Vol. 36, No. 11
ANTiMICROBLAL AGENTS AND CHEMOTHERAPY, Nov. 1992, p. 2381-2386
0066-4804/92/112381-06$02.00/0
Copyright X) 1992, American Society for Microbiology
Simian Immunodeficiency Virus (SIV) Infection of Infant Rhesus Macaques as a Model To Test Antiretroviral Drug Prophylaxis and Therapy: Oral 3'-Azido-3'Deoxythymidine Prevents SIV Infection KOEN K. A. VAN ROMPAY,l12* MARTA L. MARTHAS,1 ROSS A. RAMOS,1'2 CAROL P. MANDELL,3 ELLEN K. McGOWAN,4 STEVE M. JOYE,1 AND NIELS C. PEDERSEN2
California Regional Primate Research Center, Department of Medicine,2 and Department of Clinical Pathology,3 School of Veterinary Medicine, and Department of Medical Pathology, School of Medicine,4 University of California, Davis, California 95616 1
Received 22 May 1992/Accepted 31 August 1992
The prophylactic and therapeutic properties of 3'-azido-3'-deoxythymidine (AZT) against simian immunodeficiency virus (SIV) infection were tested in four 3-month-old rhesus macaques. The infant monkeys were inoculated intravenously with a low dose (1 to 10 100%v animal infectious doses) of uncloned SIVmac* The monkeys were treated orally with 50 mg of AZT per kg of body weight every 8 h; two animals were started on treatment 2 h prior to virus inoculation, and two animals were started on treatment 6 weeks later. All four animals were treated for a period of 6 to 10 weeks. Outward signs of AZT toxidcity were absent, but a mild macrocytic anemia occurred soon after therapy was started and resolved shortly after it was discontinued. The two infants that were begun on AZT treatment 2 h prior to virus inoculation never became infected, as demonstrated by the inability to detect cell-free or cell-associated virus in the blood, proviral DNA in peripheral blood mononuclear cells, or anti-SIV antibodies. AZT administration over a 10-week period had no detectable effect on the course of disease in the two animals that were begun on treatment after the infection had been established. In addition to demonstrating the prophylactic effect of AZT against low-dose SIV exposure, the study demonstrated the ease with which infant rhesus macaques can be used for antiretroviral drug testing.
prior to and during exposure (9). AZT did not prevent infection of SIV in monkeys (5, 15, 17, 18, 31). In the present study, infant rhesus macaques were successfully used to study the prophylactic and therapeutic potentials of oral AZT against SIV infection. AZT prevented infection with a small amount of intravenously administered virus if AZT administration was initiated shortly before exposure. However, the same AZT regimen had no demonstrable therapeutic effect once SIV infection was established.
Although simian immunodeficiency virus (SIV) infection of rhesus macaques is an excellent animal model of human immunodeficiency virus (HIV) infection in humans, its application for antiretroviral drug studies has been limited, particularly because it is not easy to obtain periodic blood samples and to administer chronic medication to macaques without some type of physical restraint, implantable device, and/or sedation. These difficulties, however, may be overcome by the use of infant rather than adolescent or adult rhesus macaques. Infant rhesus monkeys can be weaned at 2 to 3 months of age, are quite easily handled, and can be administered drugs readily by either the parenteral or the oral route without sedation and with minimal physical restraint. Rhesus infants (weight, about 1 kg) are also much smaller than adolescents or adults (weight, 5 to 10 kg) and therefore require smaller amounts of drugs, many of which are very expensive to produce in test quantities. At 3 months of age, rhesus infants already metabolize 3'-azido-3'-deoxythymidine (AZT; also called azidothymidine and zidovudine) in a manner similar to that by adult monkeys (14), and the pharmacokinetic parameters of AZT in rhesus infants equal those reported in humans (2). Although infant monkeys cannot donate as much blood for the various assays required to monitor drug toxicity and SIV infection as can adolescent or adult monkeys, many of the assay procedures have been adapted to the use of small quantities of blood. AZT has been found to have beneficial therapeutic effects when administered during several postinfection stages of HIV-related disease (6, 30). However, it is not known whether AZT can prevent HIV infection if it is administered *
MATERIALS AND METHODS Animals and virus. Infant rhesus macaques (Macaca mulatta) from the type D retrovirus-free and SIV-free colony at the California Regional Primate Research Center (University of California, Davis) were reared by their mothers until they were weaned at 2 months of age and were then housed in pairs (animals 26056 and 26069 and animals 26092 and 26093). All four animals were healthy 3-month-old weanlings at the time of virus inoculation. Animals of this age are immunocompetent (29) and are able to metabolize AZT, in a manner similar to that by adult macaques (14). Previous experiments (12) have already shown that macaques in this age group are susceptible to the development of a fatal immunodeficiency syndrome following inoculation with the same dose of the SIV stock used in the study described here. When necessary, animals were immobilized with ketamine HCI (Parke-Davis, Morris Plains, N.J.), 10 mg/kg of body weight injected intramuscularly. Animals were inoculated intravenously with 0.5 ml of a 10-4 dilution of a cell-free uncloned SWVmac virus stock propagated in human peripheral blood mononuclear cells
Corresponding author. 2381
2382
VAN ROMPAY ET AL.
