Vaccine-induced protection of chimpanzees against infection by a heterologous human immunodeficiency virus type 1.

The extraordinary genetic diversity of human immunodeficiency virus type 1 (HIV-1) is a major problem to overcome in the development of an effective vaccine. In the most reliable animal model of HIV-1 infection, chimpanzees were immunized with various combinations of HIV-1 antigens, which were derived primarily from the surface glycoprotein, gp160, of HIV-1 strains LAI and MN. The immunogens also included a live recombinant canarypox virus expressing a gp160-MN protein. In one experiment, two chimpanzees were immunized multiple times; one animal received antigens derived only from HIV-1LAI, and the second animal received antigens from both HIV-1LAI and HIV-1MN. In another experiment, four chimpanzees were immunized in parallel a total of five times over 18 months; two animals received purified gp160 and V3-MN peptides, whereas the other two animals received the recombinant canarypox virus and gp160. At 3 months after the final booster, all immunized and naive control chimpanzees were challenged by intravenous inoculation of HIV-1SF2; therefore, the study represented an intrasubtype B heterologous virus challenge. Virologic and serologic follow-up showed that the controls and the two chimpanzees immunized with the live recombinant canarypox virus became infected, whereas the other animals that were immunized with gp160 and V3-MN peptides were protected from infection. Evaluation of both cellular and humoral HIV-specific immune responses at the time of infectious HIV-1 challenge identified the following as possible correlates of protection: antibody titers to the V3 loop of MN and neutralizing antibody titers to HIV-1MN or HIV-1LAI, but not to HIV-1SF2. The results of this study indicate that vaccine-mediated protection against intravenous infection with heterologous HIV-1 strains of the same subtype is possible with some immunogens.     

Rapid CD4(+) T-cell loss induced by human immunodeficiency virus type 1(NC) in uninfected and previously infected chimpanzees

To investigate the pathogenicity of a virus originating in a chimpanzee with AIDS (C499), two chimpanzees were inoculated with a plasma-derived isolate termed human immunodeficiency virus type 1(NC) (HIV-1(NC)). A previously uninfected chimpanzee, C534, experienced rapid peripheral CD4(+) T-cell loss to fewer than 26 cells/microl by 14 weeks after infection. CD4(+) T-cell depletion was associated with high plasma HIV-1 loads but a low virus burden in the peripheral lymph node. The second chimpanzee, C459, infected 13 years previously with HIV-1(LAV), experienced a more protracted course of peripheral CD4(+) T-cell loss after HIV-1(NC) inoculation, resulting in fewer than 200 cells/microl by 96 weeks postinoculation. The quantities of viral RNA in the plasma and peripheral lymph node from C459 were below the lower limits of detection prior to inoculation with HIV-1(NC) but were significantly and persistently increased after superinfection, with HIV-1(NC) representing the predominant viral genotype. These results show that viruses derived from C499 are more pathogenic for chimpanzees than any other HIV-1 isolates described to date.     

Development of AIDS in a chimpanzee infected with human immunodeficiency virus type 1.

The condition of a chimpanzee (C499) infected with three different isolates of human immunodeficiency virus type 1 (HIV-1) for over 10 years progressed to AIDS. Disease development in this animal was characterized by (i) a decline in CD4+ cells over the last 3 years; (ii) an increase in viral loads in plasma; (iii) the presence of a virus, termed HIV-1JC, which is cytopathic for chimpanzee peripheral blood mononuclear cells; and (iv) the presence of an opportunistic infection and blood dyscrasias. Genetic analysis of the V1-V2 region of the envelope gene of HIV-1JC showed that the virus present in C499 was significantly divergent from all inoculating viruses (> or = 16% divergent at the amino acid level) and was suggestive of a large quasispecies. Blood from C499 transfused into an uninfected chimpanzee (C455) induced a rapid and sustained CD4+-cell decline in the latter animal, concomitant with high plasma viral loads. These results show that HIV-1 can induce AIDS in chimpanzees and suggest that long-term passage of HIV-1 in chimpanzees can result in the development of a more pathogenic virus.     

