Marked in vivo antiretrovirus activity of 9-(2-phosphonylmethoxyethyl)adenine, a selective anti-human immunodeficiency virus agent.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective inhibitor of the replication of human immunodeficiency virus (HIV) in vitro in human T-lymphocyte MT-4, H9, and ATH8 cells. PMEA also inhibits Moloney murine sarcoma virus (Mo-MSV)-induced transformation of murine C3H embryo fibroblasts. Moreover, PMEA causes a dose-dependent suppression of tumor formation and associated mortality in mice inoculated with Mo-MSV. At a dose of 50 or 20 mg/kg per day PMEA effected a 90-100% protection of the mice against Mo-MSV-induced tumor formation and mortality. Even with a PMEA dose as low as 1 to 5 mg/kg per day, tumor formation was significantly delayed and the survival rate was significantly enhanced. In parallel experiments, azidothymidine exhibited a comparable inhibitory effect on Mo-MSV-induced tumor formation and associated death only at a 25-fold higher dose than PMEA. Because PMEA has stronger in vivo antiretrovirus potency and selectivity than azidothymidine and various other compounds currently being subjected to clinical trials, PMEA studies should be pursued to assess the potential of this compound in the treatment of acquired immunodeficiency syndrome (AIDS) and other retrovirus infections in humans.     

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)adenine on visna virus infection in lambs: a model for in vivo testing of candidate anti-human immunodeficiency virus drugs.

The acyclic nucleoside phosphonate analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was recently found to be effective as an inhibitor of visna virus replication and cytopathic effect in sheep choroid plexus cultures. To study whether PMEA also affects visna virus infection in sheep, two groups of four lambs each were inoculated intracerebrally with 10(6.3) TCID50 of visna virus strain KV1772 and treated subcutaneously three times a week with PMEA at 10 and 25 mg/kg, respectively. The treatment was begun on the day of virus inoculation and continued for 6 weeks. A group of four lambs were infected in the same way but were not treated. The lambs were bled weekly or biweekly and the leukocytes were tested for virus. At 7 weeks after infection, the animals were sacrificed, and cerebrospinal fluid (CSF) and samples of tissue from various areas of the brain and from lungs, spleen, and lymph nodes were collected for isolation of virus and for histopathologic examination. The PMEA treatment had a striking effect on visna virus infection, which was similar for both doses of the drug. Thus, the frequency of virus isolations was much lower in PMEA-treated than in untreated lambs. The difference was particularly pronounced in the blood, CSF, and brain tissue. Furthermore, CSF cell counts were much lower and inflammatory lesions in the brain were much less severe in the treated lambs than in the untreated controls. The results indicate that PMEA inhibits the propagation and spread of visna virus in infected lambs and prevents brain lesions, at least during early infection. The drug caused no noticeable side effects during the 6 weeks of treatment.     

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) effectively inhibits retrovirus replication in vitro and simian immunodeficiency virus infection in rhesus monkeys

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective inhibitor of the in vitro replication of a number of retroviruses, including HIV-1 and HIV-2, simian immunodeficiency virus (SIV), simian AIDS-related virus (SRV), feline immunodeficiency virus (FIV) and Moloney murine sarcoma virus (MSV). PMEA causes a dose-dependent suppression of the induction of anti-SIVmacgp120 antibodies in SIV mac-infected rhesus monkeys. Complete suppression of anti-SIVmacgp120 antibodies was achieved in SIV-infected animals treated with PMEA at 2 x 10 or 2 x 5 mg/kg per day for 29 days. No toxic side-effects were noted during this treatment period. Antibodies against SIVmac gp120 appeared 1-2 weeks after PMEA treatment was stopped, but the antibody titre reached in these animals was significantly lower than in the SIVmac-infected animals who had not been treated with PMEA. Our data strongly suggest that PMEA should be pursued for its potential in the treatment of AIDS and other retrovirus infections.     

