Journal of General Virology (1992), 73, 2983-2987.

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Printed in Great Britain

Infection of macaque monkeys with a chimeric human and simian immunodeficiency virus Sayuri Sakuragi, 1 Riri Shibata, 1 Ryozaburo Mukai, 2 Toshihiko Komatsu, 3 Masashi Fukasawa, 4 Hiroyuki Sakai, 1 Jun-lchi Sakuragi, 1 Meiko Kawamura, 1 Kentaro lbuki, 1 Masanori Hayami 1 and Akio Adachi ~* 1Institute for Virus Research, Kyoto University, Kyoto 606, 2Tsukuba Primate Center for Medical Science, National Institute of Health, Tsukuba 305, 3Division of Poxviruses and Special Pathogens, National Institute of Health, Musashimurayama 190-12 and 4institute of Tropical Medicine, Nagasaki University, Nagasaki 852, Japan

Two macaque monkeys were inoculated with a chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu and env genes of human immunodeficiency virus type 1. Infectious virus was recovered from one of the monkeys at 2 and 6 weeks

post-infection. The hybrid nature of the isolated viruses was verified by Southern and Western blotting analyses. Both of the monkeys infected with the chimera elicited a humoral antibody response against the virus.

The host range of human immunodeficiency virus type 1 (HIV-1), the major causative virus of human AIDS, is narrow and other animal species susceptible to HIV-1 infection are few. However, a number of systems for the study of animal models of HIV-I infection have been reported. These include transgenic mice (Leonard et al., 1988), higher primates (Alter et al., 1984; Fultz et al., 1986; Gajdusek et al., 1985; Lusso et al., 1988; Nara et al., 1987), rabbits (Filice et al., 1988; Kulaga et al., 1989) and SCID-hu mice (Namikawa et al., 1988). Although the experimental systems described are of value for certain aspects of HIV-1 research, a new approach to investigate extensively the biology and molecular biology of HIV-1 in vivo, and to facilitate a practical study should be undertaken. The similarities between HIV-1 and simian immunodeficiency virus (SIV) in genome sequence and organization, and in their biological properties, have suggested that SIV systems would provide useful animal models for the study of AIDS (Desrosiers & Ringler, 1989). In fact, macaque monkeys experimentally infected with molecularly cloned SIVs develop the disease (Dewhurst et al., 1990; Kestler et al., 1990). The reason for the narrow host range of HIV-1 is presently unknown. However, by using chimeric clones generated between macaque monkey-tropic SIV and HIV-1, and SIV mutants, we have recently demonstrated that the vpx, vpr, tat, rev, env and nefgenes of SIV are not essential for macaque cell tropism (Shibata et al., 1991). We have also found that one of the chimeric viruses, which carries HIV-1 tat, rev, vpu and env genes, can

productively infect macaque cells in vitro (Shibata et al., 1991). In this communication, we report the inoculation of the infectious chimeric virus into macaque monkeys. The chimeric construct NM-3 used in this study (Fig. l a), which is a hybrid clone between HIV-1 NL432 (Adachi et al., 1986) and SIV MAC239 (Naidu et al., 1988), is infectious to peripheral blood mononuclear cell (PBMC) cultures from cynomolgus monkeys (Macaca fascicularis) (Shibata et al., 1991). To determine whether various PBMC cultures differ in their susceptibility to the virus, NM-3 virus obtained from transfected SW480 cells (Adachi et al., 1986) was inoculated into PBMC preparations from four cynomolgus monkeys. For controis, PBMC cultures were also infected with the parental viruses of NM-3 (NL432 and MAC239) and monitored for their replication. HIV-1 NL432 did not grow in any of the cynomolgus monkey PBMC cultures (Fig. 1b) as reported for other HIV-1 strains (McClure et al., 1987). In contrast, the NM-3 virus replicated well in all four PBMC cultures to an extent similar to that of the parental monkey cell-tropic MAC239 virus. These results indicated, together with the data presented in our previous report (Shibata et al., 1991), that the growth capability of the chimeric NM-3 virus carrying HIV-1 tat, rev, vpu and env genes in monkey PBMCs cultured in vitro is almost indistinguishable from that of SIV MAC239 virus. To determine whether NM-3 virus can establish an infection in vivo, two male cynomolgus monkeys [no. 1 (11 years old) and no. 2 (8 years old)] were inoculated intravenously with the NM-3 virus grown in M8166 cells

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Fig. 1. Growth of HIV-1/SIV chimeric virus NM-3 in macaque PBMCs. (a) Structure of NM-3 genome. Restriction enzyme sites used to construct NM-3 are indicated with nucleotide positions. (GenBank accession numbers: M19921 for HIV-1 NL432 and M33262 for SIV MAC239.) Speckled boxes represent truncated genes due to recombination. (b) Kinetics of progeny virus production (RT production; Willey et al., 1988) in parallel cultures of PBMCs from four cynomolgus monkeys (A to D) at different times post-inoculation with the chimera (11), SIV MAC239 (~), HIV-1 NL432 (~) or no virus (©). To generate virus stocks for infection, SW480 ceils (Adachi et at., 1986) were transfected with molecular clones pNL432 (Adachi et al., 1986), pMA239 (Shibata et al., 1991) or prcNM-3 (Shibata et al., 1991), and cell-free supernatants were collected on day 2. PBMCs, separated and prepared from monkeys by the standard method (Ohta et al., 1988), were infected with the same amounts of virus (RT units) as described by Folks et al. (1985).

