Biomedical subjects

J Heeney

Publications and source records attributed to J Heeney.

At least 19 recordsLinked to original sources

PubMed2001

Differences in early virus loads with different phenotypic variants of HIV-1 and SIV(cpz) in chimpanzees.

OBJECTIVE: A comparative study of the replication kinetics of different HIV-1 variants (including SIV(cpz)) was undertaken to determine which viral characteristics were associated with sustained plasma viraemia in chimpanzees. DESIGN: Plasma samples from chimpanzees infected with six different HIV-1 clade B isolates were compared with plasma samples from SIV(cpz-ant)-infected chimpanzees. METHODS: A pan-clade quantitative competitive reverse transcriptase-polymerase chain reaction assay was developed based on conserved primer sequences recognizing M, N and O human lentiviruses as well as different SIV(cpz) isolates. RESULTS: Important differences between early kinetics in the human lentivirus isolates as well as compared with the chimpanzee isolate SIV(cpz-ant) were observed. R5-dependent non-syncytium-inducing (NSI) isolates (5016, Ba-L, SIV(cpz)) were found to have relatively higher viral loads than the syncytium-inducing (SI), X4-dependent primary (SF2), T cell-adapted (IIIB) or X4/R5 (Han2, DH12) SI primary isolates. CONCLUSION: Infection of chimpanzees with NSI R5-utilizing isolates correlated with persistent viraemia (approximately 10(4) RNA equivalents/ml) in contrast to transient viraemia observed after infection with SI X4-utilizing isolates.

Animals↗

PubMed2001

Enhanced simian immunodeficiency virus-specific immune responses in macaques induced by priming with recombinant Semliki Forest virus and boosting with modified vaccinia virus Ankara.

The immunogenicity of two vector-based vaccines, either given alone or in a prime-boost regimen, was investigated. Cynomolgus macaques were immunised with modified vaccinia virus Ankara (MVA) expressing simian immunodeficiency virus (SIV)macJ5 env, gag-pol, nef, rev, and tat genes (MVA-SIVmac) or primed with a Semliki forest virus (SFV) vaccine expressing the same genes (SFV-SIVmac) and boosted with MVA-SIVmac. Generally, antibody responses, T-cell proliferative responses and cytotoxic T-cell responses remained low or undetectable in vaccinees receiving MVA-SIVmac or SFV-SIVmac alone. In contrast, monkeys who first received SFV-SIVmac twice and then were boosted with MVA-SIVmac showed increased antibody responses as well as high T-cell proliferative responses. Three of these vaccinees had cytotoxic T-lymphocytes directed against three or four of the gene products. No evidence of protection was seen against an intrarectal heterologous SIVsm challenge given 3 months after the last immunisation. The study demonstrates a prime-boost strategy that efficiently induces both humoral and cellular immune responses.

AIDS Vaccines↗

PubMed2001

An in vivo model for HIV resistance development.

Treatment of human immunodeficiency virus (HIV-1) with drugs targeted to the reverse transcriptase (RT) rapidly selects for drug-resistant virus. It is essential to develop a suitable animal model that allows the study of the emergence and reversal of drug resistance. A monkey model was previously developed on the basis of a hybrid virus (RT-SHIV) of simian immunodeficiency virus (SIV) with its RT exchanged for HIV-1 RT. In the present study cynomolgus monkeys infected with RT-SHIV were treated with varying doses of the non-nucleoside RT inhibitor nevirapine. The drug was administered for 2-3 weeks, in agreement with clinical experience of resistance development during nevirapine monotherapy. This resulted in the selection of mutants with Y181C and K103N changes in RT, which correspond to the HIV-1 mutations in nevirapine-resistant HIV-1 patients. The mutants coexisted at varying levels with wild-type virus and fluctuations in the proportion of mutants could be closely monitored. Low-dose treatment was not more efficient in induction of mutations than a virus-inhibiting dose. Structured therapy interruptions could be performed. The monkey RT-SHIV infection offers an in vivo model to determine effects of therapies on resistance development.

Animals↗

PubMed2001

Mucosal exposure to subinfectious doses of SIV primes gut-associated antibody-secreting cells and T cells: lack of enhancement by nonneutralizing antibody.

