Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light.

BACKGROUND: A photochemical treatment process has been developed for the inactivation of viruses and bacteria in platelet concentrates. This process is based on the photochemical reaction of a novel psoralen, S-59, with nucleic acids upon illumination with long-wavelength ultraviolet light (UVA, 320-400 nm). STUDY DESIGN AND METHODS: High levels of pathogens were added to single-donor platelet concentrates containing 3 to 5 x 10(11) platelets in 300 mL of 35-percent autologous plasma and 65-percent platelet additive solution. After treatment with S-59 (150 microM) and UVA (0-3 J/cm2), the infectivity of each pathogen was measured with established biologic assays. In vitro platelet function after photochemical treatment was evaluated during 7 days of storage by using a panel of 14 assays. The in vivo recovery and life span of photochemically treated platelets were evaluated after 24 hours of storage in a primate transfusion model. RESULTS: The following levels of pathogen inactivation were achieved: >10(6.7) plaque-forming units (PFU) per mL of cell-free human immunodeficiency virus (HIV), >10(6.6) PFU per mL of cell-associated HIV, >10(6.8) infectious dose (ID50) per mL of duck hepatitis B virus (a model for hepatitis B virus), >10(6.5) PFU per mL of bovine viral diarrhea virus (a model for hepatitis C virus), >10(6.6) colony-forming units of Staphylococcus epidermidis, and >10(5.6) colony-forming units of Klebsiella pneumoniae. Expression of integrated HIV was inhibited by 0.1 microM S-59 and 1 J per cm2 of UVA. In vitro and in vivo platelet function were adequately maintained after antiviral and antibacterial treatment. CONCLUSION: Photochemical treatment of platelet concentrates offers the potential for reducing transfusion-related viral and bacterial diseases.

Photochemical inactivation of pathogenic bacteria in human platelet concentrates.

Platelet concentrates (PC) may be infrequently contaminated with low levels of bacteria that can cause septicemia and death in patients receiving transfusion therapy. We evaluated the efficacy of a photochemical decontamination (PCD) technique using 8-methoxypsoralen (8-MOP) and long wavelength UV light (UVA) to inactivate bacteria in standard therapeutic PC. Twelve phylogenetically distinct pathogenic bacteria, 5 gram-positive and 7 gram-negative organisms, were seeded into PC to a final challenge dose ranging from 10(5) to 10(7) colony-forming units (CFU)/mL. Contaminated PC were treated with 8-MOP (5 micrograms/mL) and 5 J/cm2 of UVA, a PCD treatment regimen found to adequately preserve in vitro platelet function. Greater than 10(5) CFU/mL of all 5 gram-positive (Staphylococcus aureus, Streptococcus epidermidis, Streptococcus pyogenes, Listeria monocytogenes, and Corynebacterium minutissimum) and 2 of the gram-negative (Escherichia coli and Yersinia enterocolitica) organisms were inactivated. The remaining 5 gram-negative organisms were more resistant, with less than 10(1) to 10(3.7) CFU/mL inactivated under these conditions. The inactivation efficiency for this resistant group of gram-negative organisms was improved when PC were resuspended in a synthetic storage medium with reduced plasma protein concentration (15%) and an increased 8-MOP concentration (23.4 micrograms/mL). Illumination with 3 J/cm2 of UVA in this system inactivated greater than 10(5) CFU/mL of 4 resistant gram-negative organisms (Salmonella choleraesuis, Enterobacter cloacae, Serratia marcescens, and Klebsiella pneumoniae) and 10(4.1) CFU/mL of the most resistant gram-negative organism (Pseudomonas aeruginosa). This level of PCD treatment did not adversely affect in vitro platelet function. These results demonstrate that PCD using 8-MOP (5 to 23.4 micrograms/mL) effectively inactivated high levels of pathogenic bacteria in PC with adequate preservation of in vitro platelet properties.

Photochemical inactivation of cell-associated human immunodeficiency virus in platelet concentrates.

