Prions and transmissible spongiform encephalopathy (TSE) chemotherapeutics: A common mechanism for anti-TSE compounds?

No validated treatments exist for transmissible spongiform encephalopathies (TSEs or prion diseases) in humans or livestock. The search for TSE therapeutics is complicated by persistent uncertainties about the nature of mammalian prions and their pathogenic mechanisms. In pursuit of anti-TSE drugs, we and others have focused primarily on blocking conversion of normal prion protein, PrP(C), to the TSE-associated isoform, PrP(Sc). Recently developed high-throughput screens have hastened the identification of new inhibitors with strong in vivo anti-TSE activities such as porphyrins, phthalocyanines, and phosphorthioated oligonucleotides. New routes of administration have enhanced beneficial effects against established brain infections. Several different classes of TSE inhibitors share structural similarities, compete for the same site(s) on PrP(C), and induce the clustering and internalization of PrP(C) from the cell surface. These activities may represent a common mechanism of action for these anti-TSE compounds.

Inhibition of influenza virus infections in immunosuppressed mice with orally administered peramivir (BCX-1812).

Experiments were run to determine the effect of oral gavage treatment with the cyclopentane influenza virus neuraminidase inhibitor peramivir (BCX-1812, RWJ-270201) in influenza A (H1N1) virus-infected mice that had their immune system suppressed by cyclophosphamide (CP) therapy or in severe combined immune deficient (SCID) mice. Treatment of CP-immunosuppressed mice with peramivir using doses of 100, 10, or 1mg/kg/day was begun 2.5 or 8 days post-virus exposure and continued twice daily for 3 or 5 days. The 5-day therapy was more effective than the 3-day treatment, as seen by significantly increased survivor numbers, lessened decline in arterial oxygen saturation, reduced lung consolidation, and inhibition of lung virus titers. Infected SCID mice were also responsive to peramivir therapy begun 8 days after virus exposure and continued for 5 days, although antiviral effects did not include prevention of death and were dependent upon the viral challenge dose received. These data indicate that peramivir may have potential for treatment of influenza virus-infected immunosuppressed patients.

Characterization of nuclease-resistant ribozymes directed against hepatitis B virus RNA.

Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide and 1.2 million deaths each year. To explore the use of ribozymes as a novel therapy for HBV infection, nuclease-resistant ribozymes that target highly conserved regions of HBV RNA were screened in cell culture. These synthetic ribozymes have the potential to cleave all four major HBV RNA transcripts and to block the HBV lifecycle by cleavage of the pregenomic RNA. A number of the screened ribozymes demonstrate activity in cell culture systems, as measured by decreased levels of HBV surface antigen, HBV e antigen and HBV DNA. In addition, a lead anti-HBV ribozyme maintains activity against a lamivudine-resistant HBV variant in cell culture. Treatment of HBV transgenic mice with lead anti-HBV ribozymes significantly reduced viraemia compared with saline-treated animals and was as effective as treatment with lamivudine. In conclusion, the therapeutic use of a ribozyme alone or in combination with current therapies (lamivudine or interferons) may lead to improved HBV therapy.

Influence of virus strain, challenge dose, and time of therapy initiation on the in vivo influenza inhibitory effects of RWJ-270201.