It has been suggested that the presence of immunoglobulin and complement receptors on rectal epithelium may facilitate the entry of HIV complexed to nonneutralizing antibody. We tested this hypothesis using simian immunodeficiency virus (SIV) infection of rhesus macaques. First, in a pilot study, a nonneutralizing IgG fraction of macaque anti-SIV gp120 was shown to enhance the immunogenicity of SIV envelope following rectal immunization. The same antibody was then mixed with a subinfectious dose of SIV and the occurrence of rectal infection was compared with virus alone. Animals were not infected overtly and were rechallenged with a 10-fold higher dose of virus with and without addition of antibody. There was no evidence of antibody-mediated infection, since equal numbers of macaques became infected, regardless of the presence of antibody. In addition, the application of immune complexes did not alter significantly the subsequent virus load or the immune responses generated. In seronegative animals, in which virus and proviral DNA were undetectable in PBMC and tissues, SIV-specific T-cell responses and antibody-secreting cells were found in systemic and gut-associated sites. Our results show that nonneutralizing antibody neither facilitated nor enhanced rectal infection with SIV, in the small number of animals used, despite the consistent trend for this antibody to enhance antibody responses to gp120 following rectal immunization with immune-complexed antigen. However, mucosal exposure to subinfectious doses of virus primed both systemic and local immunity, regardless of addition of nonneutralizing antibody.

Animals↗

PubMed2001

Enhanced cellular immune response and reduced CD8(+) lymphocyte apoptosis in acutely SIV-infected Rhesus macaques after short-term antiretroviral treatment.

Losing the decisive virus-specific functions of both CD4(+) and CD8(+) T lymphocytes in the first weeks after immunodeficiency virus infection ultimately leads to AIDS. The SIV/rhesus monkey model for AIDS was used to demonstrate that a 4-week chemotherapeutic reduction of viral load during acute SIV infection of macaques allowed the development of a competent immune response able to control virus replication after discontinuation of treatment in two of five monkeys. Increasing SIV-specific CD4(+) T-helper-cell proliferation was found in all macaques several weeks after treatment, independent of their viral load. However, only macaques with low viral loads showed persistent T-cell reactivity of lymph node cells. In contrast to animals with higher viral loads, T-helper-cell counts and memory T-helper cells did not decline in the two macaques controlling viral replication. Lymphocyte apoptosis was consistently low in all treated macaques. In contrast, high CD8(+) lymphocyte death but only slightly increased CD4(+) lymphocyte apoptosis were observed during the first weeks after infection in untreated control animals, indicating that early apoptotic death of virus-specific CTL could be an important factor for disease development. Antiretroviral treatment early after infection obviously retained virus-specific and competent T lymphocytes, whereby a virus-specific immune response could develop in two animals able to control the viral replication after cessation of treatment.

Adenine↗

PubMed2001

Genetic variability of the V1 and V2 env domains of SIVcpz-ant and neutralization pattern of plasma viruses in a chimpanzee infected naturally.

Specific neutralizing epitope changes have been observed in a chimpanzee infected naturally with SIVcpz, which differ from HIV-1 infecting humans. To characterize further these changes, a longitudinal study of env genomic sequence variation of SIVcpz-ant isolates was undertaken in this animal. The V1 and V2 regions of the env were determined to arise from specific recombination events. To determine whether recombination of the V1 and V2 domains was possibly associated with the emergence of neutralization escape viruses, envelope sequences and gene length polymorphisms from PBMC and plasma viral variants were studied over a 7-year period. PBMCs and plasma-associated infectious virus titers as well as plasma RNA viral loads were monitored longitudinally. The first 5 viruses isolated from the plasma were found to be neutralization escape variants. Sequence analysis of their V1 and the V2 regions indicated that a 20 amino acid stretch of the V1 region had undergone recombination and was also associated with the emergence of isolates eliciting strong neutralization responses. These findings support the hypothesis that recombination of the V1 and V2 regions of the envelope play a role in neutralization escape of SIVcpz in chimpanzees infected naturally. Furthermore, the data confirm that the neutralizing antibody response plays an important role in the decline of plasma infectious virus titers in HIV-1 related SIVcpz nonpathogenic infection.

Amino Acid Sequence↗

PubMed2001

Effect of vaccination with recombinant modified vaccinia virus Ankara expressing structural and regulatory genes of SIV(macJ5) on the kinetics of SIV replication in cynomolgus monkeys.

The efficacy of a multicomponent vaccination with modified vaccinia Ankara constructs (rMVA) expressing structural and regulatory genes of simian immunodeficiency virus (SIV(mac251/32H/J5)) was investigated in cynomolgus monkeys, following challenge with a pathogenic SIV. Vaccination with rMVA-J5 performed at week 0, 12, and 24 induced a moderate proliferative response to whole SIV, a detectable humoral response to all but Nef SIV antigens, and failed to induce neutralizing antibodies. Two months after the last boost, the monkeys were challenged intravenously with 50 MID50 of SIV(mac251). All control monkeys, previously inoculated with non-recombinant MVA, were infected by week two and seroconverted by weeks four to eight. In contrast a sharp increase of both humoral and proliferative responses at two weeks post-challenge was observed in vaccinated monkeys compared to control monkeys. Although all vaccinated monkeys were infected, vaccination with rMVA-J5 appeared to partially control viral replication during the acute and late phase of infection as judged by cell- and plasma-associated viral load.

