Prem S. Sarin

INFECTION OF CHIMPANZEES BY HUMAN T-LYMPHOTROPIC RETROVIRUSES IN BRAIN AND OTHER TISSUES FROM AIDS PATIENTS

The authors report the isolation of acquired immunodeficiency syndrome (AIDS) associated retroviruses from packed leukocytes of 2 chimpanzees inoculated intracerebrally and intravenously with brain tissue suspension from 2 patients with AIDS encephalopathy on days 7 and 14 after inoculation. Antibody to the AIDS-associated retroviruses has appeared in sera of 2 chimpanzees inoculated intravenously with plasma from different AIDS patients in 1 chimpanzee inoculated intravenously with brain and thymus suspension and in 1 chimpanzee inoculated intracerebrally with brain tissue suspension from a patient with AIDS encephalopathy. 11 chimpanzees inoculated with supernatant fluids from tissue cultures infected with human T-lymphotropic virus type III (HTLV-III) lymphadenopathy-associated virus (LAV) and IDAV have acquired antibodies to the LAV/HTLV-III viral antigens 2-8 weeks after inoculation. Virus was recovered from the lymphocytes of all 6 seroconverted animals between 8-154 days after primary inoculation of HTLV-III. 2 animals have severe suppression of T-cell function. However all 23 chimpanzees that have seroconverted to HTLV-III or LAV antigen have remained clinically well for 2-15 months of follow-up. There have been no tumors lymphadenopathy or severe opportunistuc infections. Other species of non-human primates similarly inoculated with HTLV-III and LAV have not seroconverted 2-10 months postinoculation. These findings confirm the active and persistent virus infection of chimpanzees with retroviruses derived from AIDS patients. They further establish the presence of viruses in the plasma and brain of AIDS patients by direct transmission of their virus to chimpanzees.

DIFFERENTIAL IN VITRO ANTI-HIV ACTIVITY OF NATURAL LIGNANS

Abstract Two naturally occurring lignanolides, isolated from the tropical climbing shrub Ipomoea cairica, (-)-arctigen in and (-)-trachelogen in , were found to inhibit strongly replication of human immunodeficiency virus type 1 (HIV-1; strain HTLV-III B) in vitro. At a concentration of 0.5 (μм , (-)-arctigenin and (-)-trachelogenin inhibited the expression of HIV-1 proteins p 17 and p24 by 80 -90 % and 60 -70 % , respectively. The reverse transcriptase activity in the culture fluids was reduced by 80 -90 % when the cells (HTLV-III B/H 9) were cultivated in the presence of 0.5 μм (-)-arctigen in or 1 μм (-)-trachelogenin . At the same concentrations, the formation of syncytia in the HTLV-III B/H 9-Jurkat cell system was inhibited by the compounds by more than 80%. A series of other lignan type compounds displayed no anti-HIV activity. Studying the molecular mechanism of action of (-)-arctigenin and (-)-trachelogenin we found that both compounds are efficient inhibitors of the nuclear matrix-associated DNA topoisomerase II activity, particularly of the enzyme from HIV -1-infected cells. Our results suggest that both compounds prevent the increase of topoisomerase II activity, involved in virus replication, after infection of cells with HIV -1.

Preclinical and clinical studies on immunogenicity and safety of the HIV-1 p17-based synthetic peptide AIDS vaccine--HGP-30-KLH.

Immunization with a synthetic HIV-1 p17 peptide analog (HGP-30; aa 85-115 of HIV p17), coupled to a carrier protein (KLH, keyhole limpet hemocyanin) given with alum as the adjuvant induces antibodies which cross-react with both HGP-30 and HIV p17 and clones of cytotoxic and helper T-cells which recognize HGP-30 and HIV p17. Proliferation of lymphocytes in response to HGP-30 has been observed in mice, in HIV-infected individuals and in healthy HIV-seronegative volunteers vaccinated with the p17-based synthetic peptide construct. Cytotoxic T-cell responses against EBV transformed, recombinant p17 pulsed targets were observed using antigen-expanded PBLs from HGP-30-KLH immunized individuals. These results are consistent with predictions that the HGP-30 domain of HIV p17 contains both T- and B-cell epitopes that are recognized by animals and humans. In preclinical toxicology studies in animals and in initial clinical trials in humans the synthetic peptide construct (HGP-30-KLH/alum) has been shown to be safe. This paper summarizes the preclinical immunogenicity and safety data for HGP-30-KLH and presents the initial results from the first Phase 1 clinical trial.

