Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques.

A substantial risk in using live attenuated, multiply deleted viruses as vaccines against AIDS is their potential to induce AIDS. A mutant of the simian immunodeficiency virus (SIV) with large deletions in nef and vpr and in the negative regulatory element induced AIDS in six of eight infant macaques vaccinated orally or intravenously. Early signs of immune dysfunction were seen in the remaining two offspring. Prolonged follow-up of sixteen vaccinated adult macaques also showed resurgence of chronic viremia in four animals: two of these developed early signs of disease and one died of AIDS. We conclude that this multiply deleted SIV is pathogenic and that human AIDS vaccines built on similar prototypes may cause AIDS.

Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations.

Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

Autosomal trisomy in a chimpanzee: resemblance to Down's syndrome.

An infant chimpanzee (Pan troglodytes) with clinical, behavioral, and cytogenetic features similar to those in Down's syndrome is described. The infant shows retarded growth rate, congenital abnormalities, retarded neurologic and postural development, epicanthus, hyperflexibility of the joints, muscle hypotonia, and trisomy of a small acrocentric chromosome.

The chimpanzee as an animal model for investigating alcoholism.

Young chimpanzees (Pan troglodytes) will accept ethanol in quantities sufficient to produce symptoms of withdrawal when ethanol is subsequently discontinued. Mild to severe symptoms of physical dependence, including grand mal seizures, are observed when ethanol is abruptly withdrawn after 6 to 10 weeks of chronic oral intake. In addition, the rate of disappearance of ethanol in blood increased during periods of chronic ingestion, an indication of developing metabolic tolerance. These results suggest that the young chimpanzee may be a suitable model for experimental studies of alcoholism.

Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine.

Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.

Parallel evolution of CCR5-null phenotypes in humans and in a natural host of simian immunodeficiency virus.

The C-C chemokine receptor CCR5 in humans and rhesus macaques (Macaca mulatta) serves as the primary coreceptor for cellular entry by macrophagetropic strains of human immunodeficiency virus type 1 (HIV-1) and all reported strains of simian immunodeficiency virus (SIV) [1-6]. Humans homozygous for a 32 bp deletion allele of CCR5, resulting in a null phenotype, are highly resistant to infection by HIV-1 [7-9], prompting development of therapies and vaccines targeting CCR5. We now report a novel deletion allele of CCR5, with an allele frequency of 0.04, in sooty mangabey monkeys (Cercocebus torquatus atys), a natural host of SIV (SIVsmm) [10]. The mutant protein was not expressed at the cell surface and accordingly did not function as a viral coreceptor. Primary activated lymphocytes from mangabeys heterozygous for the deletion allele expressed significantly less CCR5 on the cell surface. Moreover, SIV seroprevalence and viremia were comparable among CCR5 heterozygotes and wild-type animals. Parallel evolution of CCR5-null alleles in humans and sooty mangabeys suggests that similar negative selection pressures have acted against CCR5, as would occur during epidemics of infectious agents that require CCR5 for pathogenesis. Sooty mangabeys bred to homozygosity for the deletion allele will be useful for experimental studies on the context-dependent role of CCR5 in host defense and microbial pathogenesis.

Studies on the use of nonhuman primates to determine the DR status of the human hematopoietic stem cell.

