Protection of Macaques against a SHIV with a Homologous HIV-1 Env and a Pathogenic SHIV-89.6P with a Heterologous Env by Vaccination with Multiple Gene-Deleted SHIVs

Virology 265, 252–263 (1999) Article ID viro.1999.0049, available online at http://www.idealibrary.com on

Protection of Macaques against a SHIV with a Homologous HIV-1 Env and a Pathogenic SHIV89.6P with a Heterologous Env by Vaccination with Multiple Gene-Deleted SHIVs Masahiro Ui,* Takeo Kuwata,* Tatsuhiko Igarashi,* ,1 Kentaro Ibuki,* Yasuyuki Miyazaki,* Iouly L. Kozyrev,* ,1 Yoshimi Enose,* Toshihide Shimada,† Hiromi Uesaka,* ,‡ Hiroshi Yamamoto,‡ Tomoyuki Miura,* and Masanori Hayami* ,2 *Laboratory of Viral Pathogenesis, Institute for Virus Research, and †Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan; and ‡Laboratory Animal Research Center, Toyama Medical and Pharmaceutical University, Toyama 930-0152, Japan Received May 4, 1999; returned to author for revision July 26, 1999; accepted October 13, 1999 To evaluate the potential of SHIVs as anti-HIV-1 live vaccines, we constructed two gene-deleted SHIVs, designated SHIV-drn and SHIV-dxrn. The former lacks vpr/nef and the latter lacks vpx/vpr/nef. Four macaques that had been vaccinated with SHIV-drn were challenged with SHIV-NM-3rN, which has an HIV-1 Env that is the same as that of SHIV-drn. No challenge virus was detected by DNA PCR in, or recovered from, two of the macaques. In the other two, challenge virus was detected once and twice, respectively. Plasma viral loads were much lower than those in unvaccinated controls. Another four macaques were vaccinated with SHIV-dxrn. These macaques showed resistance but less than that of SHIV-drn-vaccinated macaques. When the two SHIV-drn-vaccinated macaques were challenged with pathogenic SHIV-89.6P, which has an HIV-1 Env that is antigenically different from that of SHIV-drn, replication of the challenge virus was restricted, and the usual decrease in the number of CD4 1 cells was prevented. In this protection, it is noteworthy that protection involved not only neutralizing antibodies and killer cell activity, but also other unknown specific and nonspecific immunity elicited by the infection. © 1999 Academic Press

INTRODUCTION

Reimann et al., 1996a,b), and vaccine efficacy against challenges with these SHIVs has been evaluated (Dunn et al., 1997; Joag et al., 1998; Shibata et al., 1997; Stephens et al., 1997). Previous experience with viral vaccines, such as those for poliomyelitis and measles, has proved that live attenuated vaccines are the most effective. Efforts to develop anti-HIV-1 live vaccines have been carried out using HIV-1/chimpanzee and SIV/macaque animal model systems (for recent reviews see (Almond and Heeney, 1998; Desrosiers, 1998; Letvin, 1998). Chimpanzees infected with HIV-1 resisted superinfection with other HIV-1 strains (Fultz et al., 1987; Gibbs et al., 1991; Shibata et al., 1996). In the SIV/macaque system, attenuated SIVs protected vaccinees against challenge with not only antigenically homologous viruses (Almond et al., 1995; Connor et al., 1998; Daniel et al., 1992; Lohman et al., 1994; Marthas et al., 1990; Otsyula et al., 1996; Robinson et al., 1999; Stahl-Hennig et al., 1996; Van Rompay et al., 1996), but also heterologous viruses (Bogers et al., 1995; Clements et al., 1995; Dunn et al., 1997; Gibbs et al., 1991; Gundlach et al., 1998; Letvin et al., 1995; Miller et al., 1997; Petry et al., 1995; Putkonen et al., 1995; QuesadaRolander et al., 1996; Shibata et al., 1997; Stephens et al., 1997). Based on the success of the SIV/macaque model system, attenuated HIV-1s, which were constructed by gene deletion or which were isolated from long-term

Many attempts have been made to develop anti-human immunodeficiency virus type 1 (HIV-1) vaccines to control the AIDS pandemic. However, the lack of a suitable animal model is a major obstacle to developing anti-HIV-1 vaccines because few animal species are susceptible to HIV-1 infection. Chimpanzees are commonly used for experimental infection, but the original HIV-1 hardly develops AIDS-like disease in chimpanzees (Nara et al., 1987; Novembre et al., 1997). Another problem with chimpanzees is that they are not easily available. We previously demonstrated that HIV-1/SIVmac chimeric viruses (SHIVs) that have HIV-1 Env and that are infectious to macaque monkeys are useful for evaluating the efficacy of anti-HIV-1 vaccine candidates by using them as challenge viruses for vaccinated monkeys (Igarashi et al., 1994, 1997; Kuwata et al., 1995; Shibata et al., 1991). So far, many SHIVs have been developed from different HIV-1 strains (Joag et al., 1997; Klinger et al., 1998; Kuwata et al., 1996; Li et al., 1992; Luciw et al., 1995;

