Institute for Virus Research, Laboratory of Viral Pathogenesis, Kyoto University, 53 Shogoin-kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan
Correspondence
Eiji Ido
eido{at}virus.kyoto-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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SIV is genetically similar, but not identical, to HIV-1. The findings obtained from the SIV/monkey system cannot be applied directly to the case of HIV-1 infection in humans. Therefore, to develop a better animal model, we previously generated simian/human immunodeficiency chimeric viruses (SHIVs) that can infect monkeys. These SHIVs possessed the 3' half of several HIV-1-derived genes, including the env gene, using the SIV genome as a backbone (Kuwata et al., 1995; Li et al., 1992
; Shibata et al., 1991
). The SHIVs containing the env gene of HIV-1 were shown to be useful for evaluating the efficacy of anti-HIV-1 vaccine candidates targeting Env proteins by using them as challenge viruses for vaccinated monkeys (Ui et al., 1999
). Although these SHIVs were shown to be non-pathogenic (Hayami et al., 1999
), pathogenic SHIVs were also developed by animal passage, starting from initially non-pathogenic SHIVs (Joag et al., 1996
, 1997
; Reimann et al., 1996
). Using the SHIV/monkey system, Harouse et al. (1999)
demonstrated the role of co-receptor usage of HIV-1 Env for pathogenesis. Thus, SHIV/monkey systems have been valuable tools for understanding, at least, in part, the biological properties of HIV-1 and for developing HIV vaccines.
Most of the SHIVs reported to date, including the SHIVs generated by our group, had the 3' half of the HIV-1 genome on an SIV backbone. For instance, one of the SHIVs generated by us, which was termed NM-3rn, possessed vpr, vpu, tat, rev, env and nef of HIV-1 (Kuwata et al., 1995). These findings naturally lead us to the following question: to what extent can we replace SIV genes with those of HIV-1 without losing their infectivity to monkeys? The answer to this question may help to develop a better animal model for AIDS, one that ultimately mimics HIV-1 infection in humans.
We have been trying to construct a new SHIV chimera that has a broader HIV-1-derived region by the addition of various regions of the 5' part of HIV-1, including pol, to the previously reported SHIV containing the 3' half of HIV-1. Here, we report that we succeeded in constructing a new SHIV chimera, one which contains the reverse transcriptase (RT)-encoding region (rt region) of pol, in addition to the 3' half of the HIV-1 genome, and that this virus was able to infect and replicate in monkey peripheral blood mononuclear cells (PBMCs) and in macaque monkeys in vivo. This newly constructed SHIV has more of the HIV-1 genome, such as rt, vpr, vpu, tat, rev, env and nef, than any of the SHIVs reported so far.
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METHODS |
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Next, we introduced an XbaI site near the N-terminal end of the integrase gene for both HIV-1 and SIVmac by PCR-based site-directed mutagenesis. For HIV-1, the following oligonucleotide primer was used: NLRT-R, 5'-TTCCATCTAGAAATAGTACTTTCCTGATTCCAGC-3'. For SIVmac, the oligonuceotide primer MAIN-F, 5'-CTCTTTCTAGAAAAGATAGAGCCAGCACAAGAAG-3', was used; these primers were designed to create an XbaI site into the respective sequences (nt 4232 for HIV-1 and nt 4786 for SIVmac) without alteration to the amino acid sequences (letters in boldface represent the XbaI site and letters underlined represent the mutated nucleotides).
With the restriction sites at the junctions of the protease/RT and RT/integrase genes, two retrovirus sequences, SIVmac and HIV-1, were reassembled by conventional molecular recombinant techniques to produce a chimeric pol gene. The SpeI (nt 2026 in pMA239, gag) to NspV (nt 6131 in pMA239, vif) fragment of this chimeric gene was then inserted into the corresponding position of an HIV-1 env-possessing SHIV plasmid, pNM-3rn (Kuwata et al., 1995), to generate a novel full-genome plasmid, termed pSHIVrt/3rn.
Cell cultures.
M8166 is a subclone of C8166 (Clapham et al., 1987), a CD4+ human T-cell line. HSC-F is a cynomolgus monkey CD4+ T-cell line from a foetal splenocyte that was immortalized by infection with herpesvirus saimiri subtype C (Akari et al., 1996
). M8166 and HSC-F cells were maintained in RPMI 1640 medium containing 10 % heat-inactivated foetal bovine serum (FBS). PBMCs of healthy rhesus monkeys were separated from heparinized whole blood by Percoll density-gradient centrifugation, stimulated with 25 µg concanavalin A ml-1 for 24 h and maintained in RPMI 1640 medium containing 10 % FBS and 400 units recombinant IL-2 ml-1, as described previously (Kuwata et al., 1995
).
Transfection and infection.