Monoclonal antibodies that recognize monomorphic determinants of human DR are potentially useful for the in vitro elimination of malignant cells from marrow for use in autologous transplantation. While DR is expressed on normal hematopoietic progenitor cells and the cells of the majority of the hematologic and lymphoid malignancies, there is the possibility that DR may not be expressed on the hematopoietic stem cells responsible for marrow regeneration after transplantation. To resolve the uncertainty regarding the DR status of the human stem cell, we determined whether antihuman DR monoclonal antibodies recognized analogous antigens on nonhuman primate hematopoietic progenitor cells to determine an appropriate animal transplant model. We used antihuman DR plus C'-mediated lysis of marrow progenitor cells as an indicator of whether the analogous nonhuman primate cells express similar antigens. Using two potent C'-fixing anti-DR monoclonal antibodies separately (5F3, AMG-12), human progenitor cells are reduced by 90%-100%. The range of progenitor cell depletion varied more widely with the nonhuman primates studied: 80%-99% with cells from the chimpanzee, 48%-100% with cells from the orangutan, and 62%-100% with cells from the rhesus monkey. Despite this, the majority of animals yielded results identical to that seen with human cells. We concluded that autologous transplantation with DR-depleted rhesus bone marrow into a lethally irradiated animal would be a practical and expeditious means to determine the DR status of the cell responsible for marrow regeneration, and by inference the DR status of the human hematopoietic stem cell.

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on platelet survival and activation using a nonhuman primate model.

In humans and nonhuman primates, the in vivo administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) consistently results in marked increase of megakaryocyte ploidy and size similar to that observed with interleukin-6 (IL-6). However, whereas the administration of IL-6 also results in an increase in circulating platelets, there is no predictable corresponding increase in peripheral blood platelets following treatment with rhGM-CSF. To determine whether the failure of rhGM-CSF to produce thrombocytosis is secondary to cytokine-related increased platelet activation and consumption in vivo, we quantified autologous platelet survival time and in vivo platelet activation before and during 5 days of administration of rhGM-CSF to two rhesus monkeys. Platelet survival was measured using autologous platelets labeled with 111Indium-oxine. Platelet activation was assessed by flow cytometric determination of the expression of the major platelet membrane glycoprotein (GP) IIb/IIIa complex, and an activation-dependent epitope on GPIIb/IIIa (recognized by monoclonal antibodies [MABs] LJ-P4 and PAC1, respectively). Platelet activation was also assessed by dose-response aggregometry using adenosine diphosphate (ADP). While megakaryocyte ploidy increased during rhGM-CSF administration, peripheral platelet counts were 418 x 10(9)/L and 525 x 10(9)/L before and 402 x 10(9)/L and 508 x 10(9)/L during cytokine treatment in animals 1 and 2, respectively. No changes were observed in the mean platelet volume. 111Indium-labeled platelet recovery in circulation was similar before (94.7%, 91.8%) and during (92.9%, 92.8%) rhGM-CSF administration, which indicates that cytokine-related in vivo sequestration of platelets does not occur. Autologous platelet survival was 5.6 and 6.2 days before and 5.0 and 5.4 days during the rhGM-CSF treatment (p = 0.07), without significant change in the corresponding platelet turnover rate (derived from the platelet count and survival time). The flow cytometric analysis showed no increase in the binding of either LJ-P4 or PAC1 MABs to the platelet membrane during rhGM-CSF administration. The aggregometry studies demonstrated similar concentrations of ADP inducing half-maximal aggregation (ED50). Overall, the above data indicate that treatment with rhGM-CSF is not associated with in vivo activation, sequestration, or increased consumption of platelets. The data suggest that the failure of rhGM-CSF-stimulated megakaryocytes to increase peripheral platelet count is a manifestation of ineffective megakaryocytopoiesis resulting from inability to increase platelet delivery to the circulation.

Studies of an improved rhesus hematopoietic progenitor cell assay.

We have improved Rhesus monkey marrow cell growth in semisolid media by means of substituting supplemented calf serum for fetal bovine serum. The cloning efficiency of light-density marrow cells separated on 60% Percoll was 126 (+/- 54)/10(5) (n = 12, +/- SD), and for light-density peripheral blood cells 60 (+/- 46)/10(6) (n = 11). Thirty-five percent of the colonies were multilineage, whereas the remainder were unilineage colonies composed of erythrocytes, megakaryocytes, and neutrophilic or monocytic granulocytes. Unilineage megakaryocyte colonies comprised 12% of the total marrow progenitor cells. The [3H]TdR suicide index of marrow progenitor cells was 47% +/- 9% (n = 12). This progenitor cell assay should prove useful in preclinical studies of the effect of recombinant hematopoietic growth factors on the number and cycling status of Rhesus hematopoietic progenitor cells.