1

Present address: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland 20892-0460. 2 To whom correspondence and reprint requests should be addressed. Fax: 181 75 761 9335. E-mail: [email protected]. 0042-6822/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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nonprogressors, were proposed as vaccine candidates (Desrosiers, 1998; Johnson, 1999; Johnson and Desrosiers, 1998; Letvin, 1998). However, the efficacy and safety of these attenuated HIV-1s could not be evaluated in animal models but only in humans and chimpanzees. Recently we reported that attenuated SHIVs containing HIV-1 Env have potential as anti-HIV-1 vaccines for several reasons (Haga et al., 1998; Hayami and Igarashi, 1997; Hayami et al., 1999; Igarashi et al., 1997): (1) SHIVs are able to induce humoral and cell-mediated immunity directed to HIV-1 Env (Igarashi et al., 1997, 1994; Lekutis and Letvin, 1997; Voss and Letvin, 1996). (2) SHIVs can infect and replicate well in human peripheral blood mononuclear cells (PBMCs) as well as in macaque PBMCs (Shibata et al., 1991), and thus induction of protective immunity could be expected in humans. (3) The immunity elicited against SIV Gag may be involved in protection against HIV-1 infection since SHIVs have SIV Gag which is antigenically cross-reactive with HIV-1 Gag (Carpio et al., 1991; Voss and Letvin, 1996). (4) SIVmac, one of the parental viruses of SHIVs, is considered nonpathogenic in humans because in accidentally infected persons the infection remained silent (CDC, 1997; Khabbaz et al., 1994, 1992). (5) Their efficacy and safety can be evaluated using macaques instead of humans (Sakuragi et al., 1992; Shibata et al., 1991). Deletion of genes which were shown to be associated with disease onset in the SIV/macaque system, such as nef and vpr, is one possible approach to attenuating viruses (Desrosiers, 1998; Desrosiers et al., 1998; Kestler et al., 1991; Lang et al., 1993). We recently reported that macaques vaccinated with vpr- and/or nef-defective SHIVs elicited anti-HIV-1 humoral and cell-mediated immunity and were protected against infection when challenged with the gene-intact SHIV (Igarashi et al., 1997). Based on this success, we newly constructed SHIVs completely lacking these genes and reported their virological and immunological properties in macaques until about 20 months after infection (Igarashi et al., 1998). In this report, we further analyzed the long-term immunological status of these vaccine viruses and their protective effect against infection when challenged with two SHIVs, SHIV-NM-3rN and SHIV-89.6P. The Env of SHIVNM-3rN is homologous to that of the vaccine viruses, while the Env of SHIV-89.6P is antigenically different from that of the vaccine viruses. SHIV-89.6P is pathogenic and provokes a decrease in the number of CD4 1 cells early after infection. RESULTS Virological and immunological status before challenge Two groups, each consisting of four macaques, were vaccinated with SHIV-drn or SHIV-dxrn (Fig. 1). Their virological and immunological status up to 20 months after the vaccination was previously reported (Igarashi et

253

FIG. 1. Genomic structures of SHIVs used in this study. SHIV-drn, SHIV-dxrn, and NM-3rN were constructed from HIV-1 NL432 and SIVmac239. Open and solid boxes are from SIVmac239 and HIV-1 NL432, respectively. Stippled boxes represent deleted genes. SHIV-drn lacks vpr and nef; SHIV-dxrn lacks vpr, nef, and vpx. The genomic structures of challenge viruses are also shown. SHIV-89.6P was isolated from macaques infected with in vivo-passaged SHIV-89.6 (Reimann et al., 1996a). SHIV-89.6 was constructed from HIV-1 HXBc2 (shaded boxes), HIV-1 89.6 (lined boxes), and SIVmac239.

al., 1998). Here we report their status after an additional 6 months. Table 1 summarizes the virological and immunological status of these macaques after the vaccination just before the challenge with SHIV-NM-3rN. The SHIVdrn-vaccinated macaques showed transient viremia (at 2 weeks post primary vaccination (w.p.v.)) after vaccination but no virus was recovered just before the challenge. No virus was isolated from any of the SHIV-dxrn-vaccinated macaques from the vaccination to the challenge. The peak neutralizing antibody (NAb) titers before the challenge and the NAb titers just before the challenge (at 112 w.p.v. of the SHIV-drn-vaccinated macaques and at 116 w.p.v. of SHIV-dxrn-vaccinated macaques) are also shown in Table 1. Before the challenge, the presence of NAb in the SHIV-drn-vaccinated macaques varied. MM81 retained NAb, which neutralized not only the parental SHIV-NM-3rN (Table 1), but also its parental HIV-1 NL432 (data not shown). MM86 and MM87 lost their NAb and no NAb was detected in MM85 throughout the period before the challenge. Among the SHIV-dxrn-vaccinated macaques, MM88 and MM103 had NAb as low as the end point titer (203), and MM101 and MM102 had no

254

UI ET AL. TABLE 1 Virological and Immunological Status of SHIV-drn- and SHIV-dxrn-Vaccinated Macaques before Challenge Virus isolation a

Neutralizing antibody b

Specific killer cell activity c

Vaccine

Macaque

Anytime before challenge

Just before challenge

Peak before challenge

Just before challenge

HIV-1 Env

SIV Gag

NK activity d

SHIV-drn

MM81 MM85 MM86 MM87

1 1 1 1

2 2 2 2

5120 ,20 80 1280

1024 ,20 ,20 ,20

6.8 11.1 11.7 32.0

0 27.6 0 0

20.4 6.0 25.8 19.2

SHIV-dxrn

MM88 MM101 MM102 MM103

2 2 2 2

2 2 2 2

20 ,20 ,20 20

20 ,20 ,20 20

13.0 4.7 9.2 0

28.8 0 8.5 13.8

19.2 25.4 17.4 45.7

a (1) Vaccine virus was isolated; (2) no virus isolation. SHIV-drn was isolated from all the SHIV-drn-vaccinated macaques at 2 w.p.v. SHIV-dxrn was not isolated from any of the SHIV-dxrn-vaccinated macaques at any time during the observation period. b NAb titer against SHIV-NM-3rN. c Activities were measured at 110 w.p.v. (at 11 w.p.v. for MM86). Values show the percentage of specific lysis. The E:T ratio was 100:1. d NK activities of the SHIV-drn- and SHIV-dxrn-vaccinated macaques were measured at 83 and 87 w.p.v., respectively. Values show the percentage of lysis of K562 cells. The E:T ratio was 50:1.

NAb. Three SHIV-drn-vaccinated macaques (MM85, MM86, and MM87) and two SHIV-dxrn-vaccinated macaques (MM88 and MM103) had HIV-1 Env- and/or SIV Gag-specific killer cell activities that were more than 10% (Table 1). All vaccinated macaques except MM85 had NK cell activities that were higher than those of normal macaques, whose activities were lower than 10%. Thus, in general, SHIV-drn-vaccinated macaques, in which the vaccine virus replicated and from which it was recovered, showed higher immunity than SHIV-dxrn-vaccinated macaques, in which no evidence of vaccine virus replication was obtained. Protection of SHIV-drn-vaccinated macaques against challenge with SHIV-NM-3rN To evaluate the protective effect of SHIV-drn against a challenge with the SHIV having an HIV-1 Env homologous to that of SHIV-drn, the vaccinated macaques were intravenously challenged with SHIV-NM-3rN at about 2 years postvaccination. All of the genes of SHIV-NM-3rN were intact, and its Env was homologous to that of SHIV-drn. As unvaccinated controls, two naive macaques, MM125 and MM126, were also inoculated. For two of the four SHIV-drn-vaccinated macaques (MM85 and MM86), no infectious virus was recovered and the DNA PCR remained negative in PBMCs throughout the observation period (Table 2). In MM81, the challenge virus was detected by DNA PCR at 10 w.p.c. In MM87, the challenge virus was detected once by DNA PCR at 6 w.p.c. and isolated twice at 6 and 12 w.p.c., but the cell-associated viral load of the challenge virus was about 1/80 that in the naives. The vaccine virus was