Lack of transmission of human immunodeficiency virus from infected to uninfected chimpanzees

Four uninfected chimpanzees were each housed in separate cages with an HIV-infected chimpanzee for six to twenty-nine months. Despite close daily contact, all uninoculated chimpanzees remained seronegative for HIV, and virus was never isolated from peripheral blood mononuclear cells of the uninfected chimpanzees. These data indicate that the probability of transmission from infected animals to humans is extremely low and also provide supportive evidence for lack of transmission of HIV by casual contact.     

Virus-specific cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected chimpanzees.

Chimpanzees have been important in studies of human immunodeficiency virus type 1 (HIV-1) pathogenesis and in evaluation of HIV-1 candidate vaccines. However, little information is available about HIV-1-specific cytotoxic T lymphocytes (CTL) in these animals. In the present study, in vitro stimulation of peripheral blood mononuclear cells (PBMC) from infected chimpanzees with HIV-1 Gag peptides was shown to be a sensitive, reproducible method of expanding HIV-1-specific CD8(+) effector CTL. Of interest, PBMC from two chimpanzees had CTL activity against Gag epitopes also recognized by major histocompatibility complex class I-restricted CTL from HIV-1-infected humans. The use of peptide stimulation will help to clarify the role of CTL in vaccine-mediated protection and HIV-1 disease progression in this important animal model.     

Antibody-mediated in vitro neutralization of human immunodeficiency virus type 1 abolishes infectivity for chimpanzees.

This study was undertaken to establish whether antibody directed against the human immunodeficiency virus type 1 (HIV-1) principal gp120 type-specific neutralization determinant can abolish the infectivity of HIV-1 in chimpanzees. Challenge inocula of the IIIb virus isolate were mixed in vitro with either immunoglobulin G (IgG) from an uninfected chimpanzee, nonneutralizing IgG from an HIV-seropositive human, a virus-neutralizing murine monoclonal antibody directed against the HIV-1 IIIb isolate, or virus-neutralizing IgG from a chimpanzee infected with the IIIb isolate. Both neutralizing antibodies were directed against the principal neutralization determinant of the challenge isolate. Establishment of infection following inoculation of each virus-antibody mixture into chimpanzees was assessed by virus-specific antibody development and by virus isolation. No protective effect was noted either with the control IgG or with the nonneutralizing anti-HIV IgG. By contrast, the polyclonal chimpanzee virus-neutralizing IgG prevented HIV-1 in vivo infection, while the neutralizing monoclonal antibody notably decreased the infectivity of the challenge virus. Hence, antibody to the gp120 principal neutralization determinant is able both to prevent HIV-1 infection in vitro and to inhibit infection in vivo.     

Prevalence of GB virus type C in urban Americans infected with human immunodeficiency virus type 1

Human T-lymphotropic virus type 1 (HTLV-1) and HTLV-2 were among the first human retroviruses discovered in the early 1980's. The International Retrovirology Association is an organized effort that fostered the efforts of scientists and clinicians to form interdisciplinary groups to study this group of retroviruses and their related diseases. The Association promotes excellent science, patient education, and fosters the training of young scientists to promote "bench-to-bedside" research. The International Conference on Human Retrovirology: HTLV and Related Viruses sponsored by the Association supports clinicians and researchers in the exchange of research findings and stimulation of new research directions. This years conference will be held from June 22 to 25, in Montego Bay, Jamaica http://www.htlvconference.org.jm/. Since its inception in 1988, these conferences have provided a highly interactive forum for the global community of HTLV scientists. This is of particular importance as HTLV research enters its third decade and a new generation of scientists takes over this important work. Many of the scientists attending the meeting will be from developing countries where HTLV is endemic, consistent with the history of international collaborations that have characterized HTLV research. The International Conference on Human Retrovirology provides a unique opportunity for researchers of all disciplines interested in HTLV infections to meet their peers and to address the questions facing clinicians and scientists who study retroviruses, like HTLV.     

Variability of the env gene in cynomolgus macaques persistently infected with human immunodeficiency virus type 2 strain ben.

