagonist, reduced the dose of flurazepam required to produce convulsions. This suggests that the convulsant action of FLZ is exerted at a site other than the benzodiazepine receptor.

Convulsant component of a depressant benzodiazepine.

The convulsant influence of high doses of diazepam, in the presence of the benzodiazepine receptor antagonist Ro 15-1788, was studied in rats. Animals were implanted with permanent cortical screw electrodes for EEG recording. EEG spiking and accompanying clonic activity was observed in rats receiving greater than or equal to 200 mg/kg diazepam, followed 10 minutes later by Ro 15-1788 (20 mg/kg).

Reduction of flurazepam convulsive threshold by Ro 15-1788.

The influence of the benzodiazepine (BZ) receptor antagonist Ro 15-1788 on the convulsant properties of flurazepam (FLZ) was studied in rats. Animals were prepared with chronic epidural electrodes for EEG recording and respiratory rates were recorded via a rubber bulb connected to a pressure transducer. FLZ convulsive thresholds were determined by continuous IV infusion in the presence and absence of Ro 15-1788 pretreatment.

Increased susceptibility of audiogenic rats to barbital withdrawal convulsions.

Non-responsive progeny from Sprague-Dawley derived rats genetically susceptible to sound-induced (audiogenic) convulsions (AGS-negative) and non-responsive progeny from Sprague-Dawley derived rats not gentically susceptible to audiogenic convulsions (SD-negative) were subjected to a seven-day treatment regimen of sodium barbital. 125 mg/kg, every 12 hours. This represents a lower dose and shorter treatment period than that normally used in this laboratory to induce barbiturate dependence in rats.

Effect of ketamine enantiomers on sound-induced convulsions in epilepsy prone rats.

Epilepsy prone rats (Jobe et al, 1982) which had previously been determined to experience relatively mild seizures in response to a sound stimulus were used in this study in order to provide a model which would reveal either proconvulsant or anticonvulsant drug activity. Animals were administered dextrorotatory (+), levorotatory (-), or racemic (+/-) ketamine hydrochloride by intravenous (tail vein) injection and subsequently challenged with a 120 db broad spectrum sound stimulus. Evaluation of drug effect was based on presence/absence of seizure, seizure severity, and convulsive latency (time from onset of stimulus to onset of convulsive episode).

Lack of influence of brain catecholamines on acute effects of barbiturates.

Rats with cerebral electrode implants were tested for sensitivity to EEG burst suppression by intravenously-infused sodium methohexital following manipulation of brain catecholamine function. Although depletion of both norepinephrine (NE) and dopamine (DA) with 6-hydroxydopamine resulted in a slight increase in methohexital sensitivity (MHS), similar depletion with alpha-methyltyrosine did not alter MHS. In addition, desipramine, an agent which selectively blocks uptake of NE did not affect MHS.