detected by DNA PCR at 8 w.p.c. in MM87. In the inguinal lymph nodes (LNs) at 31 weeks postchallenge, no DNA PCR signal was detected in any of the macaques except MM87, in which the signal was proved by differential PCR to be the vaccine virus rather than the challenge virus (Table 2). Figure 2 shows the plasma viral loads in these macaques, although the vaccine viruses and the challenge viruses could not be differentiated. The plasma viral loads of all the SHIV-drn-vaccinated macaques increased after the challenge, but were from about 1/200 to 1/4000 of those of the naives (Fig. 2). To determine the host response against viral replication after the challenge, the antibody titer was measured by particle agglutination. All the SHIV-drn-vaccinated macaques except MM87 showed an anamnestic antibody reaction to the challenge (data not shown). At 2 w.p.c., MM81 and MM85 had significantly greater HIV-1 Envspecific killer cell activities (.10%) than did the naives, and at 2 and 4 w.p.c., MM85 had significantly greater SIV Gag-specific killer cell activities (.10%) than did the naives, though MM81 did not have these activities before the challenge (Table 3). Thus MM85 and MM86, which were vaccinated with SHIV-drn, showed almost complete resistance since no challenge virus was recovered from PBMCs or detected by DNA PCR in PBMCs or LNs. However, the resistance was not perfect, as low viral replication was observed by plasma RT–PCR. Protection of SHIV-dxrn-vaccinated macaques against challenge with SHIV-NM-3rN The SHIV-dxrn-vaccinated macaques were intravenously challenged with SHIV-NM-3rN at about 2 years

PROTECTIVE EFFECT OF GENE-DELETED SHIVs

255

TABLE 2 Challenge of SHIV-drn- and SHIV-dxrn-Vaccinated Macaques with SHIV-NM-3rN Weeks postchallenge Vaccine

Macaque

Method

2

4

6

8

10

12

16

24

30

LN a,c

SHIV-drn

MM 81

PCR a VL b PCR VL PCR VL PCR VL

2 ,0.71 2 ,0.38 2 ,0.11 2 ,0.56

2 ND 2 ND 2 ND 2 ND

2 ,0.56 2 ,0.45 2 ,1.6 1 8.2

2 ND 2 ND 2 ND 2d ND

1 ND 2 ND 2 ND 2 ND

2 ,1.0 2 ,2.0 2 ,4.0 2 2.1

2 ND 2 ND 2 ND 2 ND

2 ND 2 ND 2 ND 2 ND

2 ,0.76 2 ,0.19 2 ,0.95 2 ,0.56

2 ND 2 ND 2 ND 2d ND

PCR VL PCR VL PCR VL PCR VL

2 ,0.77 2 31 2 ,3.9 2 ,0.53

1 ND 1 ND 1 ND 2 ND

1 ,2.3 2 ,3.2 2 ,2.4 2 0.59

1 ND 1 ND 1 ND 2 ND

2 ND 2 ND 2 ND 2 ND

2 ,6.3 2 ,1.4 2 ,3.1 2 ,1.6

2 ND 2 ND 2 ND 2 ND

1 ND 2 ND 2 ND 2 ND

2 ,0.58 1 ,0.45 2 ,0.71 2 ,0.16

2 ND 2 ND 2 ND 2 ND

1 ND 1 ND

1 41.5 1 208

1 ND 1 ND

1 ND 1 ND

1 ,2.9 1 18

1 ND 1 ND

1 ND 1 ND

1 ,0.15 1 ,0.24

1 ND 1 ND

MM 85 MM 86 MM 87 SHIV-dxrn

MM 88 MM 101 MM 102 MM 103

Naive

MM125 MM126

PCR VL PCR VL

1 629 1 1869

Note. ND, Not determined. a The 1 and 2 refer to detection of, or failure to detect, challenge virus by DNA PCR. b Cell-associated virus loads, i.e., number of virus producing cells/1 3 10 6 CD8 1 cell-depleted PBMCs. Isolated viruses were confirmed to be the challenge viruses. c DNA PCR of inguinal LNs at 31 w.p.c. d SHIV-drn was detected.

postvaccination. The infectious challenge virus was not recovered from MM88 or MM102, but was recovered from MM101 once at 2 w.p.c. and from MM103 once at 6 w.p.c. (Table 2). Their cell-associated viral loads were

FIG. 2. Plasma viral loads of the vaccinated macaques after challenge with NM-3rN. Plasma viral loads were measured by RT–PCR as described under Materials and Methods. The vaccine viruses and the challenge virus could not be differentiated.

less than 1/20 of those of the naives. No challenge virus was detected by DNA PCR in MM103, but it was detected in MM88, MM101, and MM102 sporadically (Table 2). In the inguinal LNs of those macaques obtained at 31 w.p.c., neither the challenge virus SHIV-NM-3rN nor the vaccine virus SHIV-dxrn was detected by DNA PCR. Plasma viral RNA was detected, but the plasma viral loads were less than 1/160 of those of the naives (Fig. 2). All the SHIV-dxrn-vaccinated macaques showed an anamnestic antibody reaction against the challenge infection (data not shown). At 4 w.p.c., MM101 and MM103 showed significantly higher HIV-1 Env-specific killer cell activities (.10%), and MM102 and MM103 showed significantly higher SIV Gag-specific killer cell activities (.10%) than did the naives, though MM101 and MM102 did not have these activities before the challenge (Table 3). Thus, all the SHIV-dxrn-vaccinated macaques showed significant but not complete resistance to infection of SHIV-NM-3rN having an HIV-1 Env homologous to that of the vaccine virus. Compared with SHIV-drn, the SHIVdxrn induced less protective immunity because two of the four SHIV-drn-vaccinated macaques showed almost complete resistance.

