AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 7, 1992 Mary Ann Liebert, Inc., Publishers
Prevention of HIV-2 and SIV Infections in Cynomolgus Macaques by Prophylactic Treatment with 3'-Fluorothy irridine DISA
BÖTTIGER,1
PER
PUTKONEN,2
and BO
ÖBERG1
ABSTRACT The aim of this study was to determine the usefulness of human immunodeficiency virus type 2 (HIV-2) for in vivo evaluation of antiviral drugs in monkeys and to study if prophylactic treatment with 3'-fluorothymidine (FLT) could prevent infection against a low challenge dose of HIV-2 or simian immunodeficiency virus (SIV). Protection against infection was assessed by virus isolation and polymerase chain reaction (PCR) on monkey peripheral blood mononuclear cells (PBMC) as well as by antibody and viral antigen assays. Prophylactic treatment with FLT 3x5 mg/kg/day, starting 8 h prior to virus inoculation, prevented HIV-2 infection in 3 of 8 monkeys. In another experiment 2 of 4 monkeys resisted 2-10 monkey infectious doses (MIDS0) of SIV with the same prophylactic treatment. All control animals (HIV-2 8, SIV 4) became infected. Thus, FLT treatment prevented HIV-2 and SIV infection in 5 of 12 animals. =
INTRODUCTION
IN
HUMANS, INFECTION with HIV-1 as well as HIV-2 is associated with immunosuppression and AIDS. Both HIV-1 and HIV-2 have narrow host ranges. HIV-2 has advantages over HIV-1 with regard to the use in experimental antiviral testing and vaccine studies because of its ability to infect cynomolgus monkeys, ' which is not an endangered species. The findings that not only SIV, but also certain strains of HIV-2 can infect cynomolgus monkeys have further improved the possibilities for effective evaluation of antiviral compounds. HIV-2SBL6669 has been adapted to replicate efficiently in cynomolgus monkeys and has been shown to be a useful tool for evaluation in various vaccine strategies. '~3 HIV-2 is genetically and serologically more closely related to certain SIV strains than to HIV-1. The overall homology between HIV-2 and SIVmac/SIV,m is approximately 70%.4 We have previously used an acute SIV infection in monkeys for the in vivo evaluation of antiviral compounds such as
azidothymidine (AZT), 2',3'-dideoxyinosine (ddl), 2',3'dideoxycytidine (ddC), dideoxythymidiene (d4T), and foscarnet, all known to affect the level of HIV p24 antigen in patients. Treatment starting prior to inoculation of 50-2500 MID5U of SIV delayed the appearance of viral antigen compared with the
Departments of 'Virology, and 2Immunology.
=
controls, but did not prevent infection." Similar results were obtained for 3'-fluorothymidine (FLT) in vivo in monkeys.5 FLT is a potent inhibitor of HIV-1, HIV-2, and SIV in cell
culture.8'9
The purpose of this study was to evaluate the use of HIV-2 in non-human primate as a tool in the development of antiviral drugs and to see whether prophylactic treatment could influence HIV-2 and SIV infection when a low inoculation dose of virus was used. a
MATERIAL Animals
Cynomolgus monkeys (Macaca fascicularis), of both sexes from the Philippines, were housed in single cages in a biosafety level 3 facility. The mean body mass was 2800 g and the range was 1875 g to 3825 g. Each animal was clinically healthy at the time of virus inoculation and shown to be HIV-2 and SIV antigen and antibody negative.
Drug
and
treatment
3'-Fluorothymidine (FLT), from Medivir AB, Huddinge, was dissolved in phosphate-buffered saline to a con-
Sweden,
Karolinska Institute SBL. Stockholm, Sweden.