(PBMCs) (16). This dose was shown to be equivalent to 1 to 10 100% animal infectious doses (16). AZT preparation and administration. AZT syrup (10 mg of AZT per ml; Retrovir; Burroughs Wellcome) was supplemented with purified AZT powder (all AZT was provided by P. Sager, Division of AIDS, National Institute of Allergy and Infectious Diseases) to obtain a final concentration of 20 mg of AZT per ml, which is the maximum solubility of AZT in water at neutral pH (19). Supplementation allowed administration of 2.5 ml of AZT solution per kg of body weight, which is near the maximum volume of medication that can be chronically administered to infant rhesus monkeys (weight, -1 kg) without the use of a nasogastric tube. Tang powder (Kraft General Foods, White Plains, N.Y.) was added (4%, by weight) to the syrup to improve palatability. This final AZT solution was prepared just prior to use and was stored at room temperature under protection from light. An oral dose of 50 mg of AZT per kg of body weight (equivalent to 500 mg of AZT per m of body surface area) was given every 8 h (7:00 a.m., 3:00 p.m., 11:00 p.m.). For each dosing, animals were restrained physically and 2.5 ml of AZT solution per kg of body weight was administered orally. Animals were monitored to ensure swallowing of the drug. AZT dosages were adjusted weekly according to body weight. Blood was collected by venipuncture without sedation at various time intervals after AZT administration and following a brief fast. AZT levels were measured in plasma and serum samples by standard techniques (27) by modified high-pressure liquid chromatographic conditions (11) and with 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide as an internal standard. Determination of AZT toxicity. Complete blood counts were performed on EDTA-anticoagulated blood samples from all infants. Samples were analyzed by using an automated electronic cell counter (Baker 9000; Serono Baker Diagnostics); differential cell counts were determined manually. A standard serum chemistry profile was done by the Dacos system (Coulter Electronics, Hialeah, Fla.). Bone marrow aspirates were obtained monthly, and smears were stained with Wright-Giemsa for morphologic evaluation. Quantitative virus isolation (cell-associated and cell-free). Heparinized blood (2 ml) was collected at 1- to 2-week intervals and spun for 10 minutes at 800 x g to separate the plasma from the cells. The plasma was then centrifuged for an additional 5 min at 10,000 x g to remove residual cells. An aliquot of cell-free plasma was used for quantitation of cell-free virus (see below), and the remainder was stored at -70°C for other purposes. PBMCs were isolated by Ficoll gradient separation (Lymphocyte Separation Medium; Organon Teknika, West Chester, Pa.). Infectious virus was recovered from 1 x 106 to 10 x 106 PBMCs by cocultivation with CEMx 174 cells (kindly provided by James A. Hoxie, University of Pennsylvania, Philadelphia) by previously described methods (13). Culture supernatants were sampled regularly for SIV p27 antigen detection by antigen-capture enzyme-linked immunosorbent assay (ELISA) (13). Cultures were considered positive if culture supernatants yielded 10 ng or more of SIV p27 antigen per ml at two different time points. Negative cultures were maintained for 8 weeks before being discarded. To negate any inhibitory effects of CD8+ T cells on cell-associated virus isolation, aliquots of PBMCs were depleted of CD8+ T cells prior to cocultivation. CD8+ T cells were removed with mouse monoclonal anti-human CD8
ANTimICROB. AGENTs CHEMOTHF-R.
