Safety, immunogenicity, and efficacy of a malaria sporozoite vaccine administered with monophosphoryl lipid A, cell wall skeleton of mycobacteria, and squalane as adjuvant.

A Plasmodium falciparum circumsporozoite protein (PfCSP) recombinant fusion protein, R32NS1(81), formulated with monophosphoryl lipid A, cell wall skeleton of mycobacteria, and squalane (Detox) was administered to 12 volunteers. One volunteer had malaise and self-limited painful induration at the injection site after the second dose and declined further immunization. The other 11 volunteers tolerated the three doses of 1,230 micrograms of vaccine, but most complained of sore arms; in five cases the pain or malaise was severe enough to interfere with work or sleep. Two weeks after the third dose of vaccine, four of the 11 immunized volunteers had > or = 14 micrograms/ml of antibodies to the repeat region of the PfCSP in their serum. Two of these four volunteers did not develop P. falciparum parasitemia when challenged by the bite of five mosquitoes carrying P. falciparum sporozoites. The seven volunteers with lower levels of antibodies and 11 of 11 controls developed parasitemia. These data are consistent with other studies, and indicate that vaccine-induced antibodies against the repeat region of PfCSP can prevent effective sporozoite infection of hepatocytes in humans. The challenge is to improve the immunogenicity of PfCSP-based vaccines, and to develop methods for including PfCSP peptides as components of multitarget malaria vaccines.

Mechanism of gram variability in select bacteria.

Gram stains were performed on strains of Actinomyces bovis, Actinomyces viscosus, Arthrobacter globiformis, Bacillus brevis, Butyrivibrio fibrisolvens, Clostridium tetani, Clostridium thermosaccharolyticum, Corynebacterium parvum, Mycobacterium phlei, and Propionibacterium acnes, using a modified Gram regimen that allowed the staining process to be observed by electron microscopy (J. A. Davies, G. K. Anderson, T. J. Beveridge, and H. C. Clark, J. Bacteriol. 156:837-845, 1983). Furthermore, since a platinum salt replaced the iodine mordant of the Gram stain, energy-dispersive X-ray spectroscopy could evaluate the stain intensity and location by monitoring the platinum signal. These gram-variable bacteria could be split into two groups on the basis of their staining responses. In the Actinomyces-Arthrobacter-Corynebacterium-Mycobacterium-Propionibacterium group, few cells became gram negative until the exponential growth phase; by mid-exponential phase, 10 to 30% of the cells were gram negative. The cells that became gram negative were a select population of the culture, had initiated septum formation, and were more fragile to the stress of the Gram stain at the division site. As cultures aged to stationary phase, there was a relatively slight increase toward gram negativity (now 15 to 40%) due to the increased lysis of nondividing cells by means of lesions in the side walls; these cells maintained their rod shape but stained gram negative. Those in the Bacillus-Butyrivibrio-Clostridium group also became gram negative as cultures aged but by a separate set of events. These bacteria possessed more complex walls, since they were covered by an S layer. They stained gram positive during lag and the initial exponential growth phases, but as doubling times increased, the wall fabric underlying the S layer became noticeably thinner and diffuse, and the cells became more fragile to the Gram stain. By stationary phase, these cultures were virtually gram negative.

Freeze-fracture observations of the cell walls and peribacillary substances of various mycobacteria.

Ultrastructure of the cell wall and peribacillary substances of various mycobacteria (32 strains of 18 species) grown in vitro was studied by a freeze-fracture technique. Peribacillary substances differed in shape among species and even among strains of the same species, and were classified into five types: (1) amorphous substances; (2) multi-layered sheaths with no filamentous units; (3) structures composed of filaments of 2-4 nm diameter, which were further classified into three subtypes according to the arrangement of the filaments; (4) helical fibres; and (5) single fibres, or networks of fibrous structures, with no visible substructures. No strains revealed peribacillary structures resembling those of uncultivable Mycobacterium leprae. These results have implications for the mechanism of freeze-fracturing in mycobacterial walls.

[Bacteriophage occurrence in "Mycobacterium phlei" (author's transl)]. / Présence d'une bactériophage chez Mycobacterium phlei.

Surface growth of synchronized bacteria was obtained by means of a suspension of Mycobacterium phlei cells in pentane, the dispersion of which resulted from passage through glass (Ballotini) column. By using standardized conditions, a series of identical cultures were obtained, suitable for studying their evolution as a function of time. By counting colonies every twenty minutes, during ten hours, two doublings were observed, with a generation time of five hours. At the end of a plateau, just before the next doubling, the curve exhibited a marked decrease. Bacteriophages were found in culture medium at the time corresponding to this decrease. In thin sections of the pellicles collected at this time, condensations resembling DNA from phage heads could be noticed within the bacterial cells, as well as free phages in th close neighbourhood of burst cells. The relations between phage and bacteria, and the possible relation between the presence of the phage and the synthesis of phleates has not been determined.

The completion of the replication map of the Mycobacterium phlei chromosome.