256

UI ET AL. TABLE 3 HIV-1 Env- and SIV Gag-Specific Killer Cell Activities of SHIV-drn- and SHIV-dxrn-Vaccinated Macaques after Challenge with NM-3rN Specific killer cell activity c w.p.c.

Vaccine SHIV-drn

Macaque MM81 MM85 MM86 MM87

SHIV-dxrn

MM88 MM101 MM102 MM103

Naive

MM125 MM126

Target cell a

Before challenge b

2

4

10

30

HIV-1 Env SIV Gag HIV-1 Env SIV Gag HIV-1 Env SIV Gag HIV-1 Env SIV Gag

6.8 0.0 11.1 27.6 11.7 0.0 32.0 0.0

13.4 0.0 18.4 28.8 ND ND ND 0.0

0.0 12.1 14.1 19.1 ND ND 5.0 2.7

0.0 13.8 ND ND 9.0 23.4 ND ND

0.0 8.2 4.9 16.7 7.3 4.1 18.8 13.6

HIV-1 Env SIV Gag HIV-1 Env SIV Gag HIV-1 Env SIV Gag HIV-1 Env SIV Gag

13.0 28.8 4.7 0.0 9.2 8.5 0.0 13.8

15.3 0.0 0.0 0.0 ND ND ND ND

ND ND 32.0 7.9 0.0 31.4 9.4 20.6

3.9 19.8 5.9 2.1 5.2 9.8 8.7 15.8

1.8 21.5 15.6 5.6 18.2 5.5 14.6 11.5

HIV-1 Env SIV Gag HIV-1 Env SIV Gag

0.0 1.3 1.6 2.7

5.9 0.0 4.5 0.4

0.2 13.5 10.3 10.7

3.9 0.0 13.8 0.0

4.8 36.3 38.9 9.6

Note. ND, Not determined. a BLCL infected with a VV-expressing HIV-1 Env or SIV Gag. b At 110 w.p.v. (11 w.p.v. for MM86). c Values show the percentage of specific lysis. The E:T ratio was 100:1.

Protection of SHIV-drn-vaccinated macaques against challenge with pathogenic SHIV-89.6P having antigenically heterologous HIV-1 Env To further assess the vaccine efficacy, two of the SHIV-drn-vaccinated macaques, MM85 and MM86, were intravenously challenged with pathogenic SHIV-89.6P 36 weeks after the challenge with SHIV-NM-3rN (Fig. 1). The Env of SHIV-89.6P is antigenically heterologous to that of SHIV-drn (Montefiori et al., 1998). These two macaques previously had shown strong resistance to the challenge with SHIV-NM-3rN. Before the challenge with SHIV-89.6P, the plasmas from MM85 and MM86 were unable to neutralize SHIV-89.6P (data not shown) and the specific killer cell activity against HIV-1 89.6 Env was not examined. As a control, two naive macaques, MM142 and MM144, were also inoculated with SHIV-89.6P. The challenge virus was recovered from MM86 at 8 w.p.c. (Table 4) but was not detected by DNA PCR in the PBMCs. In MM85, the virus was detected at 3 w.p.c., which was delayed compared with the naives, and the infectious virus was recovered only twice at 2 and 3 w.p.c. Their plasma viral loads measured by RT–PCR were about 1/60,000 of that of the naives (Fig. 3a). In addition, we analyzed the change in the number of

CD4 1 cells in these macaques after the challenge, because the challenge virus SHIV-89.6P is known to be pathogenic, causing a decrease in the number of CD4 1 cells (Reimann et al., 1996a). The two unvaccinated macaques showed equally rapid decreases in the number of CD4 1 cells after the challenge with SHIV-89.6P, reaching less than 10% after 3 w.p.c. (Fig. 3b), in agreement with previous reports (Karlsson et al., 1997; Reimann et al., 1996a; Steger et al., 1998). In contrast, the vaccinated macaques did not show any decrease in the number of CD4 1 cells and were healthy during the observation period (.30 w.p.c.). Thus, the SHIV-drn-vaccinated macaques partly resisted the challenge infection of the pathogenic SHIV having HIV-1 Env heterologous to that of SHIV-drn, but did not suffer any loss of CD4 1 cells. This suggested that the vaccine virus might be effective against disease progression, even if the challenge virus escapes and infects the vaccinated macaques. DISCUSSION In this study, we examined the protective effect of two gene-deleted SHIVs against challenges with the homologous and heterologous SHIVs. When the vaccinees were challenged with homologous SHIV-NM-3rN, two of

PROTECTIVE EFFECT OF GENE-DELETED SHIVs

257

TABLE 4 Cell-Associated Viral Loads and DNA PCR in SHIV-drn-Vaccinated Macaques after Challenge with SHIV-89.6P w.p.c. Vaccine

Macaque

Method

0

1

2

3

4

6

8

10

12

SHIV-drn

MM 85

PCR a VL b PCR VL

2 ,0.19 2 ,0.95

2 ,0.71 2 ,0.38

2 9 2 ,1.0

1 726 2 ,0.71

1 ,0.71 2 ,0.83

1 ,0.71 2 ,1.0

1 ,0.83 2 1.0

1 ,0.32 2 ,3.7

1 ,0.14 2 ,0.36

PCR VL PCR VL

2 ND 2 ND

1 33 1 5

1 10 1 17

1 14 1 27

1 ND 1 ND

1 ,0.06 1 3.3

MM 86 Naive

MM142 MM144

1 2624 1 66

1 ND 1 ND

1 1094 1 14

Note. ND, Not determined. a The 1 and 2 refer to detection of, or failure to detect, proviral SHIV-89.6P by DNA PCR. b Cell-associated viral loads, i.e., number of virus producing cells/1 3 106 CD8 1 cell-depleted PBMCs.

four SHIV-drn-vaccinated macaques (MM85 and MM86) showed almost complete resistance. The other two SHIV-drn-vaccinated macaques also resisted the challenge because their plasma and cell-associated viral loads were lower than those of the unvaccinated con-

FIG. 3. Challenge of SHIV-drn-vaccinated macaques with SHIV-89.6P. (a) Plasma viral loads after the challenge. Plasma viral loads were measured by RT–PCR as described under Materials and Methods. The vaccine virus and the challenge virus could not be differentiated. (b) Changes in the number of circulating CD4 1 cells with time. Values are expressed as a percentage of the number of CD4 1 cells of each macaque just before the challenge, as described by Steger et al. (1998).