Photochemical decontamination (PCD) of platelet concentrates, with adequate preservation of platelet function, has been shown using 8-methoxypsoralen (8-MOP) and long wavelength UV light (UVA). To further evaluate this technique, models for the inactivation of pathogenic human cell-associated viruses and integrated proviral sequences are required. We have assessed the ability of the PCD technique to inactivate cell-associated human immunodeficiency virus 1 (HIV-1) in platelet concentrates. We correlated PCD inhibition of HIV-1 infectivity with 8-MOP-DNA adduct formation in contaminating nucleated cells, and measured the inhibition of polymerase chain reaction (PCR)-mediated amplification of cellular DNA sequences as a surrogate for inactivation of integrated proviral nucleic acid sequences. After PCD treatment (8-MOP 300 micrograms/mL, UVA 17 mW/cm2) for 60 minutes, 0.5 x 10(6) plaque-forming units (PFU)/mL of cell-associated HIV-1 were inactivated and no virus was detectable by infectivity assay. After 60 minutes of PCD, 15 8-MOP-DNA adducts per 1,000 bp were formed, while in the absence of UVA, no adducts were formed. PCR-mediated amplification of a 242-bp cellular DNA sequence (HLA-DQ-alpha) was inhibited when greater than eight psoralen-DNA adducts per 1,000 bp were present. These studies indicate that high titers of cell-associated HIV-1 in platelet concentrates were inactivated by PCD, and the numbers of 8-MOP-DNA adducts in nucleated cells were sufficient to inhibit amplification of DNA segments that encode for as few as 80 amino acids. Based on the frequency of 8-MOP-DNA adducts, for the 10-kb HIV-1 genome, the probability of an integrated genome without at least one 8-MOP adduct after 60 minutes of PCD was 10(-33).

Effect of dose and immunization schedule on immune response of baboons to recombinant glycoprotein 120 of HIV-1.

To evaluate the immunogenicity of purified recombinant envelope glycoprotein of HIV-1 (rgp120) as a potential vaccine for AIDS, the antibody response of 45 baboons to rgp120 was investigated using an adjuvant (alum) and route of administration (intramuscular) suitable for humans. The primary purpose was to evaluate the effects of different doses and immunization schedules on the antibody response to rgp120 in primates. A secondary objective was to evaluate possible adverse consequences of rgp120 immunization. A liquid-phase radioimmunoprecipitation (RIP) assay for detection of rgp120-reactive antibodies revealed that rgp120 doses of 30-300 micrograms per administration resulted in nearly indistinguishable serum antibody responses. However, significant enhancement of serum antibody titers was observed when the interval between the second and third administrations was increased from 4 to 20 w. Although changing the interval significantly altered the magnitude of resulting peak titers, the kinetics of antibody formation were not changed. Thus, of the three schedules of immunization tested, none resulted in a sustained humoral immune response. The significance of the RIP assay for evaluating immune responses was confirmed by analysis showing that the percentage of immunized baboons that developed in vitro HIV-1 serum neutralizing responses was greatest in groups that also exhibited high anti-rgp120 RIP titers. Immunization with rgp120 had no significant adverse effect on any clinical or laboratory parameter monitored over the course of the study.

Latency of mouse cytomegalovirus in sciatic nerve.

This report describes a mammalian model for exploring the role of virus in peripheral neuropathy. Schwann cells in culture were permissive for mouse cytomegalovirus (MCMV) replication. Intraperitoneal inoculation rarely led to sciatic nerve infection. Sciatic nerves infected by direct intraneural injection produced infectious virus and contained viral antigen at 4 days postinfection (pi). Nerves taken later, at 4 to 8 weeks pi, contained no infectious virus, but MCMV was present in a latent state because culture of nerve explants reactivated virus. The findings contrast the viral permissiveness of cultured Schwann cells to the latency observed in intact peripheral nerve.

Theiler's virus in brain cell cultures: lysis of neurons and oligodendrocytes and persistence in astrocytes and macrophages.

The mechanisms of persistence and of demyelination in Theiler's virus (TV)-induced chronic neurologic disease (a murine model for multiple sclerosis) are, as yet, disputed. We investigated the tropism and persistence of TV in brain cell culture to better understand the pathogenesis of this disease. Using anti-genic markers to identify specific cells in culture, we have demonstrated that TV infects, lytically, neurons and oligodendrocytes and persistently astrocytes and macrophages. These results suggest that host cell factors play a key role in the mechanism of demyelination and the persistence of TV in the nervous system.

Modulation of radiation-induced hemopoietic suppression by acute thrombocytopenia.

Modifications of radiation-induced hemopoietic suppression by acute thrombocytopenia were evaluated. Immediately before or after exposure to sublethal irradiation, mice were given a single injection of anti-mouse platelet serum (APS), normal heterologous serum, neuraminidase (N'ase), or saline, or no further treatment was provided. Hemopoiesis was evaluated by blood cell counts, hematocrits, and incorporation of [75Se]selenomethionine into platelets. APS and N'ase induced an acute thrombocytopenia from which there was partial recovery before the platelet count started to fall from the radiation. During the second post-treatment week, both thrombocytopoiesis and erythropoiesis were greater in mice that received APS or N'ase in addition to radiation than in control irradiated mice. Differences in leukopoiesis were not apparent. Therefore, both thrombocytopoiesis and erythropoiesis appeared to be responsive to a stimulus generated by acute thrombocytopenia in sublethally irradiated mice.