antibody-coated magnetic beads (Dynabeads M-450; Dynal, Great Neck, N.Y.), which were used according to the manufacturer's instructions. Cell-associated virus load in peripheral blood was determined by a limiting dilution assay. Tenfold serial PBMC dilutions were made in medium, and four replicates of 102 to 106 PBMCs were cultured with 2 x 105 CEMx 174 cells in a total volume of 2 ml in 24-well plates (Fisher, Santa Clara, Calif.). Cultures were maintained and tested as described above for cocultivation, except that negative cultures were discarded after 5 weeks. Cell-free virus levels were determined by a similar limiting dilution assay of the collected plasma. Serial 10-fold plasma dilutions were made in tissue culture medium, and 105 CEMx 174 cells were added to 1 ml of each dilution (representing a range of 100 pl to 1 nl of plasma) in 24-well plates, using four replicates per dilution. Cultures were maintained and tested for SIV p27 antigen as described above. Cell-associated and cell-free virus levels were calculated by the method of Reed and Muench (23) and are expressed as the number of 50% tissue culture infectious doses (TCID50s) per 106 PBMCs or per milliliter of plasma, respectively. The minimal level of cell-associated and cell-free infectious virus that could be detected reliably was 1 TCID50 per 106 PBMCs and 10 TCID50s per ml of plasma, respectively. PCR. Detection of proviral DNA in PBMC lysates was accomplished by using nested polymerase chain reaction (PCR) techniques as described elsewhere (28). Plasma p27 antigen level. Plasma p27 antigenemia was measured by using the commercial SIV Core Antigen Assay (Coulter) according to the manufacturer's instructions. Values of p27 antigen greater than or equal to 25 pg/ml were considered positive. Antibody determination. SIV-specific antibodies in plasma were detected by a whole-virus ELISA (16) and immunoblotting (26) techniques. Whole-virus ELISA antibody titers were expressed as optical density units of a 1:3,200 dilution at 450 nm. This dilution was chosen because it gave a good resolution in the linear range of plasma dilutions without reducing the sensitivity of the assay. Plasma was diluted 1:100 for immunoblotting. T-lymphocyte phenotyping. PBMCs were incubated in anti-CD4 (Leu 3a-phycoerythrin; Becton Dickinson) and anti-CD8 (Leu 2a-fluorescein isothiocyanate)-specific monoclonal antibodies according to the manufacturer's instructions and were analyzed by flow cytometry by using a FACSCAN (Becton Dickinson). Percentages of single positive cells were determined with FACSCAN software. RESULTS Pharmacokinetics of AZT in weanling rhesus macaques. AZT levels in serum following administration of a single oral dose (50 mg of AZT per kg of body weight or 500 mg/m2 of body surface area) reached a peak (12 to 22 ,M) within 30 min and then decreased rapidly to undetectable levels (
ANTiMICROBLAL AGENTS AND CHEMOTHERAPY, Nov. 1992, p. 2381-2386
0066-4804/92/112381-06$02.00/0
Copyright X) 1992, American Society for Microbiology
Simian Immunodeficiency Virus (SIV) Infection of Infant Rhesus Macaques as a Model To Test Antiretroviral Drug Prophylaxis and Therapy: Oral 3'-Azido-3'Deoxythymidine Prevents SIV Infection KOEN K. A. VAN ROMPAY,l12* MARTA L. MARTHAS,1 ROSS A. RAMOS,1'2 CAROL P. MANDELL,3 ELLEN K. McGOWAN,4 STEVE M. JOYE,1 AND NIELS C. PEDERSEN2
California Regional Primate Research Center, Department of Medicine,2 and Department of Clinical Pathology,3 School of Veterinary Medicine, and Department of Medical Pathology, School of Medicine,4 University of California, Davis, California 95616 1
Received 22 May 1992/Accepted 31 August 1992
The prophylactic and therapeutic properties of 3'-azido-3'-deoxythymidine (AZT) against simian immunodeficiency virus (SIV) infection were tested in four 3-month-old rhesus macaques. The infant monkeys were inoculated intravenously with a low dose (1 to 10 100%v animal infectious doses) of uncloned SIVmac* The monkeys were treated orally with 50 mg of AZT per kg of body weight every 8 h; two animals were started on treatment 2 h prior to virus inoculation, and two animals were started on treatment 6 weeks later. All four animals were treated for a period of 6 to 10 weeks. Outward signs of AZT toxidcity were absent, but a mild macrocytic anemia occurred soon after therapy was started and resolved shortly after it was discontinued. The two infants that were begun on AZT treatment 2 h prior to virus inoculation never became infected, as demonstrated by the inability to detect cell-free or cell-associated virus in the blood, proviral DNA in peripheral blood mononuclear cells, or anti-SIV antibodies. AZT administration over a 10-week period had no detectable effect on the course of disease in the two animals that were begun on treatment after the infection had been established. In addition to demonstrating the prophylactic effect of AZT against low-dose SIV exposure, the study demonstrated the ease with which infant rhesus macaques can be used for antiretroviral drug testing.
prior to and during exposure (9). AZT did not prevent infection of SIV in monkeys (5, 15, 17, 18, 31). In the present study, infant rhesus macaques were successfully used to study the prophylactic and therapeutic potentials of oral AZT against SIV infection. AZT prevented infection with a small amount of intravenously administered virus if AZT administration was initiated shortly before exposure. However, the same AZT regimen had no demonstrable therapeutic effect once SIV infection was established.