trols. Thus, SHIV-drn induced strong protection. All the SHIV-dxrn-vaccinated macaques showed less resistance than did the SHIV-drn-vaccinated macaques. Still, their plasma viral loads were less than 1/160 and their cell-associated viral loads were less than 1/20 of those of the unvaccinated macaques. In all these vaccinated macaques, replication of the challenge virus either did not occur or was transient, and no challenge virus was detected in the LNs. Viral replication in LNs was reported to be associated with disease progression (Pantaleo et al., 1993, 1994, 1995). The macaques vaccinated with SHIV-NM-3 and SHIVNM-3n had humoral and/or cell-mediated immunity and were protected against challenge with SHIV-NM-3rN (Igarashi et al., 1997). The more effective protection provided by SHIV-NM-3 and SHIV-NM-3n may be related to the fact that they replicated well, because these SHIVs infected rhesus macaques persistently (Igarashi et al., 1997, 1994), while SHIV-drn and SHIV-dxrn showed transient or no viremia. The present results suggested that SHIV-dxrn was less effective than SHIV-drn. Our results show that as more genes are deleted, the replication, immunogenicity, and protective effect decrease. These results are in agreement with the results of other studies of SIVs lacking one to five genes, such as nef, vpr, vpx, LTR, and vif (Desrosiers, 1998; Kestler et al., 1991; Lang et al., 1993). SHIV-dxrn, which showed no evidence of replication in the inoculated macaques, might have been overattenuated. None of the macaques inoculated with SHIV-drn or SHIV-dxrn showed any symptoms associated with disease. There is presently some controversy over whether humoral immunity (e.g., immunity provided by anti-viral neutralizing antibodies) or cell-mediated immunity (e.g., immunity provided by specific killer cells) has a more important role in protection (Johnson and Desrosiers, 1998; Shibata et al., 1997). Before being challenged, two

258

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SHIV-drn-vaccinated macaques that showed strong resistance to homologous SHIV-NM-3rN (MM85 and MM86) had no NAb, but they did have HIV-1 Env- and/or SIV Gag-specific killer cell activities. However, MM87, which also had no NAb but which had higher killer cell activities, was not as well protected as MM85 and MM86. In addition, MM81, which had a high level of NAb but no specific killer cell activity, was less protected against the challenge virus, although the degree of protection was still significant. Therefore, we could not determine in this study which kinds of immunity played important roles in protection. It is noteworthy that MM101 and MM102, which were vaccinated with SHIV-dxrn and which had neither detectable NAb nor specific killer cell activity, showed less but significant resistance. It is likely that several defense mechanisms are involved in this protection. These mechanisms would include not only well-known immunity, such as anti-viral neutralizing antibody and specific killer cell activity, but also unknown specific and nonspecific defense mechanisms, such as antibody-dependent cell cytotoxicity (ADCC), NK cell activity, and others. NK cell activity was detected in most of the macaques vaccinated with SHIV-drn and SHIV-dxrn. Complex specific and nonspecific immunities elicited by natural infection might contribute cooperatively to protection (Dittmer et al., 1999). This is one of the reasons attenuated live vaccines are expected to be most effective. Since HIV-1 is known to be highly divergent, a vaccine must be able to protect against not only homologous HIV-1 but also divergent HIV-1 strains. We showed that SHIV-drn provided some protection against challenge with SHIV-89.6P, which has an HIV-1 Env that is antigenically different from that of SHIV-drn. Since the infectious challenge virus, SHIV-89.6P, was isolated only once from one of two SHIV-drn-vaccinated macaques that were challenged with SHIV-89.6P (MM86), and the two macaques had significantly lower plasma viral loads, we conclude that SHIV-drn showed resistance against the challenge with SHIV-89.6P. As these macaques showed no NAb against SHIV-89.6P and their specific killer cell activity could not be examined in this study, we were unable to determine which kinds of immunity might be involved in this cross-resistance. In addition to the possibility of cross-reactive CTL, ADCC and other mechanisms mediated via antibodies might play a role where cross-recognition in the absence of neutralization might occur. Besides, nonspecific defense mechanisms such as NK activity and cytokines, including chemokines, might be involved in this cross-resistance. It might be also possible that different coreceptor usage between SHIV-drn, which utilizes only CXCR4 (data not shown), and SHIV-89.6P, which utilizes both CXCR4 and CCR5 (Karlsson et al., 1998), might influence the result. In any case, our results demonstrated that the gene-deleted SHIVs could provide partial protection against infection

of an SHIV having antigenically heterologous HIV-1 Env. There are several reports on protection against heterologous challenge using a SIV/SHIV combination and, as shown in this study, some of these experiments were successful (Bogers et al., 1995; Dunn et al., 1997; Miller et al., 1997; Quesada-Rolander et al., 1996; Stephens et al., 1997). Even if SHIV-drn vaccination did not provide the two macaques with complete protection against infection with SHIV-89.6P, it did prevent a decrease in the number of circulating CD4 1 cells by SHIV-89.6P in both of them. This prevention might result from restricting the replication of the challenge virus, as shown in Table 2. Joag et al. (1998) reported that immunization with vaccine SHIVs with deletion(s) of vpu and/or nef prevented the decrease in number of CD4 1 cells after intravaginal inoculation with a pathogenic SHIV KU-1 having an HIV-1 Env homologous to those of vaccine viruses. In HIV infection, a higher viral load is associated with more advanced disease progression (Iuliano et al., 1997). Watson et al. (1997) and Ten Haaft et al. (1998) reported that SIV- or SHIV-infected macaques which had higher plasma viral loads survived for shorter times. We suggest that the low viral loads of the challenge virus in the SHIV-drn-vaccinated macaques might prevent the decrease in the number of circulating CD4 1 cells. Therefore, it is possible that the gene-deleted SHIV might prevent the progression of the disease even if the challenge virus escapes and replicates. Many studies have been carried out to develop antiHIV-1 vaccines. We previously suggested that live attenuated vaccines are the most effective type of protection against infection because they can induce complex infection immunity consisting of specific humoral and cellular immunity and nonspecific immunity in macaques (Igarashi et al., 1997), as shown in this study. Previously we proposed that SHIVs are good starting materials for making anti-HIV-1 live vaccines for human use (Haga et al., 1998; Hayami and Igarashi, 1999; Hayami et al., 1999; Igarashi et al., 1997). One of the reasons is that these vaccines can be expected to induce protective immunity in humans as well as in macaques, because SHIVs can replicate not only in macaque PBMCs but also in human PBMCs. However, we must be careful in applying the data obtained in monkeys directly to humans. For attenuation, in this study we deleted two or three genes, including nef, which was reported to be associated with pathogenicity in the SIV/macaque system and in humans of the Sydney Blood Bank Cohort (Deacon et al., 1995; Kestler et al., 1991). However, similar gene-deleted SIVs were recently reported to be pathogenic in neonates and even in adult macaques that had been infected for prolonged periods of time (Baba et al., 1995, 1999). These reports claimed that attenuation by gene deletion is not satisfactory for assuring safety. But the safety of the parental SHIV-NM-3rN, from which the gene-deleted

