Live viral vectors : construction of a replication-deficient recombinant adenovirus.

Since the use of molecular biology and genetic engineering techniques has become widespread, a new generation of candidate vaccines has been developed, including live viral vectors (1, 2). The basis of using recombinant viruses as potential vaccines involves the incorporation of specific genes from a pathogenic organism into the genome of a nonpathogenic or attenuated virus. The recombinant virus can then infect specific eukaryotic cells either in vivo or in vitro, and cause them to express the recombinant protein.

Immunization of mice with plasmid DNA expressing the measles virus nucleoprotein gene.

The measles virus (MV) nucleocapsid (N) protein gene has been inserted into a plasmid vector so as to place the gene under the control of the strong constitutive human cytomegalovirus major immediate early promoter. On intramuscular injection of pMV64 DNA into C3H/He mice, seroconversion with increasing titers of N-specific serum IgG antibodies was observed over a period of 3 months. However, when 3-week-old mice were immunized by intramuscular injection of pMV64 in a two-dose schedule, and challenged intracranially with a rodent-adapted measles virus strain (CAM/RB) at 5 weeks of age, no significant protective response was seen.

Degradation of homovanillate by a strain of Variovorax paradoxus via ring hydroxylation.

A newly isolated strain of Variovorax paradoxus could grow on homovanillate and several monohydroxylated phenylacetic acids. During growth on homovanillate, the organism formed separate NAD(P)H-dependent hydroxylases with activity towards 4-hydroxyphenylacetic acid and homovanillate. Homovanillate hydroxylase catalysed a typical monooxygenase reaction and had little activity towards 4-hydroxyphenylacetic acid GC-MS and TLC analysis suggested that homovanillate was 1-hydroxylated to yield a dihydroxymonomethoxyphenylacetic acid which served as a substrate for homogentisate 1,2-dioxygenase.

The role of microbiology in models of dental caries.

Models of dental caries (laboratory, animal, and human in situ models) vary markedly in their microbiological complexity. Laboratory models range from mono-cultures of cariogenic species providing an acidic challenge to enamel, to the development of diverse mixed cultures growing on a habitat-simulating medium in an artificial mouth or chemostat. The latter systems are of value in determining either mechanisms of action or cause-and-effect relationships--e.

Constitutive expression of major histocompatibility complex class II antigens on monocytes and B cells correlates with disease in simian immunodeficiency virus-infected rhesus macaques.

Constitutive host factors that influence progression to AIDS are understood poorly. In the macaque model for AIDS, 35 animals infected with simian immunodeficiency virus (SIV) were analyzed for major histocompatibility complex class II antigen expression on blood monocytes and B cells by immunostaining and flow cytometry. Expression varied widely between animals but was constant with time.

A paint incorporating silver to control mixed biofilms containing Legionella pneumophila.

A three-stage chemostat containing a mixed consortium of microorganisms, including Legionella pneumophila, was used to determine the suitability of a silver-containing paint to control biofouling in water systems. The paint was efficient in controlling total surface colonisation by heterotrophic microorganisms and growth of the pathogen over a 2-week period. Biodiversity was limited in the presence of the silver paint and this was thought to help control L.

Chemotherapeutic tumour targeting using clostridial spores.

The toxicity associated with conventional cancer chemotherapy is primarily due to a lack of specificity for tumour cells. In contrast, intravenously injected clostridial spores exhibit a remarkable specificity for tumours. This is because, following their administration, clostridial spores become exclusively localised to, and germinate in, the hypoxic/necrotic tissue of tumours.

Human antibody response to meningococcal transferrin binding proteins: evidence for vaccine potential.

During iron-limited growth Neisseria meningitidis expresses two transferrin binding proteins, TBP1 and TBP2, with molecular masses of approximately 98 and 65-90 kDa depending on strain. Mixtures of TBP1 and TBP2 (TBP1 + 2) from three meningococcal strains were purified using affinity chromatography and used to determine anti-TBP antibodies in human sera by ELISA. Sera were obtained from healthy individuals, asymptomatic carriers of N.

Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase.

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives.

A comparison of the leakage of a monoclonal antibody from various immunoaffinity chromatography matrices.

Antibody leakage from immunoaffinity chromatography (IAC) matrices could reduce the working life of the IAC matrix and/or contaminate parenteral products, purified by IAC. There is therefore a need to measure the leakage of antibody from IAC matrices and to reduce such leakage. Using sensitive ELISAs it was found that the type of activated matrix, the buffer, the presence of proteases in the feedstock and the storage of IAC matrices between runs could all effect antibody leakage.

Efficient purification and rigorous characterisation of a recombinant gp120 for HIV vaccine studies.

A recombinant HIV-1 gp120 (rgp120) was expressed in a permanent Chinese Hamster Ovary (CHO) cell line (L761h) that constitutively secretes the product of clone p4 derived from the env gene of HIV-1 isolate GB8. The rgp120 was isolated from cell culture supernatants by a simple, rapid, non-denaturing and efficient purification procedure based on a novel combination of lectin affinity and FPLC ion-exchange chromatography. The purity of the isolated glycoprotein was rigorously confirmed by SDS-PAGE, capillary electrophoresis, laser desorption mass spectrometry, total amino acid analysis and N-terminal amino acid sequencing.