1235
1236
BOTTIGER ET AL.
centration of 5 mg/ml, sterile filtered and used for subcutaneous injections on the backs of the animals. The total daily dose was 15 mg/kg. Collection of blood was performed under ketamine anesthesia.
detected in monkey peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR) amplification using nested primers specific for regions of the gag and pol genes of HIV-2/SIV as described.3
Virus and virus dose
HIV-2SBL()669/H5 was previously titered in vivo in monkeys as was 10 monkey infectious doses (MID50). The SIV strain used, originally isolated from naturally infected sooty mangabey monkeys (SIVsm), was kindly provided by Drs. P. Fultz and H. McClure (Yerkes Regional Primate Center, Animal Resources Branch, Altanta, NIH Grant No. RR00165). The SIV was previously titrated in vivo in monkeys as described, first in tenfold dilutions and then in threefold dilutions (5, SIV pool II). Altogether 20 monkeys were used for titration. The SIVsm inoculation dose was 2-10 MID50; this number reflects a range within which the true inoculum dose is likely to be. Stocks of virus had been prepared previously and were stored at —70°C in aliquots. Virus was administered intravenously.
EXPERIMENTAL
described.2'3 The HIV-2 inoculation dose
SIV and HIV-2 antigen were analyzed by using a crossreacting kit for p24 antigen detection (Abbott. North Chicago, IL) intended for HIV-1.10 Antibodies to SIV and HIV-2 were assayed by a peptide enzyme-linked immunosorbent assay ' ' (ELISA) method. Virus isolation was performed by cocultiva¬ tion with human peripheral blood lymphocytes (PBL) using standard techniques as described earlier.3 Proviral DNA was
of
RESULTS HIV-2
METHODS
Table 1. Presence
The experiments were designed as earlier described.5 The first treatment with FLT was initiated 8 h before and the second treatment 10-15 min before virus inoculation and continued every 8 h for 10 days. The S and series of HIV-2 animals were used in two consecutive experiments. Blood specimens were collected at intervals and screened for viral antigen and antibodies. Animals negative for viral antigen and antibodies were further examined by virus isolation and PCR.
Viral Antigen
in
infection
HIV-2 inoculation resulted in the appearance of a viral antigen peak at day 14-17 in all control monkeys, followed by HIV-2 antibody production (Table 1). In 5 of the 8 HIV-2-inoculated monkeys treated with FLT no viral antigen was detected. Three of these five animals did not seroconvert and no virus could be detected by isolation or by
PCR up to 11 months after HIV-2 inoculation. However, the two animals which lacked detectable viral antigen
remaining
Serum (A), Virus Isolation (B) and Presence in Monkeys Inoculated with HIV-2
of
Viral Antibodies
in
Serum
(C)
Control animals
Monkey No.
day
0 10 14
17 21 26
30
B.
SI
+ + + +
S3
+ +
+ + -¬
S4
Tl
T2
+ +
T3
T4
S6
S7
S8
T5
T6
T7
+ +
+ + -¬
+ + + -¬
+ + -¬
+ +
+ + +
+ + +
+ + + + -¬
+ + -¬
+
+
+ + + -¬
+ + + +
+ + -¬
+
-¬
-¬
T8
+ + +
+ + +
+
-¬
+
+
+ -¬
+
+
+
+
t
O
O S7 was reinoculated with HIV-2 after 5 months. The Not tested, t the second virus inoculation. 46/90; Control monkeys tested day 46, treated =
S5
treatment
+ +
day 0
14 month 1 2 3 5 11 day 0 21 30 46/90 month 1 1
S2
FLT
=
monkey seroconverted and yielded virus 30 days after monkeys tested day 90.
1237
PREVENTION OF HIV-2 AND SIV BY FLT IN MONKEYS later shown to be infected as determined by positive virus isolation at 30 days and seroconversion. Three FLT-treated animals had detectable antigens at day 21-25 after inoculation. However, the antigen peak was signif¬ icantly delayed as compared to the control animals, where antigen appeared at day 14-17. were
SIV
infection
All control monkeys became infected and presented a tran¬ sient peak of viral antigen on day 10-14, followed by seroconversion, as shown in Table 2. In contrast, 2 of 4 FLT-treated monkeys did not become infected as evident by repeatedly negative virus isolations, negative PCR and no detectable viral antigen or seroconversion up to 11 months. The two remaining treated animals became infected by SIV, although one of these animals did not have detectable viral antigen at any time assayed.