Hematologic manifestations of feline immunodeficiency virus infection

Studies were done on 53 cats with community-acquired infection with the feline immunodeficiency virus (FIV) to determine if hematologic abnormalities were comparable with those observed in patients seropositive for the human immunodeficiency virus (HIV). Nine cats were asymptomatic, 24 had clinical symptoms equivalent to AIDS-related complex (ARC), and 20 had AIDS-like disease. Hematologic abnormalities were detected in 75% (40 of 53) of FIV-seropositive cats, and multiple concurrent cytopenias were common. Anemia, lymphopenia, neutropenia, and thrombocytopenia occurred in 36%, 53%, 34%, and 8% of FIV-seropositive cats, respectively. Cytopenias were seen only in symptomatic (ARC or AIDS) cats. The occurrence of cytopenias and the distribution of clinical stages were similar in cats with concurrent feline leukemia virus (FeLV) infection and those with FIV alone, suggesting that these abnormalities were a direct consequence of FIV infection. In addition, abnormalities were noted in 72% of marrows from symptomatic cats and included hyperplasia of individual cell lineages and dysmorphic features. Our results demonstrate that the hematologic manifestations of FIV infection are strikingly similar to those reported in HIV-seropositive patients. Thus, FIV infection in cats is an excellent animal model to study the pathogenesis of blood and marrow abnormalities in AIDS, as well as to evaluate the hematologic toxicities of drug therapies.     

In vivo infection of marrow stromal fibroblasts by feline leukemia virus.

Marrow stromal fibroblasts (FBs) likely play an important role in the regulation of hematopoiesis within the marrow microenvironment. Infection of these cells by feline leukemia virus (FeLV) might not only contribute to the pathogenesis of FeLV-induced hematologic diseases, but could provide a reservoir for virus in the infected cat. To determine the frequency of FeLV infection among marrow FB precursor cells (fibroblast colony-forming units, CFU-F) of cats viremic with FeLV-C/Sarma and FeLV-A/61E, marrow FBs and FB cell clones were isolated and assayed for expression of FeLV gag protein. From 30% to 86% and 64% to 88% of marrow FB precursors were infected with FeLV-C/Sarma and FeLV-A/61E, respectively. CFU-F from a cat viremic with FeLV-A/61E were not affected by exposure to antibody against FeLV envelope glycoprotein gp70 and heterologous complement, whereas similarly treated hematopoietic progenitors (erythroid colony-forming units, CFU-E; erythroid burst-forming units, BFU-E; and granulocyte-macrophage colony-forming units, CFU-GM) and culture-propagated, FeLV-infected marrow FBs were effectively lysed, suggesting that infected CFU-F within the marrow microenvironment do not express a significant amount of gp70 on their cell membranes. Thus, marrow FB precursor cells appear to be a major target for FeLV in vivo. Furthermore, the low level of gp70 antigen expression on the surface of these cells in vivo may allow them to escape immune surveillance and provide a reservoir of virus during active or latent infection.     

The molecular biology of feline immunodeficiency virus (FIV)

Feline immunodeficiency virus (FIV) is widespread in feline populations and causes an AIDS-like illness in domestic cats. It is highly prevalent in several endangered feline species. In domestic cats FIV infection is a valuable small animal model for HIV infection. In recent years there has been sa significant increase in interest in FIV, in part to exploit this, but also because of the potential it has as a human gene therapy vector. Though much less studied than HIV there are many parallels in the replication of the two viruses, but also important differences and, despite their likely common origin, the viruses have in some cases used alternative strategies to overcome similar problems. Recent advances in understanding the structure and function of FIV RNA and proteins and their interactions has enhanced our knowledge of FIV replication significantly, however, there are still many gaps. This review summarizes our current knowledge of FIV molecular biology and its similarities with, and differences from, other lentiviruses.     

Effect of primary-stage feline immunodeficiency virus infection on subsequent feline calicivirus vaccination and challenge in cats.

The effect of experimental primary-stage feline immunodeficiency virus (FIV) infection on feline calicivirus (FCV) vaccination and challenge in cats was studied. Clinical signs of acute FCV disease were more widespread in the cats which were infected with FIV than in those which were not. FIV infection also prolonged shedding of FCV, with more of the FIV-infected cats becoming chronic carriers. Although vaccination induced protection against acute FCV disease, this was to a lesser degree in FIV-infected cats. Vaccination by itself also appeared to enhance long-term virus shedding. There was evidence of an impaired anamnestic FCV-neutralizing antibody response in FIV-infected cats following FCV challenge.     

Molecular cloning of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a T-lymphotropic retrovirus associated with immunodeficiency and opportunistic infections in cats. The discovery of FIV provides an opportunity for the development of a small animal model for AIDS. To initiate the molecular and biological characterization of FIV, cDNA clones were synthesized and used to isolate a proviral clone of FIV. Molecular cross-hybridization analysis of FIV with five lentiviruses revealed that nucleotide-sequence similarities exist between FIV and these lentiviruses in the gag-pol genes. However, nucleotide-sequence similarities were not seen upon comparison of the FIV long terminal repeat sequence with known viral sequences. Common antigenic determinants appeared to be shared by FIV, caprine arthritis encephalitis virus, and visna virus as shown by serological cross-reactivity of rabbit antibodies to caprine arthritis encephalitis virus and visna virus with the putative FIV core protein p28. These studies demonstrated that FIV is a member of the lentivirus subfamily and is distantly related to the AIDS lentiviruses of primates. Importantly, progeny virions of our molecular clone were infectious for experimentally inoculated cats. The availability of an infectious molecular clone will make possible a detailed dissection of the molecular pathogenesis of FIV, which may facilitate the development of vaccine and therapeutic strategies for AIDS.     