The influenza virus neuraminidase inhibitor RWJ-270201 (cyclopentane carboxylic acid, 3-[cis-1-(acetylamino)-2-ethylbutyl]-4[(aminoiminomethyl)amino]-2-hydroxy-[cis, 2S, 3R, 4R]) was significantly inhibitory to an infection in mice induced by influenza A/NWS/33 (H1N1) virus when oral gavage (p.o.) treatment with 10 mg/kg per day was delayed at least 60 h after virus exposure. Treatment was 5 mg/kg twice daily for 5 days. Viral challenge doses of influenza A/Shangdong/09/93 (H3N2) virus ranging from the LD(70) to the LD(100) did not affect the marked antiviral efficacy of 12.5 mg/kg of RWJ-270201 administered p.o. twice daily for 5 days beginning 4 h pre-virus exposure; infection by an approximate 2 LD(100) dose (10(8) cell culture infectious doses/ml) was only weakly inhibited by the same treatment as seen by significant increase in mean day to death. Murine infections induced by influenza A/Bayern/57/93 (H1N1) and B/Lee/40 viruses were significantly inhibited by 100, 10, and 1 mg/kg per day of RWJ-270201 using the above treatment regimen; influenza A/PR/8/34 (H1N1) virus infections in mice were only moderately inhibited, the antiviral effects using this virus being lessening of arterial oxygen decline, reduced lung consolidation, and inhibition of lung virus titers primarily at the higher dosages.

In vivo influenza virus-inhibitory effects of the cyclopentane neuraminidase inhibitor RJW-270201.

The cyclopentane influenza virus neuraminidase inhibitor RWJ-270201 was evaluated against influenza A/NWS/33 (H1N1), A/Shangdong/09/93 (H3N2), A/Victoria/3/75 (H3N2), and B/Hong Kong/05/72 virus infections in mice. Treatment was by oral gavage twice daily for 5 days beginning 4 h pre-virus exposure. The influenza virus inhibitor oseltamivir was run in parallel, and ribavirin was included in studies with the A/Shangdong and B/Hong Kong viruses. RWJ-270201 was inhibitory to all infections using doses as low as 1 mg/kg/day. Oseltamivir was generally up to 10-fold less effective than RWJ-270201. Ribavirin was also inhibitory but was less tolerated by the mice at the 75-mg/kg/day dose used. Disease-inhibitory effects included prevention of death, lessening of decline of arterial oxygen saturation, inhibition of lung consolidation, and reduction in lung virus titers. RWJ-270201 and oseltamivir, at doses of 10 and 1 mg/kg/day each, were compared with regard to their effects on daily lung parameters in influenza A/Shangdong/09/93 virus-infected mice. Maximum virus titer inhibition was seen on day 1, with RWJ-270201 exhibiting the greater inhibitory effect, a titer reduction of >10(4) cell culture 50% infective doses (CCID(50))/g. By day 8, the lung virus titers in mice treated with RWJ-270201 had declined to 10(1.2) CCID(50)/g, whereas titers from oseltamivir-treated animals were >10(3) CCID(50)/g. Mean lung consolidation was also higher in the oseltamivir-treated animals on day 8. Both neuraminidase inhibitors were well tolerated by the mice. RWJ-270201 was nontoxic at doses as high as 1,000 mg/kg/day. These data indicate potential for the oral use of RWJ-270201 in the treatment of influenza virus infections in humans.

Ethical issues in transgenics.

The arguments of critics and concerns of the public on generating transgenic cloned animals are analyzed for the absence or presence of logical structure. Critics' arguments are symbolically compared with "genetic trespassing," "genetic speeding," or "going the wrong way," and responses are provided to these arguments. Scientists will be empowered to participate in the public discussion and to engage the critics on these issues as they consider thoughtful, plausible responses to their concerns. Temporary moratoriums are recognized as a plausible approach to dealing with possible concerns of new scientific advancements.

Utilization of transgenic mice replicating high levels of hepatitis B virus for antiviral evaluation of lamivudine.

A recently developed transgenic mouse strain which expresses high levels of hepatitis B virus (HBV) was studied as a model for evaluation of potential chemotherapeutic agents. Lamivudine ([-]2'-deoxy-3'-thiacytidine), known to reduce hepatitis B viremia in human patients, and zidovudine (3'-azido-3'-deoxythymidine), previously shown to be ineffective for HBV infections in man, were used in parallel in this transgenic animal model. Orally administered lamivudine at dosages of 100, 50, and 25 mg/kg per day given once a day for 21 days significantly decreased serum and liver HBV DNA titers in a dose-responsive manner. Zidovudine (approximately 22 mg/kg per day) administered in the drinking water for 21 days was not effective in reducing these HBV parameters as compared to transgenic placebo-treated controls. The serum HBV DNA titers rebounded to high levels 1 week after cessation of lamivudine treatment. Male and female mice responded in a similar manner to these therapies. The results using this transgenic mouse model were similar to what would be predicted from treatment of HBV-infected human patients with lamivudine and zidovudine, and indicate these mice may be useful as a small animal chemotherapeutic model for study of potential HBV inhibitors.