DISCUSSION FLT treatment initiated 8 h prior to virus inoculation pre¬ vented HIV-2 and SIV infection in 5 of 12 animals. This is to our knowledge the first demonstration of successful blocking of primate lentivirus replication with an antiviral drug in a nonhu¬ man primate. In the remaining treated monkeys treatment delayed the appearance of viral antigen compared with the controls. FLT showed similar potency in preventing SIV- and HlV-2-infection. This correlates with the results seen in cell culture where FLT is equally potent in inhibiting SIV and
HIV-2.9
We recently have shown that the amount of virus inoculated is important as to whether antiviral treatment can block virus replication or not. When a high inoculation dose of virus was used ( 100 MID,() of SIVsm) prophylactic treatment with FLT did
have any significant effect on the appearance of viral antigen.57 If the virus inoculum was reduced to 10-50 MID50 of SIV the appearance of viral antigen was markedly delayed.12 In the present experiments with HIV-2 and SIV we used a virus dose of 2-10 MID50 and this resulted in an infection which could be prevented by treatment with FLT, as earlier indicated for SIV.I2 In this experiment 4 of 4 control animals were infected. Using the same dose of virus 4 of 4 control monkeys were infected in the consecutive experiment (not shown). McClure et al. studied rhesus monkeys treated with 100 mg/kg/day of azidouridine (CS-87), AZT, d4T, and FLT, 24 hours before inoculation of approximately 75 MID5II of SIVsm. Prophylactic efficacy was not shown by virus isolation with any of the compounds, but one monkey given FLT did not seroconvert within 8 weeks. McClure et al.13 further reported that AZT prophylaxis 1 h post SIVsmmPBj 14 exposure, gave the result that 1 of 3 monkeys remained virus-negative and that 3 of 6 animals treated within 24 h after inoculation did not develop the charac¬ teristic lethal disease associated with SIVsmnPBjl4. It is not known if treatment efficacy in monkeys against this rapidly lethal isolate of SIV corresponds to treatment efficacy against the slow development of disease caused by HIV in humans. The influence of antiviral treatment on viral antigen appear¬ ance in serum has been seen in other retroviral experimental systems. SCID-hu mice represent an animal model for HIV in a laboratory animal in which therapeutic treatment with AZT was proven to suppress infection when given up to 2 h postinocula¬ tion. '4 Similar results have been reported for AZT therapy to mice infected with Raucher leukemia virus15 and in cats inocu¬ lated with FeLV."1 However, these models, apart from the SCID-hu mice model, use a virus considerably different from HIV. A clinically important issue is the problem with personnel accidentally exposed to HIV-infected blood or material. Due to the low incidence of infection after accidental exposure to HIV in the working place,17 the clinical studies on postexposure therapy using AZT are unlikely to give a clear answer. In the not
Table 2. Presence of Viral Antigen in Serum (A), Virus Isolation (B) and Presence of Viral Antibodies in Serum (C) in Monkeys Inoculated with SIV FLT
Control animals Monkey No. 0 10 14 21 B. month 1 2 3 5 II c. day 0 14 21 month 1 2 5 11
day
A.
O
=
Not tested
17 + + + + + -¬ +
+ -¬
o +
18
19
20
+ --
+ + + --
+ + + + -¬
o
o
+
+
+ --
+ +
o
o o
+
-¬
o o o o
o o o o
21
22
treatment
23
24
o o
o o o
+ + +
+ + + + +
BOTTIGER ET AL.