PROTECTIVE EFFECT OF GENE-DELETED SHIVs

SHIVs used in this study were derived, was confirmed to be nonpathogenic during 3 years of observation and because five in vivo passages failed to make the virus pathogenic. Further modification of SHIVs by other approaches will be required to make a much safer live attenuated vaccine. MATERIALS AND METHODS Vaccine viruses and challenge viruses Two gene-deleted SHIVs that had been constructed from SHIV-NM-3rN (Kuwata et al., 1995) were used for vaccine viruses (Igarashi et al., 1998). One, SHIV-drn, was lacking nef and vpr, and the other, SHIV-dxrn, was lacking nef, vpr, and vpx. Figure 1 shows the genomic structures of these gene-deleted SHIVs. They contain env, tat, rev, and vpu derived from HIV-1 NL432 (Adachi et al., 1986) and LTRs, gag, pol, vif, and/or vpx derived from SIVmac239 (Kestler et al., 1990). SHIV-drn has deletions in the 59 portion, including the initiation codons of nef and vpr. A splicing acceptor for vpr was modified so that it would not function. SHIV-dxrn was constructed by deleting the 39 portion of vpx in addition to the deletion of SHIV-drn. The initiation codon of vpx was modified to a non-sense codon. Stocks of these gene-deleted SHIVs were prepared for macaque inoculation using M8166 cells (a subclone of C8166 (Clapham et al., 1987)) as described previously (Kuwata et al., 1995). SHIV-NM-3rN (Kuwata et al., 1995) and pathogenic SHIV-89.6P (Karlsson et al., 1997; Reimann et al., 1996a) were used for challenge (Fig. 1). SHIV-89.6P was kindly provided by Dr. K. A. Reimann (Harvard Medical School). SHIV-NM-3rN, the parental SHIV from which the genedeleted SHIVs were made, is nonpathogenic, having HIV-1 NL432 Env, which is the same as that of the vaccine viruses SHIV-drn and SHIV-dxrn. SHIV-89.6P is derived from in vivo-passaged SHIV-89.6. This virus has HIV-1 89.6 Env, which is antigenically different from that of the vaccine viruses (Montefiori et al., 1998) and causes a rapid decline of CD4 1 cell numbers and AIDS-like diseases in inoculated macaques (Reimann et al., 1996b). Stocks of these challenge viruses were prepared for macaque inoculation using rhesus macaque PBMCs, as described previously (Kuwata et al., 1995). The TCID 50 of these virus stocks were determined as previously described (Igarashi et al., 1994). Animals Mature male rhesus macaques (Macaca mulatta) negative for SIV and simian T-cell lymphotropic virus type 1, were used. All the monkeys were housed throughout the experimental period and autopsies and biopsies were carried out in accordance with regulations approved by the Institutional Animal Care and Use Committee of the Institute for Virus Research, Kyoto University.

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Vaccination and challenge inoculation Two groups of four macaques were vaccinated intravenously with 1 3 10 5 TCID 50 of SHIV-drn or SHIV-dxrn, respectively. To assess the acquisition of immunological memory induced by the first inoculation, the macaques were reinoculated with 1 3 10 7 TCID 50 of each vaccine virus at 68 w.p.v. as reported previously, and three of four SHIV-drn-vaccinated macaques and none of the SHIVdxrn-vaccinated macaques showed an anamnestic antibody reaction (Igarashi et al., 1998). SHIV-drn- and SHIV-dxrn-vaccinated macaques were intravenously inoculated with 100 TCID 50 of SHIV-NM3rN at 112 and 116 w.p.v., respectively. Thirty-six weeks later, two SHIV-drn-vaccinated macaques, MM85 and MM86, were inoculated intravenously with 10 TCID 50 of SHIV-89.6P. This dose was based on preliminary experiments that showed that, for each virus, about 1/10 the original dose was the minimal amount needed to induce viremia in macaques (unpublished data). PBMCs of all of the macaques were prepared by Percoll density gradient centrifugation (Pharmacia) and used for the analyses described below. The separated plasma was subjected to RT–PCR and antibody assays. Inguinal LNs were obtained from all of the macaques for DNA PCR analysis at 31 w.p.c. with NM-3rN. LNs were obtained by biopsy of MM85 and MM86 and by autopsy of the others. Virus recovery and infectious virus loads in PBMCs The infectious cell-associated viral load was determined as described previously (Kestler et al., 1991). Briefly, serial threefold dilutions of periodically collected CD8 1 cell-depleted PBMCs were cocultured with 1 3 10 6 M8166 cells for 4 weeks in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (GIBCO-BRL), 20 mM L-glutamine, and 10 mM sodium pyruvate. Virus recovery was judged by the syncytial cytopathic effect and the expression of viral antigens detected by an indirect immunofluorescence assay. Cellassociated viral loads were expressed as the number of virus-producing cells per 1 3 10 6 CD8-depleted PBMCs. Detection and differentiation of viral genomes in PBMCs by DNA PCR To detect viruses and to differentiate vaccine or challenge viruses after the challenges, DNA PCR was carried out. The primers that were used in this study can amplify the central region of the viral genome, including vpr and vpx, which were deleted in the vaccine viruses. The structure of this region was different between SHIV-NM3rN and SHIV-89.6P (Kuwata et al., 1995; Reimann et al., 1996a). Thus, PCR amplification with the above primers can differentiate SHIV-drn, SHIV-dxrn, SHIV-NM-3rN, and SHIV-89.6P by the length of the PCR product. Cells and tissues (1 3 10 6 PBMCs, LNs at 31 w.p.c., and M8166