The sequence variability of distinct regions of the proviral env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben) isolated sequentially over 3 to 4 years from six experimentally infected macaques was studied. The regions investigated were homologous to the V1, V2, V3, V4, V5, and V7 hypervariable regions identified in the env genes of HIV-1 and simian immunodeficiency virus SIVmac, respectively. In contrast to findings with HIV-1 and SIVmac, the V1- and V2-homologous regions were found to be highly conserved during the course of the HIV-2ben infection in macaques. The V3-homologous region showed a degree of variation comparable to that of HIV-1 but not of SIV. In the V4-, V5-, and V7-homologous regions, mutation hot spots were detected in most reisolates of the infected monkeys. Most of these mutations occurred during the first 10 weeks after infection. After 50 weeks, new mutations were rarely detected. At most mutation sites, a dynamic equilibrium between the mutated viral isotype and the infecting predominant wild type was present. This equilibrium might prevent an accumulation of mutations in isolates later in the course of infection.     

Evolution of human immunodeficiency virus type 1 in perinatally infected infants with rapid and slow progression to disease.

We addressed the relationship between the origin and evolution of human immunodeficiency virus type 1 (HIV-1) variants and disease outcome in perinatally infected infants by studying the V3 regions of viral variants in samples obtained from five transmitting mothers at delivery and obtained sequentially over the first year of life from their infected infants, two of whom (rapid progressors) rapidly progressed to having AIDS. Phylogenetic analyses disclosed that the V3 sequences from each mother-infant pair clustered together and were clearly distinct from those of the other pairs. Within each pair, the child's sequences formed a monophyletic group, indicating that a single variant initiated the infection in both rapid and slow progressors. Plasma HIV-1 RNA levels increased in all five infants during their first months of life and then declined within the first semester of life only in the three slow progressors. V3 variability increased over time in all infants, but no differences in the pattern of V3 evolution in terms of potential viral phenotype were observed. The numbers of synonymous and nonsynonymous substitutions varied during the first semester of life regardless of viral load, CD4+-cell count, and disease progression. Conversely, during the second semester of life the rate of nonsynonymous substitutions was higher than that of synonymous substitutions in the slow progressors but not in the rapid progressors, thus suggesting a stronger host selective pressure in the former. In view of the proposal that V3 genetic evolution is driven mainly by host immune constraints, these findings suggest that while the immune response to V3 might contribute to regulating viral levels after the first semester of life, it is unlikely to play a determinant role in the initial viral decline soon after birth.     

Novel human endogenous sequences related to human immunodeficiency virus type 1.

Endogenous retrovirus-related sequences exist within the normal genomic DNA of all eukaryotes, and these endogenous sequences have been shown to be important to the nature and biology of related exogenous retroviruses and may also play a role in cellular functions. To date, no endogenous sequences related to human immunodeficiency virus type 1 (HIV-1) have been reported. Herein we describe the first report of the presence of nucleotide sequences related to HIV-1 in human, chimpanzee, and rhesus monkey DNAs from normal uninfected individuals. We also present the isolation and characterization of two of these endogenous HIV-1-related sequences, EHS-1 and EHS-2. With use of low-stringency Southern blot hybridization, complex banding patterns were detected in human DNA with 5' and 3' HIV-1-derived probes. When an HIV-1 env region probe was used, we detected a less complex, conserved banding pattern in human DNA as well as a related but distinct banding pattern in chimpanzee and rhesus monkey DNAs. EHS-1 and -2 were cloned from normal human genomic DNA libraries by using the env region probe. Clone EHS-1 shows sequence similarity with the domain of the envelope cellular protease cleavage site of HIV-1, while EHS-2 has sequence similarity to the overlapping reading frame for Rev and gp41. Stringent hybridization of EHS-1 back to primate genomic DNA indicates two distinct EHS-1 loci in normal human DNA, an identical band pattern in chimpanzee DNA, and a single locus in rhesus monkey DNA. Likewise, EHS-2 is present as a single highly conserved locus in all three species. An oligonucleotide derived from EHS-2 across a region of near identity to HIV-1 detects a complex banding pattern in all primates tested similar to that seen with the 3' HIV-1 probe. These data suggest that most of the HIV-1-related sequences identified in primate DNA share a common core of nucleic acid sequence found in both EHS-2 and rev and that some of these HIV-1-related sequences have additional larger regions of sequence similarity to HIV-1.     