1238
present study
showed that HIV-2 and SIV infections can be prevented by prophylactic treatment. These findings have impli¬ cations for potential postexposure treatment. The clinical and experimental situations used here are not directly comparable in terms of virus inoculum (cell-associated versus cell-free virus) and route of virus exposure. These are differences which could be addressed experimentally. It remains to be determined in future studies if postexposure treatment can block infection in this model. Prophylactic treatment with FLT did not prevent infection in all treated animals. Dosing FLT every 8 h might not be optimal to obtain sufficient serum levels of FLT, reported to have a serum half-life of 0,8-0.9 h.718 However, intracellular levels of FLT triphosphate in vivo, which has not been studied so far, are more relevant for the effect of the drug. It is not clear for how long a time virus remains replicatively competent after transmis¬ sion. In our study a treatment period of 10 days stopped virus replication in 5 of 12 monkeys. A longer treatment period might have prevented infection in a higher percentage of animals. On the other hand, it is not excluded that treatment for fewer than 10 days could have given the same results. In conclusion, we demonstrate that prophylactic treatment with FLT can block primate lentivirus replication in vivo in a nonhuman primate. HIV-2 as well as SIV infection in cynomol¬ gus monkeys represent useful experimental models for the evaluation of antiviral drugs against HIV. we
ACKNOWLEDGMENTS The study was supported by the Swedish Board for Technical Development, Stockholm and by Medivir AB, Huddinge, Swe¬ den.
6.
7.
8.
9.
10.
11.
12.
Böttiger D. Ljungdahl-Stâhle E. and Öberg : Treatment of acute SIV infection in cynomolgus monkeys with 2',3'-dideoxyinosine (ddl) and 2',3'-dideoxythymidine (d4T). Antiviral Chem Chemother 1991:6:354-367. Lundgren B. Böttiger D, Ljungdahl-Stâhle E. Norrby E, Stahle L, Wahren B, and Öberg : Antiviral effects of 3'-f]uorothymidine and 3'-azidothymidine in cynomolgus monkeys infected with sim¬ ian immunodeficiency virus. J AIDS 1991:4:489-498. Tsai CC, Follis KE, Yarnall M, Deaver LE, Benveniste RE, and Sager PR: In vitro screening for antiretroviral agents against simian immunodeficiency virus (SIV). Antiviral Res 1990;14:87-98. Vrang L, Öberg , Löwer J, and Kurth R: Reverse transcriptase from human immunodeficiency virus type I (HIV-1), HIV-2 and simian immunodeficiency virus (SIVmac) are susceptible to inhibi¬ tion by foscarnet and 3'-azido-3'-deoxythymidine triphosphate. Antimicrob Agent Chemother 1988;32:1733-1734. Goldstein S, Engle R. Olmsted RA, Hirsch VM, and Johnson PR: Detection of SIV antigen by HIV-1 antigen capture immunoassays. J AIDS 1990;3:98-102. Ljungdahl-Stâhle E, Trojnar J, Öberg . and Wahren : Early appearance of simian immunodeficiency virus (SIV) antigen and antibodies as variables in evaluating antiviral drugs in macaques. J Vir Methods. 37:43-53. Böttiger D, Vrang L. and Öberg : Influence of the infectious dose of simian immunodeficiency virus in monkeys on the effect of treatment with 3'-fluorothymidine. Antiviral Chem Chemother. In press.
13. McClure HM, Anderson DC. Ansari AA. Fultz PN, Klumpp SA, and Shinazi RF: Nonhuman primate models for evaluation of AIDS therapy. In: Georgiev VS. andMcGowan JJ(eds.). AIDS: Anti-HIV Agents. Therapies, and Vaccines. Annals New York Academy of Science. The New York Academy of Science, New York, 1990, pp. 287-298. 14. Shih CC, Kaneshima H, Rabin L, Namikawa R, Sager P, McGowan J, and McCune JM: Postexposure prophylaxis with zidovudine suppresses human immunodeficiency virus type 1 infec¬ tion in SCID-hu mice in a time-dependent manner. J Infect Dis
1991:163:625-627.