Flt3 ligand enhances the immunogenicity of a gag-based HIV-1 vaccine

Liposomes and Flt3 ligand (Flt3L), a ligand for the fms-like tyrosine kinase receptor Flt3/ FLK2, can augment the immune response to an HIV peptide vaccine. The HGP-30 peptide used in these studies is a synthetic peptide that corresponds to a highly conserved region of HIV-1 p17 gag (amino acids 86-115). Mice were immunized with HGP-30 or HGP-30 conjugated to keyhole limpet hemocyanin (KLH) and delayed-type hypersensitivity (DTH) responses, antibody (IgG) amount and antigen-specific proliferative responses by spleen cells were used to monitor the immune response. Daily injections of Flt3L prior to HGP-30 administration enhanced significantly an antigen-specific lymphocyte proliferation response when compared with Flt3L, HGP-30 alone or HGP-30 containing liposomes. Intravenous administration of HGP-30 was superior to intramuscular (i.m.) immunization for the induction of DTH responses. The HGP-30/KLH containing liposomes enhanced both DTH and antibody responses, while liposomes containing HGP-30 peptide elicited only T cell responses. In these studies, either Flt3L or liposomes increased DTH responses compared with the i.m. injection of the HGP-30 vaccine alone.

Synthesis and anti-HIV activity of oligoribonucleotides and their phosphorothioate analogs.

In previous reports, we demonstrated that replication of human immunodeficiency virus (HIV) could be inhibited by unmodified phosphodiester oligodeoxynucleotides complementary to HIV RNA.’x2 However, a relatively short half-life of unmodified oligodeoxynucleotides in serum and in cells, due to the presence of nucleases, limits their potential usefulness in vivo. To overcome this limitation, we and others have studied internucleotide phosphate backbone modified oligonucleotides, such as methyl phosphonate~,~.~ phosphor amid ate^,^ and pho~phorothioates.~-~ Several oligonucleotide conjugates such as oligonucleotide-cholesteryl conjugateslO and oligonucleotide-polylysine conjugates” have also been studied. These analogs of oligodeoxyribonucleotides are resistant to nucleases and have generally increased hydrophobicity, which provides longer survival time in vivo and may increase cell membrane permeability. Oligodeoxynucleotide phosphorothioates are effective in inhibiting HIV replication in tissue culture (ICso) at a concentration of approximately 1 x M.9 The anti-HIV activity of oligodeoxyribonucleotide phosphorothioate is found to be “relatively sequence independent” against freshly infected cells, but bear in mind that it is difficult for a computer to define a completely random 20-mer sequence against HIV and one that does not activate RNase H. There is, however, striking sequence-specific activity against HIV chronically infected cell^.^.^ The cholesteryl conjugates of oligodeoxyribonucleotide phosphorothioates were found to be more effective than their unconjugated counterparts, but cholesterol conjugation increases nonsequence-specific activity as well.]” The inhibition of HIV replication by an oligodeoxyribonucleotide phosphorothioate is length dependent, a 25 mer being more active than 15-20 mers.I2 An increase in length to 30 mer or 35 mer had little or no enhanced effect on anti-HIV activity over that of the 25 mer. Our continued interest in developing antisense oligodeoxynucleotides and their various analogs as anti-HIV agents has stimulated us to investigate oligoribonucleotides and their phosphorothioate analogs as anti-HIV agents.

Antisense oligonucleotides: gene regulation and chemotherapy of AIDS

Abstract The use of antisense oligonucleotides in control of gene regulation and inhibition of virus replication is a unique approach designed to interfere with cell and virus replication, transcription and translation machinery at the molecular level. Extensive studies ave been carried out to regulate replication of human immunodeficiency virus by synthetic oligonucleotides and their several sugar phosphate backbone modified analogues. Thus far, studies have been carried out in tissue culture, and inhibition of HIV expression has been monitored by several parameters. Oligonucleotide phosphorothioates are effective in inhibiting HIV expression in tissue culture at a concentration of 1·10 −7 M, which translates into a dose of approximately 0.6 mg/kg bodyweight. A non-toxic dose of 100 mg/kg, in preliminary acute toxicity experiments, represents an exploitable therapeutic window for ‘oligonucleotide therapy’.