Prevalence of natural infection with simian immunodeficiency virus and simian T-cell leukemia virus type I in a breeding colony of sooty mangabey monkeys.

The seroprevalence of antibodies to simian immunodeficiency virus (SIVsmm) and simian T-cell leukemia virus type I (STLV-I) in a captive breeding colony of sooty mangabey monkeys was determined, and infection by SIVsmm was confirmed in all cases by virus isolation. Among 138 animals tested, 57 and 33% were infected with SIVsmm and STLV-I, respectively. While the proportion of female mangabeys (66%) differed significantly (P less than 0.01) from the proportion of male mangabeys (42%) infected with SIVsmm, the proportions of males and females infected with STLV-I were similar, suggesting independent transmission of the two viruses. Among mangabeys less than 1 year old, none were infected with STLV-I and only five of 27 mangabeys, all of which were at least 6 months old when first tested, were infected with SIVsmm. The data document that natural infection of sooty mangabey monkeys with SIVsmm or in association with STLV-I infection does not result in increased disease or mortality, and that transmission of both SIVsmm and STLV-I appears to occur primarily through sexual activity.

Requirements for simian immunodeficiency virus antigen-specific in vitro proliferation of T cells from infected rhesus macaques and sooty mangabeys.

The measurement of cell-mediated immunity against the etiologic agent of human AIDS (HIV) in the non-human primate model of AIDS (simian immunodeficiency virus, SIV) has been difficult. In general, culture of peripheral blood mononuclear cells from HIV-1- and SIV-infected humans and monkeys, respectively, with purified inactivated HIV and SIV virus preparations has given inconsistent or negative proliferative responses. However, we describe herein an assay which consists of coculturing monocytes that have been pulsed with inactivated SIVsmm with nylon-wool-purified autologous T cells, leading to antigen-specific T-cell proliferation. The proliferative response, which predominantly occurs in CD4+ T cells, is major histocompatibility complex (MHC) class II-restricted and requires antigen processing. This assay will greatly facilitate the identification of the immunodominant epitopes recognized by T cells in sooty mangabeys, which are naturally infected but remain clinically asymptomatic, and in rhesus macaques, in which experimental infection leads to clinical symptomatology similar to human AIDS, eventually resulting in death.

Neonatal treatment with luteinizing hormone-releasing hormone analogs alters peripheral lymphocyte subsets and cellular and humorally mediated immune responses in juvenile and adult male monkeys.

We examined the effect of treatment with a LH-releasing hormone (LHRH) agonist (Ag), antagonist (Ant), or Ant and androgen (Ant/And) for the first 4 months of postnatal life on lymphocyte subsets and cellular and humorally mediated immune responses in juvenile and adult male monkeys. We also determined the effect of 9 weeks of Ant treatment on lymphocyte subsets in adult male monkeys. Adult male monkeys that had been treated neonatally with an Ag had increased levels of CD8-positive (CD8+) T-cells and reduced levels of B-cells compared to vehicle-treated controls. Lymphocytes from these animals also showed an elevated proliferative response to a variety of mitogens compared to cells from control animals. Antibody production in response to tetanus toxoid was normal in treated animals. Other neonates treated with Ant/And exhibited subnormal levels of lymphocytes, CD8+ T-cells, and B-cells at 4 months of age. Similar changes, but of lesser magnitude, were observed in animals treated with Ant alone. At 6 months of age, lymphocytes from both groups of Ant-treated monkeys exhibited an above normal proliferative response to streptolysin-O, but not to other mitogens. At 18 months of age, animals treated with Ant alone produced more antitetanus antibody in response to a tetanus toxoid booster than the controls or Ant/And-treated animals. Ant treatment was without major effect on lymphocyte subsets in adult monkeys. Serum LH and testosterone levels declined, and there was a small but significant increase in B-cells, lymphocytes expressing the interleukin-2 receptor, and the CD4+/CD8+ T-cell ratio during treatment, but these parameters normalized during the posttreatment period. The data suggest that chronic neonatal treatment with an Ag or Ant alters the development of immune system responses in male primates. The significance of these changes and their impact on the ability of these animals to respond to pathogenic agents is under investigation.