For generating infectious virus particles from a full-genome plasmid DNA, 5 µg pSHIVrt/3rn was introduced into 1·5x106 M8166 cells using the DEAEdextran method (Naidu et al., 1988). Culture medium was changed every 3 days and the supernatant was filtered (0·45 µm pore size) and stored at -80 °C. Virion-associated RT activity was measured as described previously (Willey et al., 1988
). The supernatant with the highest RT activity was used as virus stock. To determine the virus infectivity of the stocks, the TCID50 was calculated using M8166 cells, as described previously (Igarashi et al., 1994
). The virus inoculum used for in vitro infection was adjusted to contain a certain amount of RT units (typically 34x103 RT units) by adding the appropriate volume of the medium to the virus stock. M8166 cells, HSC-F cells or monkey PBMCs (1x106 cells per well) were infected with a virus and cultured in a 96-well plate. The culture supernatant was harvested every 3 days and its RT activity was monitored.
Effect of an RT inhibitor on virus replication.
MKC-442 is a non-nucleoside-type RT inhibitor (Yuasa et al., 1993) that inhibits the RT activity of HIV-1 specifically, but not that of SIV. The ability of MKC-442 to block virus replication was examined. MKC-442 was diluted from a 2 mM stock solution with the culture medium and used at 200 and 20 nM.
Inoculation of monkeys.
All animals were housed in a P3-level monkey storage facility and were treated in accordance with regulations approved by the Committee for Experimental Use of Nonhuman Primates in the Institute for Virus Research, Kyoto University, Japan. To investigate the replication competence of SHIVrt/3rn in vivo, two female adult rhesus monkeys (Macaca mulatta) were inoculated intravenously with the virus stock of SHIVrt/3rn containing 1x105 TCID50 per monkey. After inoculation, blood samples were collected periodically from the inoculated monkeys and were separated into plasma and PBMCs. Then, plasma viral RNA loads, proviral DNA and CD4+ cells were analysed. Virus isolation was attempted as described below.
Virus isolation.
We attempted to isolate infectious virus from the PBMCs of inoculated monkeys as follows. Serially threefold diluted PBMCs were co-cultured with 2·5x105 M8166 cells for 4 weeks in RPMI 1640 containing 10 % of FBS in a 48-well plate. Virus recovery was judged by the cytopathic effect (CPE) of syncytia formation and a rise in RT activity of the culture supernatants.
Detection of proviral DNA in PBMCs.
Proviral DNA in PBMCs of inoculated monkeys was detected with nested DNA PCR, as described previously (Igarashi et al., 1996). The primers used in this study were designed to amplify the V3 region of HIV-1 (NL432) env specifically, which is present in the SHIVrt/3rn genome.
Determination of plasma viral RNA loads.
Plasma viral RNA loads after inoculation were determined by quantitative RT-PCR, as described previously (Kozyrev et al., 2002).
Titration of antibody.
Antibody titres of the monkey plasma after inoculation with SHIVrt/3rn were measured by particle agglutination according to the instructions of the manufacturer (Serodia HIV-1/2, Fujirebio).
CD4+ cell kinetics.
After two rhesus monkeys were inoculated with SHIVrt/3rn, PBMCs were isolated from the animals and CD4+ cell numbers in the PBMCs were calculated, as described previously (Ui et al., 1999).
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RESULTS |
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To investigate the virus growth kinetics of SHIVrt/3rn, we infected M8166 cells, a monkey CD4+ T-cell line (HSC-F), and monkey PBMCs with the virus and monitored RT activity in culture supernatants (Fig. 2). In M8166 cells, the replication of SHIVrt/3rn reached a peak at about 19 days post-infection (p.i.) (Fig. 2a
). This profile of kinetics was similar to that of NM-3rN, except that there was a slight delay in the initial rise in RT activity, indicating that SHIVrt/3rn could replicate well in this human CD4+ cell line with almost the same replication competence as NM-3rN. In HSC-F cells, the replication of SHIVrt/3rn was delayed compared with that of NM-3rN and reached a peak at about 15 days p.i. (Fig. 2b
). In monkey PBMCs, replication of SHIVrt/3rn reached a peak at about 22 days p.i. (Fig. 2c
). Judging from the RT values, SHIVrt/3rn replicated to somewhat lower titres than NM-3rN. Nevertheless, it was clearly able to replicate in monkey PBMCs.
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Firstly, we examined whether or not there were infected PBMCs that could generate infectious virus by co-culturing with M8166 cells (Table 1). From the PBMCs of MM251, infectious virus was isolated at 2 and 3 weeks p.i., and at 4 weeks p.i. from the PBMCs of MM257. To detect proviral DNA in the isolated PBMCs, we performed DNA PCR using the extracted chromosomal DNA from the PBMCs (Table 1
). In the PBMCs from both MM251 and MM257, proviral DNA was detected constantly throughout the observation period, starting from 3 weeks p.i. until 32 weeks p.i. To determine plasma viral RNA loads, we extracted RNA from the isolated plasma samples and performed quantitative RT-PCR (Fig. 4
a). The plasma viral RNA loads of MM251 exhibited a peak during 2 to 3 weeks p.i., and those of MM257 did so during 34 weeks p.i. The number of RNA copies at the peak period was 2·6x104 and 1·2x104 copies ml-1, respectively. As for MM251, viral RNA was detected in the plasma samples up to 10 weeks p.i. On the other hand, no viral RNA was detected in the plasma samples of MM257 after 6 weeks p.i. To detect and titrate antibodies against HIV-1, we measured particle agglutination antibody titres using the Serodia HIV-1/2 kit (Fujirebio) (Table 1
). Antibodies were detected in both monkeys first at 3 weeks p.i. and were maintained with high titres (4096) up to 32 weeks p.i. In addition, we analysed the number of CD4+ cells in these rhesus monkeys after the inoculation. Both monkeys showed no significant decrease in the number of CD4+ cells up to 32 weeks p.i. (Fig. 4b
), suggesting that SHIVrt/3rn is non-pathogenic at this stage.