Animals↗

PubMed2001

Comparison of early plasma RNA loads in different macaque species and the impact of different routes of exposure on SIV/SHIV infection.

Various simian immunodeficiency virus (SIV)sm/mac and simian/human immunodeficiency virus (SHIV) strains are used in different macaque species to study AIDS pathogenesis, as well as to evaluate candidate vaccine and anti-retroviral drugs efficacy. In this study we investigated the effect of route of infection, species of macaques and nature of virus stock on early plasma viral RNA load. We monitored the plasma RNA concentrations of 63 rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) infected with well-characterised virus stocks administered either by oral, rectal, vaginal or intravenous (i.v.) routes. In SIV(mac)-infected macaques, no significant difference in plasma RNA loads was observed between the rectal, oral and i.v. routes of infection. Cynomolgus macaques developed lower steady state SIV plasma RNA concentrations compared with rhesus macaques and no significant difference was observed between rectal and i.v. routes of infection. In SHIV(89.6p)-infected macaques, no difference between species or between route of infection was observed with this particular chimeric virus.

AIDS Vaccines↗

PubMed2001

Longitudinal comparison of virus load parameters and CD8 T-cell suppressive capacity in two SIVcpz-infected chimpanzees.

In a longitudinal study we address the hypothesis that resis tance to disease progression in lentivirus-infected chimpanzees is related to potent non-cytotoxic suppression of virus replication. In a long-term follow-up, the viral suppressive capacity in two simian immunodeficiency virus (SIV)cpz-infected chimpanzees was correlated with two polymerase chain reaction (PCR)- and two culture-based virus load measurements. In both animals, quantitative virus isolation (QVI) tended to decline slowly, whereas in vitro virus suppression was sustained or increased over time. In general, plasma virus loads in SIVcpz-infected animals were maintained for extended periods of time. Based on current assays that measure virus suppressive capacity in peripheral blood, it was not possible to conclude that virus suppression played a major role in the maintenance of the disease-free state in lentivirus-infected chimpanzees.

Animals↗

PubMed2001

Characteristics of a pathogenic molecular clone of an end-stage serum-derived variant of simian immunodeficiency virus (SIV(F359)).

End-stage simian immunodeficiency virus (SIV) isolates are suggested to be the most fit of the evolved virulent variants that precipitate the progression to AIDS. To determine if there were common characteristics of end-stage variants which emerge from accelerated cases of AIDS, a molecular clone was derived directly from serum following in vivo selection of a highly virulent SIV isolate obtained by serial end-stage passage in rhesus monkeys (Macaca mulatta). This dominant variant caused a marked cytopathic effect and replicated to very high levels in activated but not resting peripheral blood lymphocytes. Furthermore, although this clone infected but did not replicate to detectable levels in rhesus monocyte-derived macrophages, these cells were able to transmit infection to autologous T cells upon contact. Interestingly, although at low doses this end-stage variant did not use any of the known coreceptors except CCR5, it was able to infect and replicate in human peripheral blood mononuclear cells homozygous for the Delta 32 deletion of CCR5, suggesting the use of a novel coreceptor. It represents the first pathogenic molecular clone of SIV derived from viral RNA in serum and provides evidence that not only the genetic but also the biological characteristics acquired by highly fit late-stage disease variants may be distinct in different hosts.

Amino Acid Sequence↗

PubMed2000

A strategy for cloning infectious molecular clones of retroviruses from serum or plasma.

To enable biological characterisation of lentiviral variants which emerge during infection and development of AIDS, a method was developed to construct molecular clones from circulating simian immunodeficiency virus (SIV) particles present in as little as 20 microl of serum from infected rhesus monkeys. This technique uses a long distance RT-PCR method optimised for the amplification of partly overlapping 5-kb SIV (half genome) amplimers. Ligation of the genome halves resulted in the construction of full-length clones which, after transfection, were able to replicate well in rhesus peripheral blood mononuclear cells (PBMCs) and in various human T-cell lines inducing syncytia. In addition to the study of molecular cloned virus quasispecies emerging in circulation as a result of immune escape, this method may also be applied to obtain entire genes or full-length molecular clones. These clones may be present in other extracellular body fluids such as urine, saliva, tears, lymph, and bronchial or cerebral spinal fluid. Genes amplified in this way can be inserted quickly in new recombinant expression vectors and may then be applied for DNA vaccination approaches.