Brain weight throughout the life span of the chimpanzee.

Studies on human postmortem material report lower brain weights in older than in younger cohorts, whereas there is no apparent change with age in the rhesus monkey. In view of these contrasting results, we examined the pattern of brain weight across the life span in the chimpanzee, one of the closest biological relatives of humans. To place the study in context of the empirical life expectancy of the chimpanzee, we first performed a survival analysis on data from 275 chimpanzees that were maintained in the colony of the Yerkes Primate Center. The survival analysis revealed the maximum life spans of female and male chimpanzees to be about 59 and 45 years, respectively. We examined fresh brain weights from 76 chimpanzees ranging in age from birth to 59.4 years of age. The brains were taken from 9 infants (birth to 1 year of age), 25 juveniles (1-7 years), 13 adolescents (7-15 years), 21 young adults (15-30 years), and 8 old adults (over 30 years). Adult brain weight was achieved by the age of 7 years. The adolescent and young adult chimpanzees had the largest brain weights; in these two age groups combined, the mean brain weight (+/- standard deviation) was 368.1 g (+/-37.3) for females (n = 17) and 405.6 g (+/-39.4) for males (n = 17). This sex difference was statistically significant (P < 0.01). Simple linear regression performed on the combined material from females and males aged 7 years and older revealed a decline in brain weight with advancing age of 1.1 g/year (P < 0.05). When the effect of sex on brain weight was statistically controlled for, the loss of brain weight with age was 0.9 g/year (P = 0.07). These results suggest that brain weight declines moderately with age in the chimpanzee as it does in humans.

Humoral response to SIV/SMM infection in macaque and mangabey monkeys.

Natural infection of sooty mangabey monkeys with simian immunodeficiency virus, designated SIV/SMM, results in long-term persistent infections with little or no disease. In contrast, experimental infection of macaques with isolates of SIV/SMM induces chronic and progressive disease that terminates in an AIDS-like illness and death in most animals. To determine whether antibodies might be important in preventing the development of disease in mangabeys or progression of disease in macaques, humoral immune responses to SIV/SMM were compared in 13 macaques infected for up to 43 months and in infected and uninfected mangabeys selected at random from among a breeding colony. Total SIV/SMM-specific antibody titers, profiles of antibodies to specific viral proteins, neutralizing antibodies that inhibited infectivity of cell-free virus or syncytia formation, antibodies that inhibited reverse transcriptase activity, and antibodies to lymphocyte cell-surface antigens were assessed. The results indicated that in macaques the magnitude of the SIV/SMM-specific antibody response and progression of disease were functions of virus load. Surprisingly, asymptomatic mangabeys also had high virus loads with, on average, lower antibody titers than macaques. In both species, the presence of neutralizing antibodies or antibodies that inhibited SIV/SMM reverse transcriptase activity did not correlate with protection from clinical disease. A correlation was observed, however, between the development of disease and the presence of antibodies to an 18-kDa protein that is found on the surface of activated lymphocytes and appears to be related to histone H2B. A similar correlation has been observed in association with HIV infection in humans, suggesting that some manifestations of both human and simian AIDS may result from autoimmune reactions.

Multi-envelope HIV vaccine safety and immunogenicity in small animals and chimpanzees.