REFERENCES 1. Putkonen P, Böttiger B, Warstedt K, Thorstensson R, Albert J, and Biberfeld G: Experimental infection of cynomolgus monkeys (Macaca fascieularis) with HIV-2. J AIDS 1989:2:366-373. 2. Putkonen P. Thorstensson R, Walther L, Albert J, Âkerblom L, Granquist O, Wadell G, Norrby E, and Biberfeld G: Vaccine protection against HIV-2 infection in cynomolgus monkeys. AIDS Res Human Retroviruses 1991;3:271-277. 3. Putkonen P. Thorstensson R, Ghavamzadeh L, Albert J, Hild K, Biberfeld G, and Norrby E: Prevention of HIV-2 and SIVsm infection by passive immunization in cynomolgus monkeys. Nature
1991;352:436-438. 4. Franchini G, Gurgo C, Guo HG, Gallo RC, Collalti E, Fargnoli KA, Hall LF, Wong-Staal F, and Reitz Jr MS: Sequence of simian immunodeficiency virus and its relationship to the human immun¬ odeficiency viruses. Nature 1987;328:539-543. 5. Lundgren B. Ljungdahl-Stâhle E. Böttiger D, Benthin R. Hedström K. Norrby E, Putkonen P. Wahren B, and Öberg B. Acute infection of cynomologus monkeys with simian immunodeficiency virus (SlVsm) as a model to evaluate antiviral compounds. Effects of 3'-azidothymidine, 3'-deoxythymidine, foscarnet and 2',3'dideoxycytidine. Antiviral Chem Chemother 1990:5:299-306.
15.
Ruprecht R, O'Brian LG. Rossini LD, and Nusinoff-Lehrman S: Suppression of mouse viremia and retroviral disease by 3'-azido3'-deoxythymidine. Nature 1986;323:467-469.
16. Tavares L, Roneker C, Johnston K, Nusinoff-Lehrman S, and de Noronha F: 3'-Azido-3'-deoxythymidine in feline leukemia virusinfected cats: a model for therapy and prophylaxis of AIDS. Cancer Res 1987;47:3190-3194. 17. Marcus R and the CDC Cooperative Needlestick Surveillance Group: Surveillance of health care workers exposed to blood from patients infected with the human immunodeficiency virus. Engl J Med 1988;319:1118-1123. 18. Shinazi RF, Boudinot FD. Doshi KJ, and McClure HM: Pharmacokinetics of 3'-fluoro-3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine in rhesus monkeys. Antimicrob Agent Chemother 1990:34:1214-1219.
Address
reprint requests to: Disa Böttiger Department of Virology
Karolinska Institute SBL S-105 21 Stockholm, Sweden
Prevention of HIV-2 and SIV Infections in Cynomolgus Macaques by Prophylactic Treatment with 3'-Fluorothy irridine DISA
BÖTTIGER,1
PER
PUTKONEN,2
and BO
ÖBERG1
ABSTRACT The aim of this study was to determine the usefulness of human immunodeficiency virus type 2 (HIV-2) for in vivo evaluation of antiviral drugs in monkeys and to study if prophylactic treatment with 3'-fluorothymidine (FLT) could prevent infection against a low challenge dose of HIV-2 or simian immunodeficiency virus (SIV). Protection against infection was assessed by virus isolation and polymerase chain reaction (PCR) on monkey peripheral blood mononuclear cells (PBMC) as well as by antibody and viral antigen assays. Prophylactic treatment with FLT 3x5 mg/kg/day, starting 8 h prior to virus inoculation, prevented HIV-2 infection in 3 of 8 monkeys. In another experiment 2 of 4 monkeys resisted 2-10 monkey infectious doses (MIDS0) of SIV with the same prophylactic treatment. All control animals (HIV-2 8, SIV 4) became infected. Thus, FLT treatment prevented HIV-2 and SIV infection in 5 of 12 animals. =
INTRODUCTION
IN