Evaluation of cytotoxic T-lymphocyte responses in human and nonhuman primate subjects infected with human immunodeficiency virus type 1 or simian/human immunodeficiency virus

Cytotoxic T-lymphocyte (CTL) responses have been implicated as playing an important role in control of human immunodeficiency virus (HIV) infection. However, it is technically difficult to demonstrate CTL responses consistently in nonhuman primate and human subjects using traditional cytotoxicity assay methods. In this study, we systematically evaluated culture conditions that may affect the proliferation and expansion of CTL effector cells and presented a sensitive method for detection of cytotoxicity responses with bulk CTL cultures. We confirmed the sensitivity and specificity of this method by demonstration of vigorous CTL responses in a simian-HIV (SHIV)-infected rhesus macaque. The expansion of epitope-specific CTL effector cells was also measured quantitatively by CTL epitope-major histocompatibility complex tetramer complex staining. In addition, two new T-cell determinants in the SIV gag region are identified. Last, we showed the utility of this method for studying CTL responses in chimpanzee and human subjects.     

Expression and processing of human immunodeficiency virus type 1 gag and pol genes by cells infected with a recombinant vaccinia virus.

Human cells infected with a recombinant vaccinia virus containing human immunodeficiency virus type 1 gag-pol genes produced large amounts of human immunodeficiency virus gag proteins beginning at 1 h and peaking at 48 h postinfection. We show that these polyproteins are processed accurately into mature forms and that the viral polymerase gene is encoded as a 160-kilodalton gag-pol fusion protein, most likely by translational frameshifting from the gag into the pol reading frame.     

Cross-reactive T-helper responses in patients infected with different subtypes of human immunodeficiency virus type 1

Immunization with a recombinant glycoprotein 160 envelope immunogen derived from a virus of genetic subtype B induced strong specific T-helper cell responses in asymptomatic human immunodeficiency virus (HIV) carriers infected with subtypes B to G. This indicates that the HIV-specific T-helper immunity, which is the basis for development of antibodies and cytotoxic T lymphocytes, can be improved by both homologous and heterologous antigens. It also suggests that a particular immunogen can be effective against many different HIV strains.     

Phenotypically Vif- human immunodeficiency virus type 1 is produced by chronically infected restrictive cells.

The permissivity of CD4+ transformed T cells for the replication of human immunodeficiency virus type 1 (HIV-1) vif mutants varies widely between different cell lines. Mutant vif-negative viruses propagate normally in permissive CD4+ cell lines but are unable to establish a productive infection in restrictive cell lines such as H9. As a consequence, elucidation of the function of Vif has been considerably hampered by the inherent difficulty in obtaining a stable source of authentically replication-defective vif-negative viral particles produced by restrictive cells. vif-negative, vpr-negative HIV-1 strain NDK stock, produced by the permissive SupT1 cell line, was used to infect restrictive H9 cells. By using a high multiplicity, infection of H9 cells was achieved, leading to persistent production of viral particles displaying a dramatically reduced infectious virus titer when measured in a single-cycle infectivity assay. Although these viral particles were unable to further propagate in H9 cells, they could replicate normally in CEM and SupT1 cells. Comparison of unprocessed and processed Gag proteins in the persistently produced vif-negative viral particles revealed no defect in the processing of polypeptide precursors, with no inversion of the Pr55gag/p24 ratio. In addition, there was no defect in Env incorporation for the vif-negative viral particles. Despite their apparently normal protein content, these particles were morphologically abnormal when examined by transmission electron microscopy, displaying a previously described abnormally condensed nucleoid. Chronically infected restrictive cell lines producing stable levels of phenotypically vif-negative HIV-1 particles could prove particularly useful in further studies on the function of Vif in the virus life cycle.     

Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins through a heterologous RNA binding site.

The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins induce cytoplasmic expression of incompletely spliced viral mRNAs by binding to these mRNAs in the nucleus. Each protein binds a specific cis-acting element in its target RNAs. Both proteins also associated with nucleoli, but the significance of this association is uncertain because mutations that inactivate nucleolar localization signals in Rev or Rex also prevent RNA binding. Here we demonstrate that Rev and Rex can function when tethered to a heterologous RNA binding site by a bacteriophage protein. Under these conditions, cytoplasmic accumulation of unspliced RNA occurs without the viral response elements, mutations in the RNA binding domain of Rev do not inhibit function, and nucleolar localization can be shown to be unnecessary for the biological response.     