Phosphoramidate, Phosphorothioate, and Methylphosphonate Analogs of Oligodeoxynucleotide : Inhibitors of Replication of Human Immunodeficiency Virus

Abstract Modified oligodeoxynucleotides complementary to RNA of human immunodeficiency virus (HIV-1) were tested for their ability to inhibit virally induced syncytium formation and expression of viral p24 protein. The modification of oligomers include replacement of phophodiester backbone with phosphorothioate, methylphosphonate and various phosphoramidates. Cells infected for four days, then treated with the antisense oligomers also showed inhibition of viral expression.

Inhibition of HIV replication by naphthalenemonosulfonic acid derivatives and a bis naphthalenedisulfonic acid compound

Several naphthalenemonosulfonic acid analogs and a bis naphthalenedisulfonic acid have been evaluated for anti-HIV activity in assays using H9 and MOLT-3 cells. Among the naphthalenemonosulfonic acids, a 4-amino-5-hydroxy compound and a 4,5-diamino compound showed low anti-HIV activity (upto 50% inhibition) at non-toxic doses. The bis naphthalenedisulfonic acid compound demonstrated significant suppression of HIV-1 antigen expression as measured by monoclonal antibodies to p17 (95%), p24 (94%) and syncytia inhibition (82%) at a dose of 20 micrograms/ml that was non-toxic to the host cells. The bis naphthalenedisulfonic acid analog represents a new class of compounds which may be effective in the treatment of HIV infected patients. The structure activity relationship and a probable mode of action of these compounds is discussed.

Booster immunization of HIV-1 negative volunteers with HGP-30 vaccine induces protection against HIV-1 virus challenge in SCID mice.

Eleven HIV-1 seronegative subjects previously injected with an HIV-1 p17 synthetic peptide vaccine (HGP-30) were given two booster immunizations to evaluate memory cell responses and the ability to boost cellular and humoral immune responses. Five of 11 subjects showed a significant increase in their antibody titres to HGP-30 or p17 and 6/11 had T-cell proliferation responses to either HGP-30 or p17. HIV-1 virus challenge studies in SCID mice demonstrated that 39 of 50 mice (78%) receiving PBMC from 5 of the HGP-30 immunized subjects were protected from infection with a different strain of HIV-1 compared to 4 of 30 mice (13%) that received PBMC from 3 non-immunized subjects (p < 0.001). These studies show that booster immunizations with HGP-30 vaccine are safe and non-toxic and induce protective cell mediated immune responses.

Thymic Hormones in the Treatment of Aids and Other Infectious Diseases

The role of the thymus in the modulation of T-cell function and in the release of various hormonelike factors (thymic hormones) has been the subject of major studies in a number of laboratories (Bach, 1983; Goldstein, 1993; Goldstein et al., 1982; Goldstein and White, 1971; Miller, 1961; Oates and Goldstein, 1991; Schulof et al., 1986,1988; Stutman, 1983; Trainin et al., 1979). It is well known that the thymus undergoes a gradual age-dependent involution during which the thymic parenchymal tissue is infiltrated with fat and adipose cells (Hammar, 1971). The thymus reaches its maximum size just before puberty and then gradually decreases in size and weight. The loss of hormone-producing epithelial cells begins early in life and by the second decade in humans, there is a substantial decrease in the number of hormone-containing medullary thymic epithelial cells. The number of hormone-containing thymic cortical epithelial cells also gradually decreases, although these cells can still be observed in the fifth decade of life (Hirokawa et al., 1982). The age-associated decrease in thymic hormone-like activity correlates with the decrease in hormone-containing thymic epithelial cells in both humans (Bach and Dardenne, 1972; Iwata et al., 1981; Lewis et al., 1978; Twomey et al., 1979; Wara and Amman, 1976) and animals (Dardenne et al., 1974; Hammar, 1971; Savino et al., 1983).