AIDS Vaccines↗

PubMed2000

Relationship between viral load in blood, cerebrospinal fluid, brain tissue and isolated microglia with neurological disease in macaques infected with different strains of SIV.

The role of the viral burden in the brain for the pathogenesis of human immunodeficiency virus-associated neurological disorders is still unclear. To address this issue, we have quantified the viral load in plasma, cerebrospinal fluid (CSF) and brain tissue of macaques infected with simian immunodeficiency virus (SIV). We discovered that the viral strain used for infection determines the replicative capacity in microglial cells as well as the extent of neuropathological lesions and the occurrence of neurological symptoms. Moreover, the viral load in the brain parenchyma correlated with the development of overt neurological disease whereas the one in plasma did not. By comparing the viral load in three different compartments, we demonstrated that the viral burden in the CSF is influenced both by the viral replication in the periphery as well as in the brain parenchyma. According to these results, it is not the absolute amount of viral load in the CSF but rather the viral antigen contributed by the viral production within the brain which correlates with the development of neurological disease. In longitudinal studies, we observed that this autochthonous virus production, as evidenced by a ratio of the viral load in CSF to the one in plasma, takes place for a prolonged period of time before overt neurological signs are manifested. This finding suggests that this ratio could be used as a prognostic marker for immunodeficiency virus-induced neurological disease.

AIDS-Related Complex↗

PubMed1999

Induction of inhibitory antibodies to the CCR5 chemokine receptor and their complementary role in preventing SIV infection in macaques.

The seven-transmembrane G-protein-linked CCR5 molecule functions as a major coreceptor for HIV or simian immunodeficiency virus (SIV) infection. Antibodies to CCR5 were studied in rhesus macaques immunized with SIV grown in human CD4(+) T cells. These macaques were completely protected against i.v. challenge with live SIV. Sera from the protected macaques showed significantly greater inhibition of SIV replication (p < 0.001) and macrophage inflammatory protein-1beta-generated CCR5-dependent chemotaxis (p < 0.01) than sera from unprotected macaques, in the absence of significant neutralizing antibodies to SIV. These two functional assays demonstrate serum antibodies to the CCR5 receptors which were specifically inhibited by CCR5-transfected HEK-293 cells. We postulate that anti-CCR5 antibodies may be complementary to beta-chemokines in blocking CCR5 coreceptors to HIV or SIV binding and fusion of CD4(+) cells.

Animals↗

PubMed1999

Differential cytotoxic T-lymphocyte (CTL) responses in HIV-1 immunised sibling chimpanzees with shared MHC haplotypes.

Cell mediated immune responses to HIV-1 and CTL responses in particular differ dramatically in infected individuals. This may largely be influenced by the immunogenetic differences of different individuals such as those encoded by the MHC. These differences may be difficult to dissect due to the immunosuppressive nature of HIV-1 infection itself. In order to reduce the variables associated with effects of the virus, one recombinant viral antigen was chosen from a particular HIV-1 variant (rgp120 of the clinical isolate HIV-1w6.1D). To minimise differences between outbred hosts, we chose two sibling chimpanzees from which the family pedigree and genetic segregation with respect to polymorphic MHC molecules was known. Immunisation induced strong antigen specific antibody and T-helper immune responses. The magnitude and persistence of the humoral and T-helper immune responses were comparable in both chimpanzees. However, CTL responses were only observed in one sibling. These responses were subsequently mapped to several distinct epitopes. The CTL response to the immunodominant epitope was found to be presented in the context of a MHC molecule which was shared by both siblings. The absence of a CTL response in the other sibling is not yet understood, but could not be attributed to MHC alleles that were not shared by these two chimpanzees. These findings suggest that other polymorphic immunoregulatory mechanisms such as those involved in antigen processing and presentation influence host CTL responses to HIV-1.

Animals↗

PubMed1999

HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates.