Resistance to Friend leukemia virus in transgenic mice expressing the native Fv-4 gene.

Fv-4 is a truncated ecotropic retrovirus gene that codes for an envelope protein under control of a cellular promoter. It confers resistance to ecotropic murine leukemia viruses. Transgenic mice were derived using the native Fv-4 gene as the construct for microinjection. Two founder mice were derived. In both founder lines, there was no detectable expression of the transgene or resistance to Friend murine leukemia virus (FrMLV) in hemizygotes. In one line, the resistance was observed in homozygotes with Fv-4 RNA formation in the thymus but not in the spleen or in other tissues. In the other founder line, a homozygous male was identified. Double integrants, derived from breeding this homozygous male to homozygous females from the other founder line, were also resistant. These results indicate that the native gene confers the resistance in homozygous transgenic mice or double integrants derived from different founders but not hemizygotes.

Transgenic mice as a chemotherapeutic model for hepatitis B virus infection.

Many transgenic mice carrying either portions of the hepatitis B virus (HBV) genome, or the complete genome, have been developed as models because HBV does not infect any other organisms besides humans and chimpanzees to cause a productive infection and disease. Some of these models have been useful in evaluating chemotherapeutic agents such as interferon-alpha, interleukins-2 and -12, other cytokines and nucleoside analogues for efficacy against HBV. A recently developed transgenic mouse (Guidotti et al., Journal of Virology 69:6158-6169.) which supports the replication of high levels of infectious HBV, provides the opportunity to evaluate the effect of antiviral drugs on various portions of the HBV life cycle in the whole animal. Evaluation of lamivudine, zidovudine and interferon-alpha B/D (IFN-alpha) in this HBV transgenic mouse model are described. Lamivudine and IFN-alpha were highly efficacious in reducing serum HBV DNA. As might be predicted, zidovudine was not efficacious. IFN-alpha was more effective in reducing virus titres in male mice as compared to female mice. This gender difference was not due to lower ability of female mice to express the virus. One anticipates that as this high level HBV transgenic-expressing mouse becomes more fully developed as a chemotherapeutic model, questions about the efficacy of different agents, routes of administration, synergy of antiviral combinations and novel drug therapies will be answered.

Interleukin 2 promoter/enhancer controlled expression of a synthetic cecropin-class lytic peptide in transgenic mice and subsequent resistance to Brucella abortus.

The addition of an antimicrobial that can be synthesized by the mammalian immune system at the point of challenge may enhance disease resistance. A possible group of agents are cecropins, broad-spectrum antimicrobial peptides, which have been described and characterized. They are relatively non-toxic to normal cells from multicellular organisms but are toxic to a wide range of bacteria, protozoa and fungi, as well as infected and abnormal cells. Twenty-six lines of transgenic mice were produced by pronuclear injection of DNA consisting of the 5'-flanking region from -593 to +110 of the mouse interleukin 2 (IL-2) gene, Shiva 1a (a synthetic cecropinclass lytic peptide), and the SV40 polyadenylation/splice signal. A reverse-transcription PCR assay determined that two lines of transgenic mice were produced whose spleen-derived lymphocytes could be induced to transcribe and mature mRNA for Shiva 1a by exposure to 3.25 mg ml-1 of Con A. Two lines were challenged with an inoculation of 5 x 10(4) Brucella abortus strain 2308. After four weeks, there were significantly fewer B. abortus organisms in the spleens of transgenic mice than in non-transgenic control mice of the same strain (p < 0.05). Since the controlling regions of the IL-2 enhancer and the amino acid sequence of the signal peptide are highly conserved among several species, it is likely that this recombinant gene will function in other mammals.