Resistance of chimpanzee T cells to human immunodeficiency virus type 1 Tat-enhanced oxidative stress and apoptosis.

CD4+ T-cell depletion in AIDS patients involves induction of apoptosis in human immunodeficiency virus (HIV)-infected and noninfected T cells. The HIV type 1 (HIV-1)-transactivating protein Tat enhances apoptosis and activation-induced cell death (AICD) of human T cells. This effect is mediated by the CD95 (APO-1/Fas) receptor-CD95 ligand (CD95L) system and may be linked to the induction of oxidative stress by Tat. Here we show that HIV-1 Tat-induced oxidative stress is necessary for sensitized AICD in T cells caused by CD95L expression. Tat-enhanced apoptosis and CD95L expression in T cells are inhibited by neutralizing anti-Tat antibodies, antioxidants, and the Tat inhibitor Ro24-7429. Chimpanzees infected with HIV-1 show viral replication resembling early infection in humans but do not show T-cell depletion or progression towards AIDS. The cause for this discrepancy is unknown. Here we show that unlike Tat-treated T cells in humans, Tat-treated chimpanzee T cells do not show downregulation of manganese superoxide dismutase or signs of oxidative stress. Chimpanzee T cells are also resistant to Tat-enhanced apoptosis, AICD, and CD95L upregulation.     

Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr.

We investigated the fate of human immunodeficiency virus type 1 (HIV-1) viral DNA in infected peripheral blood lymphocytes and immortalized T-cell lines by using a replication-defective HIV-1. We observed that integrated HIV-1 DNA and viral gene expression decrease over time. A frameshift mutation in vpr resulted in maintenance of the HIV-1 provirus and stable persistence of viral expression. Transfection of vpr together with the neomycin resistance gene in the absence of other viral genes decreased the formation of geneticin-resistant colonies, indicating either a cytotoxic or a cytostatic effect upon cells. Therefore, maintenance of HIV-1 infection within an infected proliferating population is due to two competing processes, the rate of viral spread and the degree of cell growth inhibition and/or death induced by Vpr.     

Persistent infection of chimpanzees with human T-lymphotropic virus type III/lymphadenopathy-associated virus: a potential model for acquired immunodeficiency syndrome.

The lymphadenopathy-associated virus (LAV) prototype strain of human T-lymphotropic virus type III/LAV was transmitted to juvenile chimpanzees with no prior immunostimulation by (i) intravenous injection of autologous cells infected in vitro, (ii) intravenous injection of cell-free virus, and (iii) transfusion from a previously infected chimpanzee. All five animals that received more than one 50% tissue culture infective dose were persistently infected with LAV or chimpanzee-passaged LAV for up to 18 months. During this time they developed no illnesses, but they exhibited various degrees of inguinal and axillary lymphadenopathy and significant reductions in rates of weight gain. Detailed blood chemistry and hematologic evaluations revealed no consistent abnormalities, with the exception of immunoglobulin G (IgG) hypergammaglobulinemia, which became apparent in one animal 6 months postinfection and continued at more than 1 year postinfection. Transient depressions followed by increases in the numbers of T4 cells to levels greater than normal were observed in all animals after virus inoculation. However, the number of LAV-infected peripheral blood cells decreased with time after infection. Results of enzyme immunoassays showed that all infected animals seroconverted to IgG anti-LAV within 1 month postinfection and that antibody titers remained high throughout the period of observation. In contrast, only three of the five LAV-infected chimpanzees had detectable IgM antibody responses, and these preceded IgG-specific serum antibodies by 1 to 2 weeks. Virus morphologically and serologically identical to LAV was isolated from peripheral blood mononuclear cells of all infected animals at all times tested and from bone marrow cells taken from one animal 8 months after infection. One chimpanzee that was exposed to LAV only by sharing a cage with an infected chimpanzee developed lymphadenopathy and an IgM response to LAV, both of which were transient; however, no persistent IgG antibody response to LAV developed, and no virus was recovered from peripheral blood cells during a year of follow-up. Thus, LAV readily infected chimpanzees following intravenous inoculation and persisted for extended periods despite the presence of high titers of antiviral antibodies. However, the virus was not easily transmitted from infected to uninfected chimpanzees during daily cage contact.