The specific immune mechanisms necessary and/or sufficient to elicit HIV-vaccine protection remain undefined. Utilising the SHIV rhesus macaque model the immunogenicity as well as the efficacy of ten different HIV-1 vaccine candidates was evaluated. Comparison of the immune responses induced, with the ability of the vaccine to protect from SHIV infection provided a means to determine which type of immune responses were necessary for protection. Vaccine candidates included VLPs, DNA, subunit protein with novel adjuvant formulations, ISCOMs and pox-virus vectors. Protection from SHIV infection was achieved in approximately half of the animals which received a primary intravenous cell-free challenge. The presence of CTL in the absence of other effector responses did not correlate with protection from this route and type of challenge. Virus neutralising antibodies (Nab) appeared to be necessary but alone were insufficient for protection. If Ag-specific IFN-gamma and/or IL-4 as well as lymphoproliferative (LP) responses were found with the lack of a detectable IL-2 response, then protection was not observed. Immunity correlated with the magnitude of Nab responses, beta-chemokines and as well as balanced, qualitative T-helper responses.

AIDS Vaccines↗

PubMed1999

Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine.

Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.

AIDS Vaccines↗

PubMed1998

Evolution of a simian immunodeficiency virus pathogen.

Analysis of disease induction by simian immunodeficiency viruses (SIV) in macaques was initially hampered by a lack of molecularly defined pathogenic strains. The first molecularly cloned SIV strains inoculated into macaques, SIVmacBK28 and SIVmacBK44 (hereafter designated BK28 and BK44, respectively), were cases in point, since they failed to induce disease within 1 year postinoculation in any inoculated animal. Here we report the natural history of infection with BK28 and BK44 in inoculated rhesus macaques and efforts to increase the pathogenicity of BK28 through genetic manipulation and in vivo passage. BK44 infection resulted in no disease in four animals infected for more than 7 years, whereas BK28 induced disease in less than half of animals monitored for up to 7 years. Elongation of the BK28 transmembrane protein (TM) coding sequence truncated by prior passage in human cells marginally increased pathogenicity, with two of four animals dying in the third year and one dying in the seventh year of infection. Modification of the BK28 long terminal repeat to include four consensus nuclear factor SP1 and two consensus NF-kappaB binding sites enhanced early virus replication without augmenting pathogenicity. In contrast, in vivo passage of BK28 from the first animal to die from immunodeficiency disease (1.5 years after infection) resulted in a consistently pathogenic strain and a 50% survival time of about 1.3 years, thus corresponding to one of the most pathogenic SIV strains identified to date. To determine whether the diverse viral quasispecies that evolved during in vivo passage was required for pathogenicity or whether a more virulent virus variant had evolved, we generated a molecular clone composed of the 3' half of the viral genome derived from the in vivo-passaged virus (H824) fused with the 5' half of the BK28 genome. Kinetics of disease induction with this cloned virus (BK28/H824) were similar to those with the in vivo-passaged virus, with four of five animals surviving less than 1.7 years. Thus, evolution of variants with enhanced pathogenicity can account for the increased pathogenicity of this SIV strain. The genetic changes responsible for this virulent transformation included at most 59 point mutations and 3 length-change mutations. The critical mutations were likely to have been multiple and dispersed, including elongation of the TM and Nef coding sequences; changes in RNA splice donor and acceptor sites, TATA box sites, and Sp1 sites; multiple changes in the V2 region of SU, including a consensus neutralization epitope; and five new N-linked glycosylation sites in SU.

Amino Acid Sequence↗

PubMed1998

A pathogenic threshold of virus load defined in simian immunodeficiency virus- or simian-human immunodeficiency virus-infected macaques.

To determine if a specific pathogenic threshold of plasma viral RNA could be defined irrespective of virus strain, RNA levels in the plasma of more than 50 infected rhesus macaques (Macaca mulatta) were measured. Animals were inoculated intravenously with either simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) strains of known pathogenic potential (SIV8980, SIVsmm-3, SIVmac32H/J5, SIVmac32H/1XC, reverse transcriptase-SHIV, SHIV89.6p) or with attenuated strains (SHIVW6.1D, SHIVsf13, SHIVhan-2, SIVmacDeltanef, SHIVsf33). In animals inoculated with nonpathogenic strains, shortly after the primary peak of viremia viral RNA levels declined and remained below 10(4) RNA equivalents/ml of plasma between 6 and 12 weeks postinoculation. Animals infected with documented pathogenic strains maintained viral RNA levels higher than 10(5) RNA equivalents/ml of plasma. In animals infected with strains with low virulence, a decline in plasma RNA levels was observed, but with notable individual variation. Our results demonstrate that the disease-causing potential was predicted and determined by a threshold plasma virus load which remained greater than 10(5) RNA equivalents/ml of plasma 6 to 12 weeks after inoculation. A threshold virus load value which remained below 10(4) RNA equivalents/ml of plasma was indicative of a nonpathogenic course of infection.

Animals↗