[approximately 104 TCID50 in M8166 cells (Clapham et al., 1987) per monkey]. The monkeys were examined for the presence of proviral D N A copies at 2 and 12 weeks post-inoculation. Genomic DNAs were prepared by the

Fig. 2. Presence of proviral DNA in macaque monkeys inoculated with NM-3 virus. PBMCs for PCR analysis (Saiki et al., 1988) were prepared from two cynomolgus monkeys (nos 1 and 2) infected with the NM-3 virus at 2 and 12 weeks post-inoculation. Samples (1 ixg genomic DNA) were subjected to 35 cycles of PCR. The location (arrowheads) and sequence of the primer pair used to amplify the HIV-1 NIA32 env gene are indicated in (a). (b) PCR products were run through a 3% agarose gel and subjected to Southern blotting analysis (Folks et al., 1985). A B g l l I - B g l I I DNA fragment (a) was labelled with ~2p using the random primer DNA labelling kit (Takara Shuzo) and used as a probe. The size of the specific product was predicted to be 660 bp. PBMC genomic DNA was from: monkey no. 1 at 2 weeks postinoculation (lane 1), 12 weeks post-inoculation (lane 3) and before infection (lane 5); and monkey no. 2 at 2 weeks post-inoculation (lane 2), 12 weeks post-inoculation (lane 4) and before infection (lane 6).

standard method (Folks et al., 1985) from PBMC cultures of the two monkeys and analysed by the polymerase chain reaction (PCR) (Saiki et al., 1988). Proviral DNAs were detected in the PBMC DNAs from both monkeys (Fig. 2). However, copy numbers of viral DNA appeared to decrease during the course of infection (especially in monkey no. 2). The two monkeys were also checked at intervals (2, 6, 8, 10 and 12 weeks postinoculation) for the presence of infectious virus by cocultivation of PBMCs from monkeys infected with NM-3 virus and M8166 cells, which are highly susceptible to infection by various isolates of HIV and SIV. Virus production was monitored for 1 month by extensive microscopic observation of cytopathic effects and reverse transcriptase (RT) assay. Viable virus was recovered only from monkey no. 1 at 2 and 6 weeks postinoculation. Virus was not isolated by the cocultivation of PBMCs from the infected monkeys (8 and 10 weeks post-inoculation) and PBMCs from uninfected monkeys. The general structure of the re-isolated virus was determined by Southern and Western blotting analyses of virus-producing M8166 cells (Fig. 3). Four D N A probes were used for Southern hybridization. HIV-1 sequences could be detected by probes N1 [4-3 and 1.2 kb

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26K-Fig. 3. (a) Structure of virus re-isolated from macaque monkey inoculated with NM-3 virus. M8166 cells persistently infected with PBMC-derived NM-3 virus or transfected SW480-derived HIV-1 NL432 and SIV MAC239 were subjected to Southern (b) and Western (c) blotting analyses (Folks et al., 1985; Willey et al., 1988). (b) Southern hybridization analysis of unintegrated virus D N A s obtained from infected M8166 cells. Viral DNAs were prepared by the method of Hirt (1967). DNAs cleaved with HindlII (positions are indicated by nucleotide nos above bars) were subjected to Southern blotting analysis using four DNA probes. The DNA fragments expected to be observed in our experimental conditions are indicated. Solid triangles represent the restriction sites used to make the chimeric clone prcNM-3. Size markers (kb) are shown on the left. Lanes: c, negative control; h, HIV-I NL432; s, SIV MAC239; 3, chimeric virus NM-3 (isolated from monkeyno. 1 at 6 weeks post-inoculation). (c) Western immunoblotting analysis of viral proteins. Lysates prepared from M8166 cells infected with viruses were subjected to Western blotting analysis with either anti-HIV-I human serum (upper panel) or anti-SIV macaque serum (lower panel). Size markers are indicated on the left. Lanes: c, negative control; h, HIV-1 NL432; s, SIV MAC239; 3, chimeric virus NM-3 (isolated from monkey no. 1 at 6 weeks post-inoculation). The same results (for both Southern and Western blotting analyses) were obtained with NM-3 virus isolated from monkey no. 1 at 2 weeks postinoculation (data not shown).

bands (fragments A and C, respectively, in Fig. 3) by HindlII digestion] and N2 [4-3 and 2.1 kb (fragments A and B, respectively)] (lanes h). Probes M1 and M2 recognize 6-2 kb (fragment a) and 3.5 kb (fragment b) HindlII cleavage products of SIV MAC239 DNA, respectively (lanes s). The Southern hybridization profile of NM-3 virus, obtained by cocultivation of PBMCs and M8166 ceils, was exactly as predicted from its initial structure (Fig. 3b). HIV-1 2.1 kb and SIV 6.2 kb bands were detected by probes N2 and M1, respectively (lanes 3). In addition, no specific signals were observed with probes N1 and M2 (lanes 3). The hybrid nature of the