New facts about the replication map of Mycobacterium phlei chromosome are summarized. Replication positions of two genes located in marginal regions of the replication map, ile close to the origin and ser near the terminus, were determined. Known positions of replication of some genes were defined with more precision within 2.5--5-min intervals using the method of sequential mutagenesis in synchronized cultures (leu, met, bac, pyr, stm, tet, cyc, his). Replication positions of genes responsible for the biosynthesis of thiamine and resistance to tetracycline and vancomycin were further identified. The contemporary replication map contains replication positions of 24 genes.

Mycobacterium phlei PN-bb colonies: a morphological characterisation by scanning and transmission electron microscopy.

Colonies of a non-acid-fast mutant of Mycobacterium phlei--termed "PN--bb"--were examined by scanning electron microscopy of gold-sputted whole colonies and by transmission electron microscopy of thin sections. Inspection of the colonies before and after preparation showed that the fixed colonies had retained their original appearance. Colonies are about 2-5 mm in height and width. They are made up of converging ridges forming a cone. These ridges consists of rounded bodies which are made up of clustered cells. The top of the cone consists of small, rugged, irregular structures. Thin sections show that the cells are arranged close together, without interconnections but with some electron-dense material between them. The colony is covered with a layer of unknown composition.

The replication map of the chromosome of Mycobacterium phlei.

It was the aim of the present work to construct the replication map of the chromosome of Mycobacterium phlei. The method of mutagenesis of the replication point by N-methyl-N-nitroso-N'-nitroguanidine in synchronously dividing populations and the method of analysis of gene frequency were applied. The order of replication of 19 genes on the chromosome was determined by means of induction of back mutations and forward mutations in auxotrophic mutants PA leu and PA met and in double auxotrophic mutants with methionine as a reference marker.

Immunotherapy of cancer: tumor suppression and regression by cell walls of Mycobacterium phlei attached to oil droplets.

Components of mycobacterial cell wall(s) (CW) attached to oil droplets were evaluated for their ability 1) to inhibit the growth of line-10 tumor transplants in the skin of syngeneic guinea pigs when inoculated together with 10(6) tumor cells (suppression experiments) and 2) to regress established 7-day-old intradermal tumors and eradicate microscopic lymph node metastases upon injection into the tumors (regression experiments). CW and cell-wall skeleton (CWS) preparations from Mycobacterium phlei, a fast-growing saprophyte of group IV of the atypical mycobacteria, suppressed tumor growth in essentially all animals when 37.5-mug doses were administered; at a dose of 300 mug, they cured 50-60% of the animals in regression tests. The addition of 300 mug of a purified trehalose mycolate, isolated from M. tuberculosis strain Aoyama B, to 300 mug M. phlei CW or CWS preparations significantly increased their tumor regressive potency to provide cure rates to about 90%. Because M. phlei can be propagated more readily, it can be used advantageously in place of BCG to prepare stable, non-living immunologic adjuvants of defined composition and consistently high potency to meet the need for standards with minimal residual malignant disease.

Ultrastructure of L-phase variants isolated from a culture of Mycobacterium phlei.

Relatively stable L-phase colonies were isolated from old cultures of a selected clone of Mycobacterium phlei. The colonies grew at 52 degrees C and were composed of rod-shaped, oval or spherical cells. Large amoeba-like cells were occasionally present. These were usually limited by a double-layered membrane and devoid of normal cell-wall components such as bacteriophage receptors. The large amoeba-like bodies sometimes showed both outer and inner double-layered membranes, especially in pseudopodium-like cellular extensions. An unusual feature of rod-shaped cells was retention of the original shape despite the loss of their cell walls. Two types of walled cells occurred during successive transfers of L colonies. One was the true revertant which had characteristics in common with the wild-type M. phlei, such as growth at 52 degrees C and ultrastructural organisation. The other, designated as the "atypical-cell-wall variant", was characterised by growth at 52 degrees C, thick cell walls, and disordered septation. Wild-type M. phlei, L variants, revertants and atypical-cell-wall variants released mycobacteriophage particles. These bacteriophages were almost identical in respect to morphology, host range, and neutralisation by antiserum. The results obtained suggest strongly that all types of cells examined were derived from M. phlei.

Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei.

The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.

Purification and properties of membrane-bound coupling factor-latent ATPase from Mycobacterium phlei.

The membrane bound coupling factor-latent ATPase was solubilized from the membrane vesicles of Mycobacterium phlei by using 0.25 M sucrose or low ionic strength buffer. Purification of the solubilized enzyme by use of Sepharose-ADP conjugate gel yielded a homogenous preparation of latent ATPase which was purified about 216-fold in a single step with an 84% yield. The enzyme exhibits a specific activity of 39 mumoles of ATP hydrolyzed per min per mg protein. The purified enzyme exhibits coupling factor activity. Electrophoresis in two dissociating solvent systems indicates that the enzyme contains at least three major polypeptides of molecular weights 56,000, 51,000, and 46,000 daltons, and two minor polypeptides of 30,000 and 17,000 daltons. Equilibrium binding studies of ADP with purified coupling factor-latent ATPase reveal the presence of two nucleotide binding sites per molecule with an apparent Ka of 8.1 X 10(-5) M. By use of affinity chromatography, another latent ATPase has been isolated from the solubilized enzyme, which does not exhibit coupling factor activity.