Biomedical subjects

M D Daniel

Publications and source records attributed to M D Daniel.

At least 19 recordsLinked to original sources

PubMed1996

Cytotoxic T-lymphocyte responses to cytomegalovirus in normal and simian immunodeficiency virus-infected rhesus macaques.

Disseminated cytomegalovirus (CMV) infection is a frequent occurrence in human immunodeficiency virus-infected humans and in simian immunodeficiency virus (SIV)-infected rhesus macaques. Rhesus macaques are a suitable animal model with which to study in vivo interactions between CMV and AIDS-associated retroviruses. Since cytotoxic T lymphocytes (CTL) play a major role in control of viral infections, we have characterized CMV-specific CTL responses in SIV-infected and uninfected rhesus macaques. Autologous fibroblasts infected with rhesus CMV were used to stimulate freshly isolated peripheral blood mononuclear cells from CMV-seropositive animals. Following in vitro stimulation, specific CTL activity against CMV-infected autologous fibroblasts was detected in CMV-seropositive but not in CMV-seronegative normal macaques. CMV-specific CTL activity comparable to that in normal animals was also detected in two CMV-seropositive macaques infected with a live attenuated SIV strain (SIVdelta3) and in two of three macaques infected with pathogenic SIV strains. The CMV-specific CTL response was class I major histocompatibility complex restricted and mediated by CD8+ cells. An early CMV protein(s) was the dominant target recognized by bulk CTL, although the pattern of CTL recognition of CMV proteins varied among animals. Analysis of CMV-specific CTL responses in macaques should serve as a valuable model for CMV immunopathogenesis and will facilitate prospective in vivo studies of immune interactions between CMV and SIV in AIDS.

Animals↗

PubMed1995

Identification of a nef allele that causes lymphocyte activation and acute disease in macaque monkeys.

Residues 17 and 18 in nef of SIVmac239 were changed from RQ to YE to create a translated sequence of SRPSGDLYERLLRARGETYGRLLGEVEDGYSQSP from residues 10-43. The YXXL motifs in this context match very well with consensus sequences for SH2 binding domains and are similar to ones present in nef of the acutely lethal pathogen SIVpbj14. The YE variant of SIVmac239, unlike SIVmac239 but like SIVpbj14, replicated well in resting peripheral blood mononuclear cell cultures, caused extensive T lymphocyte activation, and produced an acute disease in rhesus and pigtailed monkeys characterized by severe diarrhea, rash, and extensive lymphoid proliferation in the gastrointestinal tract. The YEnef gene transformed NIH 3T3 cells in culture. Both 239nef and YEnef were found to associate with src in cotransfected COS cells, and both 60 kDa src and 34 kDa nef were phosphorylated at tyrosine in these cells. The extent of tyrosine phosphorylation of 239nef was considerably less than that of YEnef in these assays. These findings identify an important determinant of the SIVpbj14 phenotype, and they provide evidence of a role for nef in signal transduction and cellular activation.

3T3 Cells↗

PubMed1995

A comparative study of higher primate foamy viruses, including a new virus from a gorilla.

Few foamy (spuma) retroviruses have been investigated in molecular detail, despite their previous isolation from several mamalian species, including ten neutralization serotypes from various primates. Here, we have studied a new gorilla foamy virus (SFV-Gg) and investigated its functional and phylogenetic relationship to the human (HFV) and other primate foamy viruses, including that recently described in orangutans (SFV-11). Nucleotide sequencing of PCR products obtained from the R/U5 region of the LTR, gag, and pol genes revealed a close relationship between HFV and three chimpanzee isolates (SFV-6, SFV-7, and SFV-cpz). The SFV-Gg, SFV-11, rhesus macaque (SFV-1), and African green monkey (SFV-3) isolates were more divergent. To explore functional relationships, primate foamy virus transactivation of HFV LTR driven beta-galactosidase expression in a newly constructed cell line, BHLL, was investigated. HFV, SFV-6, and SFV-7 potently transactivated HFV LTR driven lacZ gene expression, SFV-Gg induced expression approximately 10-fold less efficiently, and SFV types 1, 2, 3, and 11 did not significantly transactivate the HFV LTR. It was, thus, possible to assay serum neutralizing activity in SFV-infected primates against HFV, SFV-6, and SFV-7 by reduction of beta-galactosidase activity following infection of the indicator cell line. Sera from infected chimpanzees and gorillas neutralized, to varying degrees, each of these three viruses, whereas orangutan sera did not. Our results, based on DNA sequences and functional assays, support the conclusion that HFV is closely related to foamy viruses of chimpanzee origin.