HUMANS, INFECTION with HIV-1 as well as HIV-2 is associated with immunosuppression and AIDS. Both HIV-1 and HIV-2 have narrow host ranges. HIV-2 has advantages over HIV-1 with regard to the use in experimental antiviral testing and vaccine studies because of its ability to infect cynomolgus monkeys, ' which is not an endangered species. The findings that not only SIV, but also certain strains of HIV-2 can infect cynomolgus monkeys have further improved the possibilities for effective evaluation of antiviral compounds. HIV-2SBL6669 has been adapted to replicate efficiently in cynomolgus monkeys and has been shown to be a useful tool for evaluation in various vaccine strategies. '~3 HIV-2 is genetically and serologically more closely related to certain SIV strains than to HIV-1. The overall homology between HIV-2 and SIVmac/SIV,m is approximately 70%.4 We have previously used an acute SIV infection in monkeys for the in vivo evaluation of antiviral compounds such as
azidothymidine (AZT), 2',3'-dideoxyinosine (ddl), 2',3'dideoxycytidine (ddC), dideoxythymidiene (d4T), and foscarnet, all known to affect the level of HIV p24 antigen in patients. Treatment starting prior to inoculation of 50-2500 MID5U of SIV delayed the appearance of viral antigen compared with the
Departments of 'Virology, and 2Immunology.
=
controls, but did not prevent infection." Similar results were obtained for 3'-fluorothymidine (FLT) in vivo in monkeys.5 FLT is a potent inhibitor of HIV-1, HIV-2, and SIV in cell
culture.8'9
The purpose of this study was to evaluate the use of HIV-2 in non-human primate as a tool in the development of antiviral drugs and to see whether prophylactic treatment could influence HIV-2 and SIV infection when a low inoculation dose of virus was used. a
MATERIAL Animals
Cynomolgus monkeys (Macaca fascicularis), of both sexes from the Philippines, were housed in single cages in a biosafety level 3 facility. The mean body mass was 2800 g and the range was 1875 g to 3825 g. Each animal was clinically healthy at the time of virus inoculation and shown to be HIV-2 and SIV antigen and antibody negative.
Drug
and
treatment
3'-Fluorothymidine (FLT), from Medivir AB, Huddinge, was dissolved in phosphate-buffered saline to a con-
Sweden,
Karolinska Institute SBL. Stockholm, Sweden.
1235
1236
BOTTIGER ET AL.
centration of 5 mg/ml, sterile filtered and used for subcutaneous injections on the backs of the animals. The total daily dose was 15 mg/kg. Collection of blood was performed under ketamine anesthesia.
detected in monkey peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR) amplification using nested primers specific for regions of the gag and pol genes of HIV-2/SIV as described.3
Virus and virus dose
HIV-2SBL()669/H5 was previously titered in vivo in monkeys as was 10 monkey infectious doses (MID50). The SIV strain used, originally isolated from naturally infected sooty mangabey monkeys (SIVsm), was kindly provided by Drs. P. Fultz and H. McClure (Yerkes Regional Primate Center, Animal Resources Branch, Altanta, NIH Grant No. RR00165). The SIV was previously titrated in vivo in monkeys as described, first in tenfold dilutions and then in threefold dilutions (5, SIV pool II). Altogether 20 monkeys were used for titration. The SIVsm inoculation dose was 2-10 MID50; this number reflects a range within which the true inoculum dose is likely to be. Stocks of virus had been prepared previously and were stored at —70°C in aliquots. Virus was administered intravenously.