Exogenous glucocorticoids alter parameters of early feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV), a lentivirus, causes progressive immunosuppression and neurologic dysfunction in cats. Glucocorticoids are common therapeutic agents that are also immunosuppressive, and their use might enhance the pathogenic effects of lentivirus infections. Methylprednisolone acetate, a long-acting glucocorticoid, was administered to cats before FIV inoculation, and the course of early infection was monitored. The humoral immune response to FIV was not affected by corticosteroid treatment, but CD8+ cell-mediated antiviral activity was poor in cultures from FIV-infected cats treated with methylprednisolone. Steroid-treated cats had higher plasma viral RNA levels than untreated cats during acute viremia. In contrast, FIV-associated changes in brain stem auditory-evoked potentials were slow to develop in the methylprednisolone-treated cats. Methylprednisolone treatment of cats with established FIV infections appeared to reverse these neurophysiologic changes. These results emphasize the complexity of host-lentivirus interactions and suggest potential advantages and drawbacks of using glucocorticoids in lentivirus infections.     

Experimental vaccine protection against feline immunodeficiency virus.

Infection of domestic cats with the feline immunodeficiency virus (FIV) represents an important veterinary health problem and a useful animal model for the development of vaccines against acquired immunodeficiency syndrome (AIDS). Two experimental FIV vaccines have been developed; one consisting of fixed infected cells (Vaccine 1), the other of inactivated whole virus (Vaccine 2). After 4-6 immunizations over 2-5 months, both vaccines induced a strong FIV-specific immune response including neutralizing antibody and T-cell proliferation. Vaccine 1 protected 6 of 9 and Vaccine 2 protected 5 of 6 recipient cats against any detectable infection with a low dose (10 animal ID50) of FIV given intraperitoneally 2 weeks after the final boost. One additional cat in each vaccine group had a transient infection at 5-7 weeks postchallenge following which virus could no longer be detected. Thus, a total of 13 of 15 vaccinated cats were protected against persistent infection. By contrast, 13 of 13 controls were persistently infected by this challenge. The infected cell vaccine failed to protect against a higher dose (5 x 10(4) ID50) of FIV. These results indicate that vaccine prophylaxis against natural FIV infection should be achievable and enhance optimism of the prospect of developing an effective AIDS vaccine for humans.     

Productive infection of T-helper lymphocytes with feline immunodeficiency virus is accompanied by reduced expression of CD4.

An antigen-specific feline T-lymphocyte cell line (Q201) was generated and infected in vitro with the feline immunodeficiency virus (FIV). Syncytium formation and the release of the viral core protein p24 into culture fluid were accompanied by a reduction in expression of the CD4 surface antigen. The reduction in CD4 expression was transient, the resulting persistently infected population of cells expressing levels of CD4 comparable to those observed prior to infection. Persistently infected cells gradually lost expression of major histocompatibility antigen (MHC) class II while maintaining pre-infection levels of expression of CD4, MHC class I, CD18 or CD29.     

Immunodiagnosis of feline immunodeficiency virus infection using recombinant viral p17 and p24.

The coding sequences of p17 and p24 of the Glasgow-8 strain of feline immunodeficiency virus (FIV) were amplified using the polymerase chain reaction and cloned into plasmid vectors. The predicted amino-acid sequences of FIV/Glasgow-8 p17 and p24 were compared with those of the Petaluma and PPR isolates of FIV. As seen with other retroviruses, these gag gene products are highly conserved, indicating that the protein products would be suitable antigens to detect anti-FIV antibodies in an immunoassay. Both p17 and p24 were stably expressed in Escherichia coli as fusion proteins with glutathione S transferase. A pure preparation of each fusion protein was obtained from induced bacterial lysates by affinity chromatography using glutathione-agarose beads. These recombinant proteins were used in an enzyme-linked immunosorbent assay to detect antibodies directed against FIV p17 and p24 in cat sera. This assay allows the identification of seropositive cats following infection with FIV and has greater sensitivity and specificity than a currently available immunodiagnostic test.     