Animals↗

PubMed1995

Progression to AIDS in the absence of a gene for vpr or vpx.

Rhesus monkeys (Macaca mulatta) were experimentally infected with strains of simian immunodeficiency virus (SIV) derived from SIVmac239 lacking vpr, vpx, or both vpr and vpx genes. These auxiliary genes are not required for virus replication in cultured cells but are consistently conserved within the SIVmac/human immunodeficiency virus type 2/SIVsm group of primate lentiviruses. All four rhesus monkeys infected with the vpr deletion mutant showed an early spike in plasma antigenemia, maintained high virus burdens, exhibited declines in CD4+ lymphocyte concentrations, and had significant changes in lymph node morphology, and two have died to date with AIDS. The behavior of the vpr deletion mutant was indistinguishable from that of the parental, wild-type virus. Rhesus monkeys infected with the vpx deletion mutant showed lower levels of plasma antigenemia, lower virus burdens, and delayed declines in CD4+ lymphocyte concentrations but nonetheless progressed with AIDS to a terminal stage. The vpr+vpx double mutant was severely attenuated, with much lower virus burdens and no evidence of disease progression. These and other results indicate that vpr provides only a slight facilitating advantage for wild-type SIVmac replication in vivo. Thus, progression to AIDS and death can occur in the absence of a gene for vpr or vpx.

Animals↗

PubMed1994

High-titer immune responses elicited by recombinant vaccinia virus priming and particle boosting are ineffective in preventing virulent SIV infection.

Eighteen rhesus monkeys were vaccinated with recombinant vaccinia viruses expressing SIVmac antigens in 3 separate rounds of experiments. Twelve of the monkeys were primed with a trivalent vaccinia virus recombinant expressing Gag, Pol, and Env polypeptides that can assemble into SIV pseudovirion particles and boosted with SIV particles in adjuvant. Four of the monkeys were primed with different vaccinia virus recombinants expressing env or gag+env followed by SIV particle boosts; two received vaccinia virus recombinants alone (env or env+gag). Despite the induction of vigorous immune responses, 17 of 18 rhesus monkeys became infected on challenge with a low dose of virulent SIVmac. The single protected animal was one of three challenged with homologous cloned SIV exactly matched to the clone used for construction of trivalent vaccinia virus recombinant and particles. Vaccination may have diminished SIV burdens and rates of CD4+ cell declines in some of the animals, but vaccinated/challenge/infected animals eventually developed fatal disease similar to control animals. These results highlight the extreme difficulty in achieving vaccine protection against virulent SIVmac infection even under idealized laboratory conditions.

Animals↗

PubMed1994

Reactivity of primate sera to foamy virus Gag and Bet proteins.

In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M(r) and the cytoplasmic 60K M(r) Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin. This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies. Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.

Animals↗

PubMed1994

The role of upstream U3 sequences in the pathogenesis of simian immunodeficiency virus-induced AIDS in rhesus monkeys.

The nef reading frame overlaps about 70% of the U3 region of the 3' long terminal repeat (LTR) in primate lentiviruses. We investigated the functional role of these overlapping U3 sequences by analyzing the properties of three mutant forms of the pathogenic SIVmac239 clone. In mutant UScon, 90 of 275 bp in the upstream sequences (US) of U3 were changed in a conservative fashion without changing the predicted nef coding sequence. In mutant USnon, 101 of 275 bp in this region were changed in a nonconservative fashion, again without changing the predicted nef coding sequence. In mutant delta US, 275 bp in this region were deleted. Full-size, immunoreactive nef protein was synthesized in cells infected with the UScon and USnon mutants. The USnon and delta US mutants replicated with similar kinetics and to similar extents as wild-type, parental SIVmac239 in primary rhesus monkey peripheral blood mononuclear cell (PBMC) cultures. The UScon mutant replicated with slightly delayed kinetics in rhesus monkey PBMC cultures. In the CEMx174 cell line, the delta US mutant replicated similarly to the wild type, but the UScon and USnon mutants replicated with significantly delayed kinetics. Analysis of LTR-driven chloramphenicol acetyltransferase (CAT) activity and the effects of 5-azacytidine on virus replication suggested that the growth defect of the point mutants in CEMx174 cells was due in whole or in part to the introduction of multiple CG methylation sites in proviral DNA. Rhesus monkeys were experimentally infected with the UScon and USnon mutants, and the characteristics of the infection were compared with those of the parental SIVmac239. Analysis of the levels of plasma antigenemia, virus load, and CD4+ cells in PBMC revealed no decreased virulence of the mutant viruses. Analysis of lymph node biopsies taken from animals that received mutant viruses revealed histologic changes and levels of virus expression indistinguishable from those of the wild type. Furthermore, the wild-type behavior of the mutant viruses in rhesus monkeys occurred without any specific reversional events through at least 20 weeks of infection. These results, and the recent results of Kirchhoff et al. (F. Kirchoff, H. W. Kestler III, and R. C. Desrosiers, J. Virol. 68:2031-2037, 1994), suggest that these upstream sequences in U3 are primarily or exclusively nef coding sequence.