Progression of Aleutian disease in natural and experimentally induced infections of mink.

OBJECTIVE: To study temporal changes in amounts of viral DNA in blood leukocytes over long periods, and to determine whether severity of the disease is greater in experimentally induced, compared with natural, infection. ANIMALS: 18 naturally and 6 experimentally infected black mink; 26 naturally infected brown mink. PROCEDURE: Polymerase chain reaction amplification to detect viral DNA in blood and counter-immune electrophoresis to detect serum antibody were performed at regular intervals. RESULTS: In naturally infected black mink, amounts of viral DNA were initially high, but after the appearance of antibody, viral DNA fluctuated and, in some instances, was undetectable. In other mink, small amounts of viral DNA were infrequently detected during the course of the infection. Amounts of viral DNA in leukocytes in late stages of the disease correlated with renal lesions in brown mink, but black mink had more severe lesions associated with smaller amounts of viral DNA. Severity of the disease was not enhanced in experimentally inoculated black mink. CONCLUSIONS: After infection, leukocyte viral DNA is initially present in large amounts, but, in most mink, decreases markedly in association with the appearance of antibody. There is no difference in the progression and severity of the disease between black mink infected experimentally or naturally. Transmission of the disease may be enhanced by use of contaminated toenail clippers for blood collection.

Effect of the combination of interferon-alpha and stavudine on Friend virus infections in (B10.A x A.By)F1 mice.

The anti-Friend leukemia virus (FLV) effects of interferon-alpha-A/D (IFN-alpha) and 2',3'-didehydro-2',3'-dideoxythymidine (stavudine) used alone and in combination were examined in Mus dunni cells using a checkerboard-type experiment design. Strong antiviral synergy and a suggested cytotoxic synergy were seen. In two experiments to evaluate the effect of combining therapy with IFN-alpha and stavudine against FLV disease in the hybrid mouse strain (B10.A x A.By)F1, which is a strong producer of cytotoxic T cells, the drug combination resulted in better inhibition of FLV disease than did either drug used alone. Combination therapy inhibited splenomegaly, splenic virus infectious centers, plasma virus, and the virus-induced increase in hematocrit to a greater degree than did either drug alone. These data indicate that combination therapy with stavudine and IFN-alpha is effective in the treatment of murine retrovirus infections and may be of value in the treatment of human AIDS.

Importance of primer selection in the application of PCR technology to the diagnosis of bovine leukemia virus.

The polymerase chain reaction (PCR) was used to detect bovine leukemia virus in bovine blood samples. When applied to leucocytes extracted from the blood samples, the standard method of DNA extraction gave good correlation with agar gel immunodiffusion, but a method in which 5 microliters of blood was the starting material was unreliable. Selection of the primers was important, and differences in results were observed when the PCR method was applied to blood samples from different geographic areas. The sensitivity varied from 50% to 90%, depending on the primer set applied to the gag gene of proviral nucleic acid. This variation was based on geographic origin of the cattle, suggesting an influence of viral strain. In some areas, more than 1 primer may needed to optimize results.

Activation of the human immunodeficiency virus long terminal repeat by abrasion of the skin in transgenic mice.

Mechanical wounding was shown to activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in the skin of transgenic mice. Both noninvasive rubbing and scratching of the skin resulted in a range of 4- to 44-fold increased levels of luciferase reporter gene activities when assayed 24-48 h after wounding. Moreover, long-term noninvasive rubbing each day for 17 days resulted in similar increased levels of luciferase activity. Experiments were done to determine whether the HIV-1 LTR-luciferase transgene might be activated when pups were nursed on the mammary tissues of transgenic mice. Luciferase reporter gene activity in mammary glands skin following nursing was significantly higher than in skin from non-pregnant transgenic mice or transgenic mice 20 days post-conception, which suggests that the natural abrasive action of nursing resulted in activation of the LTR. These results may have implications for sexual transmission and maternal-to-infant transmission of HIV-1.