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cells infected with the isolated virus) were lysed by incubating them in 100 ml of Proteinase K buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, pH 8.0, 0.001% Triton X-100, 0.0001% SDS, 600 mg/ml Proteinase K) for 1 h at 56°C, followed by incubation for 15 min at 95°C. These lysates were used for detection of the proviral genome. The PCR was carried out using AmpliTaq DNA polymerase (Perkin–Elmer) in a total volume of 50 ml. The primers were DF1 (59-ATCCCACCTGGAAACAGTGGAGAAGAGACA-39) and DR1 (59-CAAGCAGTTTTAGGCTGACTTCCTGGATGC-39) for the first PCR and DF2 (59-CGGTAAACCACCTACCAAGGGAGCTAATTT-39) and DR2 (59-GTAACGCCTATTCTGCTATGTCGACACCCA-39) for the nested PCR. Samples were first denatured at 94°C for 9 min, followed by 35 cycles consisting of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and elongation at 72°C for 1 min, with a final elongation at 72°C for 5 min. The PCR products were analyzed on a 2% agarose gel.

each macaque were infected with vaccinia viruses (VVs) expressing the Env of HIV-1 (HIV-1IIIb) or SIV Gag (from SIVmac239) and were used for target cells to measure Env- or Gag-specific killer cell activities, respectively. Parental vaccinia virus-infected and noninfected cells were used as control targets. To measure NK activity, K562 cells were used as target cells. Env- or Gag-specific killer cell activity was expressed as the percentage of specific lysis when the effector:target cell ratio was 100:1. This was the percentage of parental VV-infected BLCL that were lysed subtracted from the that of the HIV-1 Env- or SIV Gag-expressing BLCL that were lysed. NK activity was expressed as the percentage of K562 cells that were lysed when this ratio was 50:1. The activity was regarded as significant when more than 10% of the cells were lysed.

Quantitation of plasma viral loads

CD4 1 cell numbers in PBMCs isolated from the macaques were calculated after the challenge with SHIV89.6P. PBMCs were treated with anti-CD4 antibody (NuTH/I-PE; NICHIREI) and examined on a FACScan analyzer (Becton–Dickinson) according to the manufacturer’s recommendations. Absolute lymphocyte counts in the blood were determined with an automated blood cell counter (F-820; Sysmex). The percentages of CD4 1 cells remaining were calculated as described by Steger et al. (1998). The number of CD4 1 cells of each macaque just before the challenge was defined as 100%.

Plasma viral loads after the challenges were determined by RT–PCR. RT reactions and PCRs were performed by a Taqman RT–PCR kit (Perkin–Elmer) (Suryanarayana et al., 1998) using RNA samples (corresponding to 40 ml of plasma) prepared by Viral RNA kit (QIAGEN) and the primers SIVII-696F (59-GGAAATTACCCAGTACAACAAATAGG-39) and SIVII-784R (59-TCTATCAATTTTACCCAGGCATTTA-39). A labeled probe, SIVII731 (59-Fam-TGTCCACCTGCCATTAAGCCCG-Tamra-39, Perkin–Elmer), was used for detection of 59-nuclease activity. For each run, a standard curve was generated from dilutions of an in vitro transcript containing gag. The RT–PCR system amplifies both the challenge and vaccine viruses and cannot differentiate these viruses. Under these conditions, the detection limit was 250 copy/ ml. Detection and titration of neutralizing antibody and particle-agglutinating antibody Neutralizing antibody was assessed by the method of Igarashi et al. (1994). Samples of SHIV-NM-3rN or SHIV89.6P (100 TCID 50) were treated with twofold serial dilutions of the plasma samples. The virus was considered neutralized when RT activity was reduced to no more than half that in normal macaque plasma. Antibody titer after the challenge with SHIV-NM-3rN was measured by particle agglutination according to the manufacturer’s instructions (Serodia HIV, Fujirebio). HIV-1 Env- or SIV Gag-specific killer cell activities and NK cell activity HIV-1 Env- or SIV Gag-specific killer cell activities were measured by the method of Yamamoto et al. (1990) as modified by Igarashi et al. (1997). Herpesvirus papiotransformed B cell lines (BLCL) derived from PBMCs of

Lymphocyte phenotyping

ACKNOWLEDGMENT This work was supported by a grant from the Organization for Drug ADR Relief, R&D Promotion and Product Review, Japan.

REFERENCES Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., and Martin, M. A. (1986). Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59(2), 284–291. Almond, N., Kent, K., Cranage, M., Rud, E., Clarke, B., and Stott, E. J. (1995). Protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells. Lancet 345(8961), 1342–1344. Almond, N. M., and Heeney, J. L. (1998). AIDS vaccine development in primate models. AIDS 12(Suppl. A), S133–S140. Baba, T. W., Jeong, Y. S., Pennick, D., Bronson, R., Greene, M. F., and Ruprecht, R. M. (1995). Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science 267(5205), 1820– 1825. Baba, T. W., Liska, V., Khimani, A. H., Ray, N. B., Dailey, P. J., Penninck, D., Bronson, R., Greene, M. F., McClure, H. M., Martin, L. N., and Ruprecht, R. M. (1999). Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques. Nature Med. 5(2), 194–203. Bogers, W. M., Niphuis, H., ten Haaft, P., Laman, J. D., Koornstra, W., and Heeney, J. L. (1995). Protection from HIV-1 envelope-bearing chimeric simian immunodeficiency virus (SHIV) in rhesus macaques infected

