Improved methods of retroviral vector transduction and production for gene therapy.

To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses. The purpose of this study was to optimize methods of retroviral vector production and transduction. Studies were conducted using 22 retroviral vector producer cell lines. Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C. A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor. Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system. Retroviral supernatants were successfully lyophilized. The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83%. In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses. The combination of the above methods has significantly increased the growth and transduction by this vector system.

Fatal infection of silvered leaf monkeys with a virus-like infectious agent (VLIA) derived from a patient with AIDS.

Four silvered leaf monkeys, inoculated with a virus-like infectious agent (VLIA) derived from transformed NIH/3T3 cells (sb51) transfected with Kaposi's sarcoma DNA of an AIDS patient, showed wasting syndromes and died in 7-9 months. Two monkeys had a transient lymphadenopathy in earlier stages. Two moribund animals showed lymphopenia. Although 3 of the VLIA inoculated monkeys had persistent low grade fever early in the infection, the animals became afebrile in the later stages. One VLIA inoculated animal had a prominent antibody response, which occurred 7 months after VLIA inoculation. The other 3 monkeys had a transient or poor antibody response in the later stages. These 3 animals revealed periodic VLIA antigenemia during the course of the experiment. A control monkey was killed 8 months after the last VLIA inoculated monkey succumbed and showed neither an antibody response nor evidence of antigenemia. VLIA-specific DNA could be directly detected in necropsy tissues of all 4 monkeys inoculated with VLIA using the polymerase chain reaction method. VLIA infection was identified in all 4 spleens, 2 of 4 livers, 1 of 2 kidneys, and all 3 brains tested from these 4 animals, but not in the tissues from the control monkey. The necropsy examination of the 4 VLIA inoculated animals revealed no opportunistic infections, acute inflammatory lesions, malignancy or cause of death other than VLIA infection. We believe that the VLIA caused a fatal systemic infection in these monkeys.

Virus-like infectious agent (VLIA) is a novel pathogenic mycoplasma: Mycoplasma incognitus.

The newly recognized pathogenic virus-like infectious agent (VLIA), originally reported in patients with AIDS but also known to be pathogenic in previously healthy non-AIDS patients and in non-human primates, was cultured in cell-free conditions using a modified SP-4 medium and classified as a member of the order Mycoplasmatales, class Mollicutes. The infectious microorganism is tentatively referred to as Mycoplasma incognitus. M. incognitus has the unique biochemical properties of utilizing glucose both aerobically and anaerobically, as well as having the ability to metabolize arginine. Among all known human mycoplasmas, these specific biochemical characteristics were found previously only in a rarely isolated species, M. fermentans. In comparison with M. fermentans, M. incognitus appears to be even more fastidious in cultivation requirements and fails to grow in all tested mycoplasma media other than modified SP-4 medium. In addition, M. incognitus grows much more slowly, has a smaller spherical particle size and occasional filamentous morphology, and forms only irregular and very small colonies with diffuse edges on agar plates. Antigenic analysis using polyclonal and monoclonal antibodies and DNA analysis of sequence homology and restriction enzyme mappings in M. incognitus, M. orale, M. hyorhinis, M. hominis, M. pneumoniae, M. fermentans, M. arginini, M. genitalium, M. salivarium, Ureaplasma urealyticum, and Acholeplasma laidlawii revealed that M. incognitus is distinct from other mycoplasmas, but is most closely related to M. fermentans.

Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy non-AIDS patients.