Amino Acid Sequence↗

PubMed1993

Targeted inhibition of immunodeficiency virus replication in lymphocytes through retroviral mediated gene transfer.

To test the feasibility of gene therapy for AIDS patients, an animal model is needed to evaluate the efficacy and safety of this approach. Antiviral genes (encoding antisense RNA or viral protein) derived from Simian immunodeficiency virus (SIV) were efficiently targeted into CD4+ lymphocytes through retroviral-mediated gene transfer. After challenging with infectious viruses, the transduced lymphocytes that received antiviral genes were not only protected from SIV infection, but also from infection with HIV, for at least 25 days. Furthermore, little or no cytolytic effect (syncytium formation) was observed in the protected cells. These data demonstrated that SIV or HIV replication could be effectively blocked by antisense sequence(s) or negative dominant factors which were introduced into targeted cells through retroviral-mediated gene transfer.

Animals↗

PubMed1993

Disseminated B virus infection in a cynomolgus monkey.

A cynomolgus monkey (Macaca fascicularis) was euthanatized 1 week following dystocia because of severe peritonitis. Histologic examination revealed lesions characteristic of herpesvirus infection in lungs, liver, spleen, bone marrow, uterus, and adrenal gland, and on the serosal surface of intestines, pancreas, and reproductive tract. Immunohistochemical studies on liver and lungs revealed Herpes B-like antigens in the lesions. B virus was isolated from serum. As systemic B-virus infection was not diagnosed before death of the monkey, these findings underscore the need for universal precautions when handling blood, fluids, or tissues from macaques.

Animals↗

PubMed1992

Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene.

Vaccine protection against the human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV) in animal models is proving to be a difficult task. The difficulty is due in large part to the persistent, unrelenting nature of HIV and SIV infection once infection is initiated. SIV with a constructed deletion in the auxiliary gene nef replicates poorly in rhesus monkeys and appears to be nonpathogenic in this normally susceptible host. Rhesus monkeys vaccinated with live SIV deleted in nef were completely protected against challenge by intravenous inoculation of live, pathogenic SIV. Deletion of nef or of multiple genetic elements from HIV may provide the means for creating a safe, effective, live attenuated vaccine to protect against acquired immunodeficiency syndrome (AIDS).

Animals↗

PubMed1992

Differences in neutralization of simian lentivirus (SIVMAC) in lymphocyte and macrophage cultures.

Comparison of neutralization of SIVmac251 in primary macrophage cultures with neutralization in lymphocytes (CEM174 cells) showed that neutralizing antibodies induced by SIV251 in infected rhesus macaques protected both macrophages and T lymphocytes against infection when the virus was preincubated with the antibodies. In macrophages, the neutralizing antibodies also protected against infection when added 1 hour after the virus. Addition of antisera to macrophages between 24 and 48 hours after virus inoculation resulted in infection with continuous release of small amounts of p24 into the supernatant fluids but these antibody-treated cultures failed to exhibit cytopathic virus replication. In contrast, the same neutralizing antisera did not protect lymphocytes against infection and subsequent cytopathic replication of the virus when added only 1 hour after virus inoculation. This distinction in the effect that neutralizing antibodies had on the development of cytopathic infection in lymphocytes and macrophages when added after virus inoculation, suggests that they could alter the dynamics of virus replication and therefore the pathogenesis of disease.

Animals↗

PubMed1992

Genetic analysis of simian virus 40 from brains and kidneys of macaque monkeys.