A significant obstacle to HIV vaccine development lies in the remarkable diversity of envelope proteins, the major targets of neutralizing antibody. That envelope diversity must be targeted is demonstrated by results from nonhuman primate studies in which single-envelope vaccines have protected against homologous, but rarely against heterologous virus challenges. Similarly, in clinical trials, single-envelope vaccines have failed to prevent break-through infections when challenge viruses were inevitably mismatched with the vaccine. To protect humans from infection by any isolate of HIV, we have prepared vaccine cocktails combining multiple envelopes from distinct viral isolates. We have tested several vehicles for vaccine delivery in small animals and have shown that successive immunizations with envelope, presented first as a DNA recombinant, then as a vaccinia virus (VV) recombinant, and finally as purified protein elicited strong neutralizing antibody responses. We have also tested the VV recombinant vaccine in chimpanzees. Pairs of animals received either single- or multi-envelope VV recombinant vaccines administered by the subcutaneous route. Results showed that the multi-envelope vaccine was safe, immunogenic, and superior to the single-envelope vaccine in eliciting HIV-specific antibody measurable in a standard clinical, immune assay. The promise of this system has led to the initiation of clinical trials, with which the hypothesis that cocktail vaccines will prevent human HIV infections may ultimately be tested.

Chronic fatal disease in gorillas seropositive for simian T-lymphotropic virus I antibodies.

Sera from 10 gorillas were tested by indirect immunofluorescent antibody assay, Western blotting and ELISA to Human T-lymphotropic viruses for cross-reacting with antibodies to Simian T-lymphotropic virus I (STLV-I). Four were antibody positive. Of the 4 seropositive gorillas, one has remained healthy, while 3 have died with similar disease problems as reconstructed from clinical records. It is not known whether a causal relationship exists between these diseases and STLV-I retrovirus infection.

Simian immunodeficiency virus SIVsmmPBj 1.9 induces multinucleated giant cell formation in human peripheral blood monocytes.

SIVsmmPBJ 1.9 is an extremely virulent clone of the simian immunodeficiency virus SIVsmmPBj 14 that causes an acute lethal disease in pigtail macaques, with death occurring 6 to 8 days after infection. The disease is characterized by bloody mucoid diarrhea, lymphoid hyperplasia, and giant cell pneumonia. We have developed an in vitro model for the production of multinucleated giant cells (MGCs) in which peripheral blood monocytes rapidly fuse to form MGCs when cultured in lymphocyte-conditioned medium and antibody against class II MHC. We have tested the effect of SIVsmmPBj on monocytes in our MGC model system. Peripheral blood mononuclear cells (PBMCs) from normal healthy human subjects, when cultured in the presence of anti-class II MHC monoclonal antibody and SIVsmmPBj 1.9, but not either alone, resulted in the formation of MGCs within 4 days. Experiments using Transwell chambers indicated that such MGCs are formed by fusion of monocytes, not by virus-induced fusion of lymphocytes. SIVsmmPBj 1.9 is unique in inducing MGC formation in that other SIV and HIV isolates do not induce MGCs. Whereas SIVsmmPBj 1.9 grown in PBMCs was a potent inducer of MGCs in the presence of anti-class II MHC antibody, SIVsmmPBj 1.9 grown in CEMx174 failed to do so. Antibodies against IFN-gamma and TNF-alpha significantly inhibited SIVsmmPBj/anti-class II-induced formation of MGCs. These results indicate that cytokines released in response to SIVsmmPBj 1.9, in conjunction with antibodies to class II MHC, caused fusion of monocytes.

Identification and biologic characterization of an acutely lethal variant of simian immunodeficiency virus from sooty mangabeys (SIV/SMM).