We studied 6 patients from 6 different geographic areas who presented with acute flu-like illnesses. The patients developed persistent fevers, lymphadenopathy or diarrhea, pneumonia, and/or heart, liver, or adrenal failure. They died in 1-7 weeks. These patients had no serological evidence of HIV infection and could not be classified as AIDS patients according to CDC criteria. The clinical signs as well as laboratory and pathological studies of these patients suggested an active infectious process, although no etiological agent was found despite extensive infectious disease work-ups during their hospitalization. Post-mortem examinations showed histopathological lesions of fulminant necrosis involving the lymph nodes, spleen, lungs, liver, adrenal glands, heart, and/or brain. No viral inclusion cells, bacteria, fungi, or parasites could be identified in these tissues using special tissue stains. We report that immunohistochemistry using rabbit antiserum raised against VLIA, the virus-like infectious agent previously identified in patients with AIDS and shown to cause fatal systemic infection in primates, revealed VLIA antigens in these necrotizing lesions. In situ hybridization using an 35S labeled VLIA-specific DNA probe also detected VLIA genetic material in the areas of necrosis. Furthermore, virus-like particles closely resembling VLIA were identified ultrastructurally in these histopathological lesions. VLIA was associated with the systemic necrotizing lesions in these previously healthy non-AIDS patients with an acute fatal disease.

Identification of Mycoplasma incognitus infection in patients with AIDS: an immunohistochemical, in situ hybridization and ultrastructural study.

Monoclonal antibodies (Mabs) were developed against antigens from a pure culture of Mycoplasma incognitus grown in modified SP-4 medium. All the Mabs obtained were shown to react only with M. incognitus, and not with other species of human mycoplasma. The Mabs identified M. incognitus immunohistologically in thymus, liver, spleen, lymph node, or brain from 22 patients with AIDS, as well as in 2 placentas delivered by patients with AIDS. Using an 35S-labeled DNA probe specific for M. incognitus and in situ hybridization technique, we also identified M. incognitus-specific genetic material in these tissues. Furthermore, ultrastructural studies of the specific areas of tissues which were highly positive for M. incognitus antigens revealed characteristic structures of mycoplasma organisms. These mycoplasma-like particles could be identified intracellularly and extracellularly. Histopathology of the tissues infected by M. incognitus varied from no pathological changes to fulminant necrosis with or without an associated inflammatory reaction. M. incognitus, a novel pathogenic mycoplasma, was cytopathic and cytocidal.

Fatal systemic infections of nonhuman primates by Mycoplasma fermentans (incognitus strain).

Four silvered leaf monkeys inoculated with Mycoplasma fermentans (incognitus strain) showed wasting syndromes and died in 7-9 months. Infected animals had a late and transient antibody response to mycoplasmal infection. Three monkeys revealed periodic mycoplasmal antigenemia. The one that had the most persistent antigenemia failed to mount a detectable antibody response and was the first to die of the infection. The control monkey was killed 8 months later, after the last of the infected animals had died, and revealed no evidence of seroconversion or antigenemia. Polymerase chain reaction, immunohistochemical, and electron microscopic studies identified systemic infections of M. fermentans in the infected animals. No other opportunistic infection or neoplastic disease was found. It is interesting to note the absence of an inflammatory reaction to the large number of mycoplasmas in the infected tissues. M. fermentans (incognitus strain) apparently suppressed normal inflammatory or immune responses, produced wasting syndromes, and caused a fatal systemic infection in these monkeys.

Adhesion onto and invasion into mammalian cells by mycoplasma penetrans: a newly isolated mycoplasma from patients with AIDS.

The newly identified mycoplasma, Mycoplasma pentrans shows remarkable pathobiologic properties: it adheres to cell surfaces, deeply penetrates into the cell, strongly hemadsorbs human red blood cells, and cytadsorbs human CD4+ lymphocytes and monocytes. These in vitro biologic activities of mycoplasmas have been previously shown to be associated with pathogenic virulence in vivo. Both adhesion and invasion clearly involve the organism's unique tip-like structure. Invading mycoplasmas often have their tip-like structure deeply buried in the cytoplasm of infected mammalian cells. Extensive invasion of the mycoplasma into the cytoplasm may kill the cells. The same pathobiologic processes of adhesion and invasion using the specialized tip-like structure are found on the epithelium in the patient's urogenital tract infected by M. penetrans. Both in vitro and in vivo findings suggest a possible pathogenic role of this newly discovered human mycoplasma and call for careful evaluation of its role in human diseases.