Simian virus 40 (SV40) was isolated from the brains of three rhesus monkeys and the kidneys of two other rhesus monkeys with simian immunodeficiency virus-induced immunodeficiency. A striking feature of these five cases was the tissue specificity of the SV40 replication. SV40 was also isolated from the kidney of a Taiwanese rock macaque with immunodeficiency probably caused by type D retrovirus infection. Multiple full-length clones were derived from all six fresh SV40 isolates, and two separate regions of their genomes were sequenced: the origin (ori)-enhancer region and the coding region for the carboxy terminus of T antigen (T-ag). None of the 23 clones analyzed had two 72-bp enhancer elements as are present in the commonly used laboratory strain 776 of SV40; 22 of these 23 clones were identical in their ori-enhancer sequences, and these had only a single 72-bp enhancer element. We found no evidence for differences in ori-enhancer sequences associated with tissue-specific SV40 replication. The T-ag coding sequence that was analyzed was identical in all clones from kidney. However, significant variation was observed in the carboxy-terminal region of T-ag in SV40 isolated from brain tissues. This sequence variation was located in a region previously reported to be responsible for SV40 host range in cultured cell lines. Thus, SV40 appears to be an opportunistic pathogen in the setting of simian immunodeficiency virus-induced immunodeficiency, similarly to JC virus in human immunodeficiency virus-infected humans, the enhancer sequence organization generally attributed to SV40 is not representative of natural SV40 isolates, and sequence variation near the carboxy terminus of T-ag may play a role in tissue-specific replication of SV40.

Amino Acid Sequence↗

PubMed1991

Importance of the nef gene for maintenance of high virus loads and for development of AIDS.

When rhesus monkeys were infected with a form of cloned SIVmac239 having a premature stop signal at the 93rd codon of nef, revertants with a coding codon at this position quickly and universally came to predominate in the infected animals. This suggests that there are strong selective forces for open functional forms of nef in vivo. Although deletion of nef sequences had no detectable effect on virus replication in cultured cells, deletion of nef sequences dramatically altered the properties of virus in infected rhesus monkeys. Our results indicate that nef is required for maintaining high virus loads during the course of persistent infection in vivo and for full pathologic potential. Thus, nef should become a target for antiviral drug development. Furthermore, the properties of virus with a deletion in nef suggest a means for making live-attenuated strains of virus for experimental vaccine testing.

Animals↗

PubMed1991

Priming with T helper cell epitope peptides enhances the antibody response to the envelope glycoprotein of HIV-1 in primates.

The induction of a memory immune response to HIV, mediated by any kind of effector mechanism, requires the induction of T cell help. In previous studies performed in different murine MHC haplotypes, three immunodominant T cell epitopes (T1, T2, and TH4.1) had been identified in the HIV envelope glycoprotein. Moreover, these peptides were proliferative T cell epitopes in humans. In this study, rhesus monkeys, Macaca mulatta, were primed with these three peptides either in combination or given separately. Half of the monkeys had a proliferative response to one or more of the priming peptide(s). Those monkeys who had a T cell proliferative response also had a high antibody response after one boost with a suboptimal dose of the native protein gp 160, whereas three of four control monkeys who had received only the native protein immunization gave no detectable antibody response, and one displayed a very weak response. For reasons that are unclear, antibodies only to the gp41 portion of gp 160 could be detected in the sera. Thus, the peptides can prime Th cells in primates for an enhanced antibody response on first exposure to the whole protein. The three peptides belong to highly conserved and nonglycosylated regions of the envelope protein. The fact that the peptides acted as immunogenic T cell proliferative and helper epitopes in nonhuman primates is very encouraging for including them in future vaccine studies in humans.

Amino Acid Sequence↗

PubMed1991

Macrophage-tropic variants of SIV are associated with specific AIDS-related lesions but are not essential for the development of AIDS.

The importance of macrophage infection for the development of acquired immune deficiency syndrome (AIDS) was investigated. Molecularly cloned simian immunodeficiency virus (SIV)mac239 replicates very poorly in cultured macrophages yet it causes AIDS in rhesus monkeys. Three of five rhesus monkeys that died with AIDS following SIVmac239 infection showed no disease manifestations directly associated with macrophage infection, such as encephalitis and granulomatous interstitial pneumonia. Simian immunodeficiency virus recovered from the peripheral blood of these three animals at or near the time of death replicated very poorly if at all in cultured macrophages, and tissues taken at autopsy showed little or no infection of macrophages by immunohistochemical staining. However two of the five rhesus monkeys that died with AIDS following SIVmac239 infection displayed a characteristic SIV-related meningoencephalitis and/or granulomatous pneumonia, lesions associated with macrophage infection. Simian immunodeficiency virus recovered from the peripheral blood of these two animals near the time of death replicated extremely well in cultured macrophages, indicating the emergency of macrophage-tropic variants in vivo. Furthermore tissues taken at autopsy from these two showed many infected macrophages by immunohistochemical staining. These results indicate that AIDS and death can occur without obvious involvement of macrophage infection. However the presence of macrophage-tropic viral strains appears to influence the disease course and disease manifestations.