A virus pool isolated from lymphoid tissue of a macaque (PBj) infected for 14 months with SIV/SMM was found to be associated with acute disease and death. Six of six pig-tailed macaques, one of three rhesus macaques, and three of four SIV/SMM-seronegative mangabeys developed acute disease within 5 days and died from 7 to 13 days postinoculation; however, neither of two SIV/SMM-infected mangabeys died or developed disease. The virus associated with acute disease and death was shown by electron microscopy to be a lentivirus and was serologically indistinguishable from SIV/SMM by immunofluorescence and radioimmunoprecipitation assays. A biologic clone generated from lymphoid tissue of an animal that died 7 days after inoculation of the lethal pool resulted in death within 8 days of three of three pig-tailed macaques. Comparison of the lethal virus, designated SIV/SMM(PBj14), with the parent virus, SIV/SMM-9 (the isolate with which macaque PBj was originally inoculated), showed that although the kinetics of replication in peripheral blood mononuclear cells (PBMC) from pig-tailed macaques and mangabey monkeys were similar, SIV/SMM(PBj14) replicated more efficiently than SIV/SMM-9 in human PBMC and also replicated in chimpanzee PBMC whereas SIV/SMM and other SIV isolates did not. In addition, the variant was shown to replicate efficiently in some established cell lines whereas replication of SIV/SMM-9 in cell lines could be demonstrated only occasionally. That parental SIV/SMM-9, but not SIV/SMM(PBj14), was neutralized by serum from macaque PBj suggests that the variant may have been generated by immune selection. Comparison of sequential virus isolates from macaque PBj for host range and the ability to be neutralized and of sequential serum samples for neutralization activity indicated that changes in biologic properties were detected in virus isolates and serum obtained at 6 months after infection and later. Normal macaque PBMC infected in vitro with SIV/SMM(PBj14), but not with SIV/SMM-9 or other virus pools from PBj, formed syncytia with Sup-T1 cells, whereas all isolates formed syncytia with MOLT-4 clone 8 cells. These data suggest that, relative to SIV/SMM-9, SIV/SMM(PBj14) acquired multiple mutations, at least one (or more) of which is in the gene coding for the envelope glycoprotein. Continued analysis of this series of SIV/SMM isolates with diverse properties may lead to the identification of specific regions of the viral genome that influence defined biologic properties. Furthermore, the availability of a strain of SIV that induces rapid onset of disease and death may facilitate screening of drugs for antiviral activity against lentiviruses.

Sensitive and robust one-tube real-time reverse transcriptase-polymerase chain reaction to quantify SIV RNA load: comparison of one- versus two-enzyme systems.

Plasma viral RNA load is a key parameter in disease progression of lentiviral infections. To measure simian immunodeficiency virus (SIV) RNA loads, we have established a quantitative one-tube assay based on TaqMan chemistry. This real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has advantages compared with previous methods, such as higher sensitivity, shorter time consumption, and low risk of cross-contamination. The sensitivity of the assay was optimized by comparing different enzyme systems. The one-enzyme protocol using rTth DNA polymerase was superior to two assays employing two enzymes. It detects 100% of the samples containing four copies of RNA transcript and allows quantification of viral RNA loads over an 8-log unit dynamic range. As few as 50 copies per milliliter of plasma can be detected within RNA extracted from 140 microl of plasma. This is especially relevant in studies employing neonatal macaques, from which only small volumes of blood can be sampled, and in studies in which low viral RNA loads are expected. Because of the use of rTth DNA polymerase, DNA contamination can be avoided by carryover prevention with uracil N-glycosylase (UNG). We demonstrate that for optimization of real-time PCR sensitivity, not only concentrations of different reagents but also different enzyme systems must be evaluated. Our assay facilitates and enhances the quantification of plasma RNA loads, a critical parameter for many studies, including evaluations of vaccine candidates or antiviral regimens.

Active and passive immunization against HIV type 1 infection in chimpanzees.

Several vaccine strategies have been developed to prevent primary infection with human immunodeficiency virus (HIV), and some of the candidate vaccines have been tested in chimpanzees to determine their safety, efficacy, and to delineate immune correlates of protection. To date, more than 25 vaccines representing active and passive immunization strategies have been evaluated in the chimpanzee model. Efficacy of a given vaccine was based on protection against primary infection with HIV after intravenous or mucosal challenge with cell-free or cell-associated virus. Based on the results from a majority of the studies, neutralizing antibodies appear to play a major role in preventing primary infection with HIV.