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DISCUSSION |
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Since the newly constructed SHIVrt/3rn has the RT of HIV-1, we confirmed the inhibition of the replication of SHIVrt/3rn by an HIV-1-specific non-nucleoside-type RT inhibitor, MKC-442, in vitro. The 90 % effective concentration (EC90) and the 50 % effective concentration (EC50) of MKC-442 against HIV-1 were reported to be 98 and 15 nM, respectively (Baba et al., 1994). EC90 and EC50 values were defined as the concentrations at which 90 and 50 % of HIV-1 induced CPE in MT-4 cells were protected (Pauwels et al., 1988
). In this study, the replication of SHIVrt/3rn was inhibited completely by MKC-442 at 200 nM. In addition, the initial rise in RT activity of SHIVrt/3rn was delayed by 20 nM MKC-442, which is considered to be a consequence of incomplete inhibition. (Fig. 3b
). These results indicate that the sensitivity to MKC-442 of SHIVrt/3rn is similar to that of HIV-1, although it should be noted that different cells were employed for this inhibition assay. Today, highly active antiretroviral therapy, in which combinations of RT inhibitors and protease inhibitors are used, has shown satisfactory clinical benefits. Moreover, new anti-HIV drugs targeting other virus components, such as Env, are being developed. Since monotherapy resulted, in most cases, in failure, such entry blockers should be prescribed in combination with other drugs such as RT inhibitors. Having the RT and Env of HIV-1 and having a sensitivity to an RT inhibitor similar to that of HIV-1, SHIVrt/3rn can be used for the in vivo evaluation of a new combination therapy of HIV-1-specific RT inhibitors and entry blockers, such as CXCR4 antagonists, in monkeys.
Two rhesus monkeys were inoculated with 1x105 TCID50 SHIVrt/3rn and both monkeys were infected consequently. The in vivo replication of NM-3rn, the parental molecular clone of SHIVrt/3rn, was reported previously (Bogers et al., 1997). According to that report, eight rhesus monkeys were inoculated with NM-3rn at six different virus titres, ranging from 6·3x103 to 6·3x10-1 TCID50. Seven of the eight monkeys were infected with NM-3rn. Viruses were isolated continuously from 2 to 12 weeks p.i. from five of the seven infected monkeys. In this study, we also performed virus isolation. However, we could isolate viruses only at 2 and 3 weeks p.i. from one of the two monkeys and at 4 weeks p.i. from the other (Table 1
). In the case of NM-3rn infection, the results of DNA PCR showed the presence of proviral DNA from all seven monkeys infected with NM-3rn at 2 and 4 weeks p.i. and from all three monkeys analysed out of the seven at 8 weeks p.i. In this study, we also performed DNA PCR and could detect proviral DNA constantly, starting at 3 weeks p.i., from both of the monkeys, although we could not detect it at 2 weeks p.i. (Table 1
). These results suggest that SHIVrt/3rn possesses a slightly lower replication competence in vivo than NM-3rn. This weak replication competence was also observed in vitro, as shown by the growth kinetics in monkey PBMCs (Fig. 2c
). The replacement of the rt region of SIV with that of HIV-1 might have affected the replication potential of the virus.
Überla et al. (1995) constructed a SHIV having the rt region of HIV-1 and the rest of the genome from SIVmac (RT-SHIV). Two rhesus monkeys inoculated with RT-SHIV exhibited a systemic infection and one of them developed an AIDS-like symptom at approximately 6 months p.i. This result suggested that RT-SHIV possessed a higher replication competence in vivo than SHIVrt/3rn. This difference may be because SHIVrt/3rn and RT-SHIV use different strains of HIV-1 for the rt region, or because SHIVrt/3rn has a broader HIV-1-derived region, one that covers the region from vpr to nef. Although the reason for the lower replication competence of SHIVrt/3rn is not clear at this stage, we expect that it can be improved by serial animal passages and/or by using different strains of HIV-1.
We are now attempting to make new SHIVs in which the HIV-1-derived region is as broad as possible, without losing the infectivity of the virus to monkeys. We hope this will allow us to establish a novel animal model that ultimately mimics HIV-1 infection in humans and to identify the virus determinants of the species tropism of HIV-1, namely, the virus factors that restrict HIV-1 replication in monkey cells.
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ACKNOWLEDGEMENTS |
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Received 20 September 2002;
accepted 25 February 2003.