Immunomodulator effects on the Friend virus infection in genetically defined mice.

The disease induced by the Friend virus complex (FV) in F1 hybrid mice containing the Rfv-3r/s genotype in the presence of H-2a/a was used to evaluate a variety of immunomodulating substances. In these genetically defined mice, the FV disease results in splenomegaly, early production of high titers of cell-associated and plasma virus, high levels of splenic viral RNA, increased hematocrit, and eventual death. As the disease progresses, reduced levels of infectious virus correlate with development of specific antibody; reduction in T cell populations, increase in B cells, and decrease in T-cell function also occur. The following immunomodulators were evaluated, listed in the order of their ability to inhibit the FV disease: imexon > MVE-2 > human recombinant IFN-alpha A/D > AS101 > ampligen > AM-3 = oxamisole > ImuVert > bropirimine. In fact, bropirimine, used with certain treatment regimens, appeared to enhance the FV disease. These data suggest that certain immunomodulators may have potential value in the treatment of HIV disease, but also indicate that caution should be exercised in their clinical use.

Early detection of bovine leukemia virus in cattle by use of the polymerase chain reaction.

A study was performed to determine whether experimentally induced bovine leukemia virus (BLV) infection in cattle could be detected earlier by use of polymerase chain reaction (PCR) amplification of genomic DNA extracted from leukocytes than by use of conventional agar-gel immunodiffusion (AGID). The PCR primers were designed to amplify a 375-base-pair region of the proviral gag gene. Five cows were identified that were BLV-negative on the basis of AGID and PCR results. At day 0, these cows were inoculated IM with blood pooled from 3 naturally infected cows. Blood samples were taken on days 0, 1, and 7, and every 2 weeks thereafter until 3 months after inoculation. Three of the cows were BLV-positive by AGID test results 3 weeks after inoculation, and the remaining 2 seroconverted at 5 weeks. In contrast, all 5 cows were BLV-positive by PCR results 7 days after inoculation and remained positive for the duration of the study. Five cows that were BLV-positive by AGID test and PCR results on day 0 and from which samples were obtained at the same times as those from the other 5 cows, remained BLV-positive by results of both tests during the course of the study. Results indicate that under experimental conditions, BLV infection in cattle can be detected as much as 2 to 4 weeks earlier by use of PCR than by use of the AGID test.

Activation of the human immunodeficiency virus type 1 long terminal repeat by skin-sensitizing chemicals in transgenic mice.

Topical dinitrochlorobenzene (DNCB) is often used for evaluating contact skin hypersensitivity in immunocompromised patients. We have determined, in this study, that topical application of DNCB alone, even without induction of contact skin hypersensitivity, was sufficient to observe activation of the human immunodeficiency virus promoter (long terminal repeat) in the skin of an HIV-1 long terminal repeat-luciferase transgenic mouse model. Such treatment might be contra-indicative in patients infected with the human immunodeficiency virus, because in earlier studies DNCB-exposed skin dendritic cells might migrate into draining lymph nodes which play an important role in AIDS pathogenesis.

Effect of immunomodulators in the hu-PBL-SCID mouse model.