Repair and evolution of nef in vivo modulates simian immunodeficiency virus virulence.

Experimental evidence from the simian immunodeficiency virus (SIV) model of AIDS has shown that the nef gene is critical in the pathogenesis of AIDS. Consequently, nef is of considerable interest in both antiviral drug and vaccine development. Preliminary findings in two rhesus macaques indicated that a deletion of only 12 bp found in the overlapping nef/3' long terminal repeat (LTR) region (9501 to 9512) of the SIVmacC8 molecular clone was associated with reduced virus isolation frequency.

Expression of the measles virus nucleoprotein gene in Escherichia coli and assembly of nucleocapsid-like structures.

To investigate the use of fusion systems to aid the purification of recombinant proteins for structure/function studies and potential uses as diagnostic reagents, the measles virus (MV) gene encoding the nucleoprotein was cloned and expressed in Escherichia coli in three forms: as a full-length intact protein and as two fusion proteins. Expression of the intact N gene under the control of the tac promoter in the pTrc99c plasmid produced a protein of the correct size (60 kDa) which represented approx. 4% of the total cellular protein, and was recognised by known measles positive human sera.

High-level expression of the measles virus nucleocapsid protein by using a replication-deficient adenovirus vector: induction of an MHC-1-restricted CTL response and protection in a murine model.

Replication-deficient adenovirus (Ad) vectors provide an efficient technology for direct DNA delivery to cells both in vitro and in vivo. We have inserted the measles virus nucleoprotein (N) gene under the control of the strong constitutive CMV major IE promoter into an Ad type 5 E1- vector to produce the recombinant virus RAd68. Following infection of human fibroblasts with RAd68 in vitro, recombinant N protein was synthesized as a 60-kDa protein that represented up to 20% total soluble cell protein.

The simian immunodeficiency virus transmembrane protein is poorly immunogenic in inactivated virus vaccine.

The transmembrane proteins (TMP) of immunodeficiency lentiviruses are primary candidates for inclusion in AIDS vaccines, the design and testing of which is facilitated by the SIV-macaque infection model. Antibody responses to linear determinants in the SIVmac TMP were investigated in rhesus macaques either infected with the SIVmac J5 molecular clone or vaccinated with partially purified, formalin-inactivated SIVmac. Infected animals were shown to recognise predominantly four regions in the external domain and three regions in the internal domain of the TMP defined by a series of nominally 20mer overlapping peptides.

Experimental Legionnaires' disease in SCID-Beige mice reconstituted with human leucocytes.

A new small animal model of experimental Legionnaires' disease is described in which the reconstitution of SCID-Beige mice with human peripheral blood leucocytes permits the in-vivo growth of Legionella pneumophila in the lungs of aerosol-challenged mice. Following infection, viable bacterial counts within the lungs of mice increased from 10(5) cfu/lung at the time of inoculation to a maximum of 10(10) cfu/lung by 48 h post-inoculation. Two types of disease were detected in the lungs of infected SCID-Beige mice.

Protection of mice from Bordetella pertussis respiratory infection using microencapsulated pertussis fimbriae.

Conditions have been established which allow the efficient entrapment of Bordetella pertussis fimbriae in poly(lactide-co-glycolide) microspheres. Fimbriae released from the matrix were found to have retained some degree of conformational structure, as determined by assessing the capacity of fimbrial protein to bind to antibodies mapping to either conformational or denatured structures on the fimbriae, either encapsulated in microspheres with a mean diameter of 24 microns and an estimated in vitro protein release rate of approximately 42 days, or conventionally adjuvanted with alhydrogel, elicited vigorous immune responses in mice. The encapsulated fimbriae appear to elicit marginally lower serum antibody levels than those induced by equivalent amounts of alhydrogel-adjuvanted fimbriae.

Catalytic-rate improvement of a thermostable malate dehydrogenase by a subtle alteration in cofactor binding.

The nucleotide-binding fold of many NAD(+)-dependent dehydrogenases contains a conserved acidic amino acid residue which hydrogen-bonds with the 2'- and 3'-hydroxy groups of the adenine-ribose of the cofactor. This residue is highly conserved as aspartate in malate dehydrogenases, except in the thermophilic enzyme from Thermus aquaticus B (TaqMDH), which has glutamic acid-41 in the equivalent position. The catalytic mechanism was dissected to investigate the functional significance of this difference in TaqMDH with respect to a mutant enzyme where glutamic acid-41 was replaced by aspartic acid.

Characterisation of Legionella pneumophila antigen in urine of guinea pigs and humans with Legionnaires' disease.

To study and develop the urinary antigen detection assay, urine was obtained from aerosol infected guinea pigs. The appearance of Legionella pneumophila antigen in guinea pig urine was then compared with that detected in urine from human Legionnaires' Disease (LD) cases. This study demonstrated that the antigen expressed in the experimental model of LD was of identical molecular weight, reacted almost identically in an ELISA based assay and in Western blots with the antigen found in human urine.