EXPERIMENTAL
described.2'3 The HIV-2 inoculation dose
SIV and HIV-2 antigen were analyzed by using a crossreacting kit for p24 antigen detection (Abbott. North Chicago, IL) intended for HIV-1.10 Antibodies to SIV and HIV-2 were assayed by a peptide enzyme-linked immunosorbent assay ' ' (ELISA) method. Virus isolation was performed by cocultiva¬ tion with human peripheral blood lymphocytes (PBL) using standard techniques as described earlier.3 Proviral DNA was
of
RESULTS HIV-2
METHODS
Table 1. Presence
The experiments were designed as earlier described.5 The first treatment with FLT was initiated 8 h before and the second treatment 10-15 min before virus inoculation and continued every 8 h for 10 days. The S and series of HIV-2 animals were used in two consecutive experiments. Blood specimens were collected at intervals and screened for viral antigen and antibodies. Animals negative for viral antigen and antibodies were further examined by virus isolation and PCR.
Viral Antigen
in
infection
HIV-2 inoculation resulted in the appearance of a viral antigen peak at day 14-17 in all control monkeys, followed by HIV-2 antibody production (Table 1). In 5 of the 8 HIV-2-inoculated monkeys treated with FLT no viral antigen was detected. Three of these five animals did not seroconvert and no virus could be detected by isolation or by
PCR up to 11 months after HIV-2 inoculation. However, the two animals which lacked detectable viral antigen
remaining
Serum (A), Virus Isolation (B) and Presence in Monkeys Inoculated with HIV-2
of
Viral Antibodies
in
Serum
(C)
Control animals
Monkey No.
day
0 10 14
17 21 26
30
B.
SI
+ + + +
S3
+ +
+ + -¬
S4
Tl
T2
+ +
T3
T4
S6
S7
S8
T5
T6
T7
+ +
+ + -¬
+ + + -¬
+ + -¬
+ +
+ + +
+ + +
+ + + + -¬
+ + -¬
+
+
+ + + -¬
+ + + +
+ + -¬
+
-¬
-¬
T8
+ + +
+ + +
+
-¬
+
+
+ -¬
+
+
+
+
t
O
O S7 was reinoculated with HIV-2 after 5 months. The Not tested, t the second virus inoculation. 46/90; Control monkeys tested day 46, treated =
S5
treatment
+ +
day 0
14 month 1 2 3 5 11 day 0 21 30 46/90 month 1 1
S2
FLT
=
monkey seroconverted and yielded virus 30 days after monkeys tested day 90.
1237
PREVENTION OF HIV-2 AND SIV BY FLT IN MONKEYS later shown to be infected as determined by positive virus isolation at 30 days and seroconversion. Three FLT-treated animals had detectable antigens at day 21-25 after inoculation. However, the antigen peak was signif¬ icantly delayed as compared to the control animals, where antigen appeared at day 14-17. were
SIV
infection
All control monkeys became infected and presented a tran¬ sient peak of viral antigen on day 10-14, followed by seroconversion, as shown in Table 2. In contrast, 2 of 4 FLT-treated monkeys did not become infected as evident by repeatedly negative virus isolations, negative PCR and no detectable viral antigen or seroconversion up to 11 months. The two remaining treated animals became infected by SIV, although one of these animals did not have detectable viral antigen at any time assayed.