Hematopoiesis in asymptomatic cats infected with feline immunodeficiency virus.

Anemia and neutropenia often develop in cats that are infected with the feline immunodeficiency virus (FIV), a lentivirus biologically similar to the human immunodeficiency virus (HIV). To assess the role of FIV in the pathogenesis of these abnormalities, marrow culture studies were performed on nine asymptomatic, hematologically normal cats that were chronically infected with FIV. In these experiments, the frequencies of granulocyte/macrophage progenitors (CFU-GM) and early and late erythroid progenitors (CFU-E and BFU-E, respectively) were equivalent to progenitor frequencies in simultaneously studied uninfected control cats. Asymptomatic FIV infection was not associated with a change in the cell-cycle kinetics of CFU-E, BFU-E, or CFU-GM, nor was there an alteration in the dose-response of BFU-E or CFU-GM to hematopoietic growth factors present in fibroblast-derived conditioned medium. Sera from FIV-infected cats supported progenitor growth in vitro as well as normal cat sera. Furthermore, there was no evidence that these sera contained complement-fixing antibodies that recognized hematopoietic progenitors. Therefore, these data show that the in vitro behavior of hematopoietic progenitors is not affected by FIV infection alone, and they are in agreement with recent evidence that human progenitors are not a major target of HIV infection. It is likely that factors associated with progressive immunodeficiency, opportunistic infections, nutritional deficiencies, or malignancies play significant roles in the cytopenias that develop during the symptomatic disease induced by FIV, and by analogy, HIV. Prospective marrow culture studies of FIV-infected cats that develop hematologic abnormalities should provide a valuable animal model of acquired immunodeficiency syndrome-associated hematologic disorders.     

RNase inhibition of human immunodeficiency virus infection of H9 cells.

Onconase and bovine seminal RNase, two members of the RNase A superfamily, inhibit human immunodeficiency virus type 1 replication in H9 leukemia cells 90-99.9% over a 4-day incubation at concentrations not toxic to uninfected H9 cells. Two other members of the same protein family, bovine pancreatic RNase A and human eosinophil-derived neurotoxin, have no detectable antiviral activity, demonstrating a strikingly selective antiviral activity among homologous ribonucleases. The antiviral RNases do not appear to affect viral particles directly but inhibit replication in host cell cultures. Onconase, already in clinical trials for cancer therapy, and bovine seminal RNase have potential as antiviral therapeutics.     

NIH conference. Immunopathogenic mechanisms in human immunodeficiency virus (HIV) infection.

An understanding of the immunopathogenic mechanisms of infection with human immunodeficiency virus (HIV) is fundamental in developing successful approaches to designing effective therapeutic and vaccine strategies. In this regard, we have investigated the mechanisms by which HIV inserts itself into the human immune system and uses the elaborate cytokine network to its own replicative advantage. We have also shown that the burden of HIV in CD4+ T cells is directly associated with a decline in this cell population in vivo and a progression to disease. Mononuclear phagocytes may play a role in the pathogenesis of HIV infection by serving as reservoirs of the virus. Of note is the fact that monocytes in the peripheral blood of HIV-infected individuals are rarely infected in vivo, whereas infected-tissue macrophages may play a role in organ-specific HIV-related pathogenesis. The role of HIV-specific humoral and cell-mediated immunity in HIV infection is not well understood. However, fine specificity of responses against HIV have been delineated in some in-vitro systems. It is unclear why these responses, particularly HIV-specific cytolytic T-cell responses, diminish over the course of infection and are unable to contain progression of infection.     

(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA]: a purine analogue with trypanocidal activity in vitro and in vivo

The unique features of purine salvage systems of pathogenic haemoflagellates render them selectively susceptible to the cytotoxic effects of purine analogues. A series of acyclic nucleoside phosphonates were evaluated for activity against pathogenic haemoflagellates in vitro. One of the phosphonylmethoxyalkylpurines, namely (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], was active in vitro against bloodstream forms of Trypanosoma brucei rbodesiense, T. b. gambiense, multidrug-resistant T. b. brucei, T. congolense and T. evansi, but not against intracellular T. cruzi or Leishmania donovani. Cytotoxic effects against mammalian cells were observed at 4900-27 300-fold higher concentrations than those necessary to inhibit T. b. rbodesiense. (S)-HPMPA was able to eliminate T. b. rbodesiense and multidrug-resistant T. b. brucei in an acute rodent model with two administrations of 10 mg/kg each.