Although simian immunodeficiency virus (SIV) infection of rhesus macaques is an excellent animal model of human immunodeficiency virus (HIV) infection in humans, its application for antiretroviral drug studies has been limited, particularly because it is not easy to obtain periodic blood samples and to administer chronic medication to macaques without some type of physical restraint, implantable device, and/or sedation. These difficulties, however, may be overcome by the use of infant rather than adolescent or adult rhesus macaques. Infant rhesus monkeys can be weaned at 2 to 3 months of age, are quite easily handled, and can be administered drugs readily by either the parenteral or the oral route without sedation and with minimal physical restraint. Rhesus infants (weight, about 1 kg) are also much smaller than adolescents or adults (weight, 5 to 10 kg) and therefore require smaller amounts of drugs, many of which are very expensive to produce in test quantities. At 3 months of age, rhesus infants already metabolize 3'-azido-3'-deoxythymidine (AZT; also called azidothymidine and zidovudine) in a manner similar to that by adult monkeys (14), and the pharmacokinetic parameters of AZT in rhesus infants equal those reported in humans (2). Although infant monkeys cannot donate as much blood for the various assays required to monitor drug toxicity and SIV infection as can adolescent or adult monkeys, many of the assay procedures have been adapted to the use of small quantities of blood. AZT has been found to have beneficial therapeutic effects when administered during several postinfection stages of HIV-related disease (6, 30). However, it is not known whether AZT can prevent HIV infection if it is administered *
MATERIALS AND METHODS Animals and virus. Infant rhesus macaques (Macaca mulatta) from the type D retrovirus-free and SIV-free colony at the California Regional Primate Research Center (University of California, Davis) were reared by their mothers until they were weaned at 2 months of age and were then housed in pairs (animals 26056 and 26069 and animals 26092 and 26093). All four animals were healthy 3-month-old weanlings at the time of virus inoculation. Animals of this age are immunocompetent (29) and are able to metabolize AZT, in a manner similar to that by adult macaques (14). Previous experiments (12) have already shown that macaques in this age group are susceptible to the development of a fatal immunodeficiency syndrome following inoculation with the same dose of the SIV stock used in the study described here. When necessary, animals were immobilized with ketamine HCI (Parke-Davis, Morris Plains, N.J.), 10 mg/kg of body weight injected intramuscularly. Animals were inoculated intravenously with 0.5 ml of a 10-4 dilution of a cell-free uncloned SWVmac virus stock propagated in human peripheral blood mononuclear cells
Corresponding author. 2381
2382
VAN ROMPAY ET AL.
(PBMCs) (16). This dose was shown to be equivalent to 1 to 10 100% animal infectious doses (16). AZT preparation and administration. AZT syrup (10 mg of AZT per ml; Retrovir; Burroughs Wellcome) was supplemented with purified AZT powder (all AZT was provided by P. Sager, Division of AIDS, National Institute of Allergy and Infectious Diseases) to obtain a final concentration of 20 mg of AZT per ml, which is the maximum solubility of AZT in water at neutral pH (19). Supplementation allowed administration of 2.5 ml of AZT solution per kg of body weight, which is near the maximum volume of medication that can be chronically administered to infant rhesus monkeys (weight, -1 kg) without the use of a nasogastric tube. Tang powder (Kraft General Foods, White Plains, N.Y.) was added (4%, by weight) to the syrup to improve palatability. This final AZT solution was prepared just prior to use and was stored at room temperature under protection from light. An oral dose of 50 mg of AZT per kg of body weight (equivalent to 500 mg of AZT per m of body surface area) was given every 8 h (7:00 a.m., 3:00 p.m., 11:00 p.m.). For each dosing, animals were restrained physically and 2.5 ml of AZT solution per kg of body weight was administered orally. Animals were monitored to ensure swallowing of the drug. AZT dosages were adjusted weekly according to body weight. Blood was collected by venipuncture without sedation at various time intervals after AZT administration and following a brief fast. AZT levels were measured in plasma and serum samples by standard techniques (27) by modified high-pressure liquid chromatographic conditions (11) and with 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide as an internal standard. Determination of AZT toxicity. Complete blood counts