The effects of two immunomodulators were investigated in severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID mice). Both immunomodulators, maleic anhydride divinyl ether (MVE-2) and 4-imino-1,3-diazobicyclo-(3.1.0)-hexan-2- one (imexon), have been previously studied by us in retrovirus-infected mice. To determine the effects of these compounds as they may function in humans, 24 SCID mice were each reconstituted with 20 x 10(6) ficoll-purified lymphocytes from a single donor. Five weeks after reconstitution, the mice received 16 mg/kg/day of MVE-2 intraperitoneally (i.p.) on days 0, 7, and 14 or 110 mg/kg/day of imexon i.p. daily for 14 days. Spleens were removed and splenocytes labeled with monoclonal antibodies for T- and B-cell enumeration as determined by flow cytometry 24 h after final treatment. Imexon-treated mice demonstrated a slight increase in total T cells and T cell subsets compared to control mice. T helper/T suppressor cell ratios in imexon-treated mice were brought to a normal 3:2 ratio compared to placebo-treated mice. Human immunoglobulin levels were markedly increased in imexon-treated mice. MVE-2-treated hu-PBL-SCID mice had significantly reduced numbers of total T cells compared to controls. The T-cell population results using human cells in SCID mice were similar to the effects of these immunomodulators on murine cells in immunologically competent mice.

HIV-1 LTR activation model: evaluation of various agents in skin of transgenic mice.

Mice containing the HIV-1 long terminal repeat (LTR) regulating the expression of firefly luciferase reporter gene were investigated for their use as a model for activation of the LTR. As a limited test of this model, a number of different factors were screened for their ability to affect reporter gene activities in the skin. Reporter gene levels were increased in the skin by topical treatment of dimethylsulfoxide, retinoic acid, phorbol ester, ultraviolet light, and hydrogen peroxide, all of which have previously been shown to cause increased HIV production in cultured human cells. Topically applied arachidonic acid, histamine, ethanol, acetone, and methanol did not increase reporter gene activities. A lack of published reports on activation of HIV-1 in human cells by these agents suggests that they do not activate viral expression in human cells, which corroborates with the findings of this report. Minor forms of skin wounding and intraperitoneally administered psoralen plus ultraviolet light also increased reporter gene activities in skin. Control and test treatments could be performed on the same mouse and repetitive samples could be obtained from each treatment area. These transgenic mice might be useful as predictive models for regulation of the LTR in epidermal or dendritic cells.

Suppression of murine retroviral disease by 2',3'-didehydro-2',3'-dideoxythymidine (D4T).

The thymidine analog, 2',3'-didehydro-2',3'-dideoxythymidine (D4T), and 3'-azido-3'-deoxythymidine (AZT) were evaluated for activity against Friend virus complex (FV) in Mus dunni cells using a focal immunoenzyme assay. The 50% effective doses were, respectively, 1.2 and 0.1 microM for the two compounds; the 50% cytotoxic doses using trypan blue dye exclusion were 25.4 and > 100 microM. Four FV inhibition experiments with D4T were run in F1 hybrid mice containing the Rfv-3r/s genotype. This mouse strain allows the study of treatment effects on development of specific neutralizing antibodies and on splenomegaly, splenic and plasma virus titers, and splenic viral RNA. In the first experiment, D4T was given by oral gavage (p.o.) three times daily (t.i.d.) for 14 days beginning 4 h post-virus inoculation. All dosages used (187.5, 375, 750 mg/kg/day) significantly inhibited all viral parameters. Other experiments used D4T p.o. twice daily, with dosages of 46.9, 93.8, 187.5 and 375 mg/kg/day or four times daily with a dose of 375 mg/kg/day. No significant disease inhibition was seen using the twice daily treatment schedule, but efficacy was apparent using the four times daily treatment. The final experiment repeated the initial study, extending the t.i.d. treatments to 25 days and using dosages of 46.9, 93.8, 187.5 and 375 mg/kg/day. All but the lowest dose reduced each virus parameter. None of the D4T treatment regimens caused death in toxicity controls, although moderate host weight loss or less weight gain was seen, and variable hematocrit decreases occurred, particularly in mice receiving the highest drug dosage. Inhibition of natural killer (NK) cell activity also was seen in these same animals, but in infected mice, FV-induced decrease in NK cell activity was prevented by D4T treatment. Virus-specific neutralizing antibodies developed in all infected, treated animals. These data indicate D4T has potential as a possible candidate for anti-human immunodeficiency virus evaluations in the clinic.