DISCUSSION FLT treatment initiated 8 h prior to virus inoculation pre¬ vented HIV-2 and SIV infection in 5 of 12 animals. This is to our knowledge the first demonstration of successful blocking of primate lentivirus replication with an antiviral drug in a nonhu¬ man primate. In the remaining treated monkeys treatment delayed the appearance of viral antigen compared with the controls. FLT showed similar potency in preventing SIV- and HlV-2-infection. This correlates with the results seen in cell culture where FLT is equally potent in inhibiting SIV and
HIV-2.9
We recently have shown that the amount of virus inoculated is important as to whether antiviral treatment can block virus replication or not. When a high inoculation dose of virus was used ( 100 MID,() of SIVsm) prophylactic treatment with FLT did
have any significant effect on the appearance of viral antigen.57 If the virus inoculum was reduced to 10-50 MID50 of SIV the appearance of viral antigen was markedly delayed.12 In the present experiments with HIV-2 and SIV we used a virus dose of 2-10 MID50 and this resulted in an infection which could be prevented by treatment with FLT, as earlier indicated for SIV.I2 In this experiment 4 of 4 control animals were infected. Using the same dose of virus 4 of 4 control monkeys were infected in the consecutive experiment (not shown). McClure et al. studied rhesus monkeys treated with 100 mg/kg/day of azidouridine (CS-87), AZT, d4T, and FLT, 24 hours before inoculation of approximately 75 MID5II of SIVsm. Prophylactic efficacy was not shown by virus isolation with any of the compounds, but one monkey given FLT did not seroconvert within 8 weeks. McClure et al.13 further reported that AZT prophylaxis 1 h post SIVsmmPBj 14 exposure, gave the result that 1 of 3 monkeys remained virus-negative and that 3 of 6 animals treated within 24 h after inoculation did not develop the charac¬ teristic lethal disease associated with SIVsmnPBjl4. It is not known if treatment efficacy in monkeys against this rapidly lethal isolate of SIV corresponds to treatment efficacy against the slow development of disease caused by HIV in humans. The influence of antiviral treatment on viral antigen appear¬ ance in serum has been seen in other retroviral experimental systems. SCID-hu mice represent an animal model for HIV in a laboratory animal in which therapeutic treatment with AZT was proven to suppress infection when given up to 2 h postinocula¬ tion. '4 Similar results have been reported for AZT therapy to mice infected with Raucher leukemia virus15 and in cats inocu¬ lated with FeLV."1 However, these models, apart from the SCID-hu mice model, use a virus considerably different from HIV. A clinically important issue is the problem with personnel accidentally exposed to HIV-infected blood or material. Due to the low incidence of infection after accidental exposure to HIV in the working place,17 the clinical studies on postexposure therapy using AZT are unlikely to give a clear answer. In the not
Table 2. Presence of Viral Antigen in Serum (A), Virus Isolation (B) and Presence of Viral Antibodies in Serum (C) in Monkeys Inoculated with SIV FLT
Control animals Monkey No. 0 10 14 21 B. month 1 2 3 5 II c. day 0 14 21 month 1 2 5 11
day
A.
O
=
Not tested
17 + + + + + -¬ +
+ -¬
o +
18
19
20
+ --
+ + + --
+ + + + -¬
o
o
+
+
+ --
+ +
o
o o
+
-¬
o o o o
o o o o
21
22
treatment
23
24
o o
o o o
+ + +
+ + + + +
BOTTIGER ET AL.
1238
present study
showed that HIV-2 and SIV infections can be prevented by prophylactic treatment. These findings have impli¬ cations for potential postexposure treatment. The clinical and experimental situations used here are not directly comparable in terms of virus inoculum (cell-associated versus cell-free virus) and route of virus exposure. These are differences which could be addressed experimentally. It remains to be determined in future studies if postexposure treatment can block infection in this model. Prophylactic treatment with FLT did not prevent infection in all treated animals. Dosing FLT every 8 h might not be optimal to obtain sufficient serum levels of FLT, reported to have a serum half-life of 0,8-0.9 h.718 However, intracellular levels of FLT triphosphate in vivo, which has not been studied so far, are more relevant for the effect of the drug. It is not clear for how long a time virus remains replicatively competent after transmis¬ sion. In our study a treatment period of 10 days stopped virus replication in 5 of 12 monkeys. A longer treatment period might have prevented infection in a higher percentage of animals. On the other hand, it is not excluded that treatment for fewer than 10 days could have given the same results. In conclusion, we demonstrate that prophylactic treatment with FLT can block primate lentivirus replication in vivo in a nonhuman primate. HIV-2 as well as SIV infection in cynomol¬ gus monkeys represent useful experimental models for the evaluation of antiviral drugs against HIV. we
ACKNOWLEDGMENTS The study was supported by the Swedish Board for Technical Development, Stockholm and by Medivir AB, Huddinge, Swe¬ den.
6.
7.
8.
9.
10.
11.
12.
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