NM-3 virus isolated from monkey no. 1 was further confirmed by Western blotting (Fig. 3c). The human antiserum used here (Fig. 3 c, upper panel) can react with HIV- 1 env-gp120, gag-p24 (lane h) and SIV gag-p26 (lane s). The monkey serum (Fig. 3 c, lower panel) recognizes SIV env-gp41, env-gpl30 and gag-p26 (lane s). As demonstrated in Fig. 3 (b), NM-3 virus had SIV gag and HIV-1 env in agreement with the chimeric structure (lanes 3). To investigate the humoral antibody response to NM-3 virus infection, sera were sampled from the two monkeys at 2, 4, 6, 8, 10 and 12 weeks post-inoculation,

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26 Fig. 4. Antibody response of macaque monkeys to infection by NM-3 virus, gag- and env-encoded proteins of HIV-I NL432 and SIV MAC239 detected by Western blotting with sera collected from two cynomolgus monkeys (nos 1 and 2) infected with the chimeric NM-3 virus are shown. Lysates prepared from M8166 cells infected with HIV-1 NL432 (HIV-1, upper three panels) or SIV MAC239 (SIVMAc, lower two panels) were resolved by PAGE and electrophoretically transferred onto nitrocellulose membranes (Willey et al., 1988). The membranes were cut vertically into strips, incubated with various sera and with 12SI-labelled Protein A, then visualized by autoradiography (Willey et al., 1988). Samples were run on the same gels. The positions of gag p24 (24) and env gpl20 (120) of HIV-1, and of gag p26 (26), env gp41 (41) and gp130 (130) of SIVuAc are indicated. Lanes: p, preinfection serum (100-fold dilution); 2 to 12, sera collected at 2 to 12 weeks post-inoculation (100-fold dilution), respectively; e, monoclonal anti-HIV-1 env gpl20 serum (Epitope Inc.); g, monoclonal antiHIV-1 gag p24 serum (Cellular Products Inc.); s, anti-SIV macaque serum.

and monitored for reactivity with virus antigens by Western blotting (Fig. 4). In sera of two infected monkeys, antibodies appeared 6 to 8 weeks postinoculation and persisted during the observation period. Consistent with the structure of NM-3 virus, only antiHIV-1 env and anti-SIV gag antibodies were detected. However, the extent and the appearance of these antibodies during the course of infection varied considerably between the monkeys. Anti-HIV-1 env gpl20 activity in monkey no. 2 was clearly detected at 6 weeks post-inoculation and thereafter. In contrast, a relatively

low level of activity was detected at 12 weeks postinoculation in monkey no. 1. Although no recognizable anti-SIV gag antibody was present in the sera of monkey no. 2, a high titre was detected in the sera of monkey no. 1. Of note is the failure to recover virus from monkey no. 2 despite the presence of viral DNA. In this study, two cynomolgus monkeys were inoculated with the HIV-1/SIV chimeric NM-3 virus, and virus recovery and host response were monitored. Both monkeys demonstrated high antibody responses to the virus following inoculation. High titres of the antibodies were still detected in the sera of the two monkeys at 28 weeks post-inoculation, though the titres declined thereafter (data not shown). The HIV-1 sequence was detected by the PCR method in DNAs prepared from PBMC cultures of the monkeys. Although virus was recovered from the inoculated monkeys only on some occasions, these data strongly suggest that the NM-3 virus is replication-competent in vivo in cynomolgus monkeys. In addition to enabling basic studies, this macaque system with a chimeric virus may facilitate practical approaches to, for instance, HIV-I env vaccine research. Apart from small changes that might occur, the chimeric structure of NM-3 virus was maintained during the course of infection in vivo. The infection process of NM-3 virus in vivo appeared less efficient than in vitro. The infected monkeys were apparently healthy 1 year post-inoculation. Under similar experimental conditions, progeny virus was easily isolated from monkeys inoculated with the parental monkey cell-tropic MAC239 virus (Kestler et al., 1991; S. Sakuragi et al., unpublished observation). The basis for the difference between the in vitro and in vivo activities of NM-3 virus is presently unknown. It is worth mentioning that the n e f gene of SIV MAC239 is reportedly required for the maintenance of high virus loads and for the development of AIDS in monkeys (Kestler et al., 1991). The NM-3 virus in this study lacks an intact n e f gene. It is also possible that the HIV-1 sequence in the chimeric virus affects the in vivo activity of the virus. It is clear that more chimeric clones should be constructed and analysed. This work was supported in part by a grant-in-aid for AIDS research from the Ministry of Education, Science and Culture of Japan.

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(Received 24 April 1992; Accepted 27 July 1992)

Infection of macaque monkeys with a chimeric human and simian immunodeficiency virus.

Two macaque monkeys were inoculated with a chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu and env genes of human immunode...
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