Animals↗

PubMed1991

Cytokine influence on simian immunodeficiency virus replication within primary macrophages. TNF-alpha, but not GMCSF, enhances viral replication on a per-cell basis.

The control of HIV-1 or SIV replication within macrophages is probably influenced by a variety of viral and cellular factors. Of the cellular factors, the authors have studied cytokine influence on SIV replication in vitro utilizing simian alveolar macrophages and uncloned SIVmacMTV, a macrophage-tropic variant. The approach allowed quantification of viral replication on a per-cell basis. As reported for HIV-1 replication in macrophages, TNF-alpha significantly increased SIV production in these macrophage cultures. GMCSF also resulted in marked increases in SIV gag protein in culture supernatants. However, after correcting for differences in total cell numbers and numbers of gag-containing cells in the treated and untreated cultures, GMCSF did not upregulate SIV production on a per-cell basis. IL-6 increased SIV replication little if at all but induced significantly greater cytopathic changes in the treated cultures compared with infected, untreated cultures. In contrast, IFN-gamma greatly decreased replication. Our results for GMCSF, IFN-gamma, and IL-6 are in contrast to previously published reports of cytokine control of HIV-1 growth in target cells, and they stress the importance of cell number analyses and the use of primary cultures in the study of lentiviral replication kinetics in macrophages.

Animals↗

PubMed1990

Complement-mediated, infection-enhancing antibodies in plasma from vaccinated macaques before and after inoculation with live simian immunodeficiency virus.

Rhesus monkeys vaccinated against infection with simian immunodeficiency virus (SIV) were examined, in retrospect, for the presence of infection-enhancing antibodies and possible consequences associated with the presence of these antibodies. At the time of experimental inoculation with live virus, complement-mediated, infection-enhancing antibodies were detected in plasma specimens from six of six animals vaccinated with detergent-inactivated whole virus, from nine of nine animals vaccinated with Formalin-inactivated whole virus, and from seven of eight animals immunized with two SIV subunit preparations. The presence of infection-enhancing antibodies at the time of viral challenge gave no indication of predicting vaccine success or failure. After live-virus challenge, titers of infection-enhancing antibodies, like enzyme-linked immunosorbent assay titers, increased in unprotected animals and decreased or became undetectable in animals protected by vaccination. Thus, vaccine protection against SIV infection can still be achieved in the presence of detectable complement-mediated, infection-enhancing antibodies.

Animals↗

PubMed1990

Study of long-term cultures of simian immunodeficiency virus (SIVmac 251)-infected peripheral blood lymphocytes.

A culture of rhesus monkey peripheral blood lymphocytes was divided into two parts; one was kept as an uninfected control, and the other was infected with a strain of simian immunodeficiency virus (SIVmac251) originally isolated from a rhesus monkey that died of a malignant lymphoma associated with acquired immune deficiency syndrome. Both cultures were sampled at successive intervals from 1 to 40 days postinfection. Each sample was subjected to in situ hybridization for detection of viral mRNA, immunocytochemical detection of viral core protein (p27), reverse transcriptase assay, electron microscopy, and immunophenotypic characterization of infected cells. These techniques were used to define viral growth kinetics of this novel lentivirus in peripheral blood lymphocytes. The first evidence of SIVmac251 replication was obtained by an in situ hybridization signal for viral mRNA at 2 days postinoculation. This was followed by detection of viral p27 core protein by immunocytochemistry on day 4. Reverse transcriptase activity above control values was not detected until day 8. Budding particles were not found in the infected cultures until 14 days postinfection. Results of in situ hybridization, immunocytochemistry, and reverse transcriptase assay indicated that two bursts of viral replication occurred during the course of this study. The first, at 3 weeks postinfection, was due to infection and subsequent depletion of CD4+ lymphocytes, while the second, 3 weeks later, resulted from a cycle of replication in CD8+ lymphocytes and the remaining CD4+ cells, culminating in the death of all cells on day 39 postinoculation.

Animals↗