were performed on EDTA-anticoagulated blood samples from all infants. Samples were analyzed by using an automated electronic cell counter (Baker 9000; Serono Baker Diagnostics); differential cell counts were determined manually. A standard serum chemistry profile was done by the Dacos system (Coulter Electronics, Hialeah, Fla.). Bone marrow aspirates were obtained monthly, and smears were stained with Wright-Giemsa for morphologic evaluation. Quantitative virus isolation (cell-associated and cell-free). Heparinized blood (2 ml) was collected at 1- to 2-week intervals and spun for 10 minutes at 800 x g to separate the plasma from the cells. The plasma was then centrifuged for an additional 5 min at 10,000 x g to remove residual cells. An aliquot of cell-free plasma was used for quantitation of cell-free virus (see below), and the remainder was stored at -70°C for other purposes. PBMCs were isolated by Ficoll gradient separation (Lymphocyte Separation Medium; Organon Teknika, West Chester, Pa.). Infectious virus was recovered from 1 x 106 to 10 x 106 PBMCs by cocultivation with CEMx 174 cells (kindly provided by James A. Hoxie, University of Pennsylvania, Philadelphia) by previously described methods (13). Culture supernatants were sampled regularly for SIV p27 antigen detection by antigen-capture enzyme-linked immunosorbent assay (ELISA) (13). Cultures were considered positive if culture supernatants yielded 10 ng or more of SIV p27 antigen per ml at two different time points. Negative cultures were maintained for 8 weeks before being discarded. To negate any inhibitory effects of CD8+ T cells on cell-associated virus isolation, aliquots of PBMCs were depleted of CD8+ T cells prior to cocultivation. CD8+ T cells were removed with mouse monoclonal anti-human CD8
ANTimICROB. AGENTs CHEMOTHF-R.
antibody-coated magnetic beads (Dynabeads M-450; Dynal, Great Neck, N.Y.), which were used according to the manufacturer's instructions. Cell-associated virus load in peripheral blood was determined by a limiting dilution assay. Tenfold serial PBMC dilutions were made in medium, and four replicates of 102 to 106 PBMCs were cultured with 2 x 105 CEMx 174 cells in a total volume of 2 ml in 24-well plates (Fisher, Santa Clara, Calif.). Cultures were maintained and tested as described above for cocultivation, except that negative cultures were discarded after 5 weeks. Cell-free virus levels were determined by a similar limiting dilution assay of the collected plasma. Serial 10-fold plasma dilutions were made in tissue culture medium, and 105 CEMx 174 cells were added to 1 ml of each dilution (representing a range of 100 pl to 1 nl of plasma) in 24-well plates, using four replicates per dilution. Cultures were maintained and tested for SIV p27 antigen as described above. Cell-associated and cell-free virus levels were calculated by the method of Reed and Muench (23) and are expressed as the number of 50% tissue culture infectious doses (TCID50s) per 106 PBMCs or per milliliter of plasma, respectively. The minimal level of cell-associated and cell-free infectious virus that could be detected reliably was 1 TCID50 per 106 PBMCs and 10 TCID50s per ml of plasma, respectively. PCR. Detection of proviral DNA in PBMC lysates was accomplished by using nested polymerase chain reaction (PCR) techniques as described elsewhere (28). Plasma p27 antigen level. Plasma p27 antigenemia was measured by using the commercial SIV Core Antigen Assay (Coulter) according to the manufacturer's instructions. Values of p27 antigen greater than or equal to 25 pg/ml were considered positive. Antibody determination. SIV-specific antibodies in plasma were detected by a whole-virus ELISA (16) and immunoblotting (26) techniques. Whole-virus ELISA antibody titers were expressed as optical density units of a 1:3,200 dilution at 450 nm. This dilution was chosen because it gave a good resolution in the linear range of plasma dilutions without reducing the sensitivity of the assay. Plasma was diluted 1:100 for immunoblotting. T-lymphocyte phenotyping. PBMCs were incubated in anti-CD4 (Leu 3a-phycoerythrin; Becton Dickinson) and anti-CD8 (Leu 2a-fluorescein isothiocyanate)-specific monoclonal antibodies according to the manufacturer's instructions and were analyzed by flow cytometry by using a FACSCAN (Becton Dickinson). Percentages of single positive cells were determined with FACSCAN software. RESULTS Pharmacokinetics of AZT in weanling rhesus macaques. AZT levels in serum following administration of a single oral dose (50 mg of AZT per kg of body weight or 500 mg/m2 of body surface area) reached a peak (12 to 22 ,M) within 30 min and then decreased rapidly to undetectable levels (