PROTECTIVE EFFECT OF GENE-DELETED SHIVs with attenuated SIV: consequences of challenge. AIDS 9(12), F13– F18. Carpio, E., Duarte, C., Hinkula, J., Broliden, P. A., Rosen, J., Campal, A., Gavilondo, J., Wahren, B., and Jondal, M. (1991). Monoclonal antibodies to conserved regions of the major core protein (gag24) of HIV-1 and HIV-2. AIDS. Res. Hum. Retroviruses 7(1), 97–101. CDC. (1997). Nonhuman primate spumavirus infections among persons with occupational exposure—United States, 1996. MMWR Morb. Mortal Wkly. Rep. 46(6), 129–131. Clapham, P. R., Weiss, R. A., Dalgleish, A. G., Exley, M., Whitby, D., and Hogg, N. (1987). Human immunodeficiency virus infection of monocytic and T-lymphocytic cells: Receptor modulation and differentiation induced by phorbol ester. Virology 158(1), 44–51. Clements, J. E., Montelaro, R. C., Zink, M. C., Amedee, A. M., Miller, S., Trichel, A. M., Jagerski, B., Hauer, D., Martin, L. N., Bohm, R. P., and Murphey-Corb, M. (1995). Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus. J. Virol. 69(5), 2737– 2744. Connor, R. I., Montefiori, D. C., Binley, J. M., Moore, J. P., Bonhoeffer, S., Gettie, A., Fenamore, E. A., Sheridan, K. E., Ho, D. D., Dailey, P. J., and Marx, P. A. (1998). Temporal analyses of virus replication, immune responses, and efficacy in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine. J. Virol. 72(9), 7501–7509. Daniel, M. D., Kirchhoff, F., Czajak, S. C., Sehgal, P. K., and Desrosiers, R. C. (1992). Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 258(5090), 1938–1941. Deacon, N. J., Tsykin, A., Solomon, A., Smith, K., Ludford-Menting, M., Hooker, D. J., McPhee, D. A., Greenway, A. L., Ellett, A., Chatfield, C., Lawson, V. A., Crowe, S., Maerz, A., Sonza, S., Learmont, J., Sullivan, J. S., A., C., Dwyer, D., Dowton, D., and Milles, J. (1995). Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 270(5238), 988–991. Desrosiers, R. C. (1998). Prospects for live attenuated HIV. Nature Med. 4(9), 982. Desrosiers, R. C., Lifson, J. D., Gibbs, J. S., Czajak, S. C., Howe, A. Y., Arthur, L. O., and Johnson, R. P. (1998). Identification of highly attenuated mutants of simian immunodeficiency virus. J. Virol. 72(2), 1431– 1437. Dittmer, U., Brooks, D. M., and Hasenkrug, K. J. (1999). Requirement for multiple lymphocyte subsets in protection by a live attenuated vaccine against retroviral infection. Nature Med. 5(2), 189–193. Dunn, C. S., Hurtrel, B., Beyer, C., Gloeckler, L., Ledger, T. N., Moog, C., Kieny, M. P., Mehtali, M., Schmitt, D., Gut, J. P., Kirn, A., and Aubertin, A. M. (1997). Protection of SIVmac-infected macaque monkeys against superinfection by a simian immunodeficiency virus expressing envelope glycoproteins of HIV type 1. AIDS. Res. Hum. Retroviruses 13(11), 913–922. Fultz, P. N., Srinivasan, A., Greene, C. R., Butler, D., Swenson, R. B., and McClure, H. M. (1987). Superinfection of a chimpanzee with a second strain of human immunodeficiency virus. J. Virol. 61(12), 4026–4029. Gibbs, C. J., Jr., Peters, R., Gravell, M., Johnson, B. K., Jensen, F. C., Carlo, D. J., and Salk, J. (1991). Observations after human immunodeficiency virus immunization and challenge of human immunodeficiency virus seropositive and seronegative chimpanzees. Proc. Natl. Acad. Sci. USA 88(8), 3348–3352. Gundlach, B. R., Reiprich, S., Sopper, S., Means, R. E., Dittmer, U., Matz-Rensing, K., Stahl-Hennig, C., and Uberla, K. (1998). Env-independent protection induced by live, attenuated simian immunodeficiency virus vaccines. J. Virol. 72(10), 7846–7851. Haga, T., Kuwata, T., Ui, M., Igarashi, T., Miyazaki, Y., and Hayami, M. (1998). A new approach to AIDS research and prevention: The use of gene-mutated HIV-1/SIV chimeric viruses for anti-HIV-1 live-attenuated vaccines. Microbiol. Immunol. 42(4), 245–251. Hayami, M., and Igarashi, T. (1997). SIV/HIV-1 chimeric viruses having

261

HIV-1 env gene: A new animal model and a candidate for attenuated live vaccine. Leukemia 11(Suppl. 3), 95–97. Hayami, M., Igarashi, T., Kuwata, T., Ui, M., Haga, T., Ami, Y., Shinohara, K., and Honda, M. (1999). Gene-mutated HIV-1/SIV chimeric viruses as AIDS live attenuated vaccines for potential human use. Leukemia 13(Suppl. 1), S42–S47. Igarashi, T., Ami, Y., Yamamoto, H., Shibata, R., Kuwata, T., Mukai, R., Shinohara, K., Komatsu, T., Adachi, A., and Hayami, M. (1997). Protection of monkeys vaccinated with vpr- and/or nef-defective simian immunodeficiency virus strain mac/human immunodeficiency virus type 1 chimeric viruses: A potential candidate live-attenuated human AIDS vaccine. J. Gen. Virol. 78(Pt. 5), 985–989. Igarashi, T., Kuwata, T., Yamamoto, H., Moriyama, H., Ui, M., Miyazaki, Y., and Hayami, M. (1998). Infectivity and immunogenicity of SIVmac/ HIV-1 chimeric viruses (SHIVs) with deletions in two or three genes (vpr, nef and vpx). Microbiol. Immunol. 42(1), 71–74. Igarashi, T., Shibata, R., Hasebe, F., Ami, Y., Shinohara, K., Komatsu, T., Stahl-Hennig, C., Petry, H., Hunsmann, G., Kuwata, T., and Hayami, M. (1994). Persistent infection with SIVmac chimeric virus having tat, rev, vpu, env and nef of HIV type 1 in macaque monkeys. AIDS. Res. Hum. Retroviruses 10(8), 1021–1029. Iuliano, R., Forastieri, G., Brizzi, M., Mecocci, L., Mazzotta, F., and Ceccherini-Nelli, L. (1997). Correlation between plasma HIV-1 RNA levels and the rate of immunologic decline. J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 14(5), 408–414. Joag, S. V., Li, Z., Foresman, L., Pinson, D. M., Raghavan, R., Zhuge, W., Adany, I., Wang, C., Jia, F., Sheffer, D., Ranchalis, J., Watson, A., and Narayan, O. (1997). Characterization of the pathogenic KU-SHIV model of acquired immunodeficiency syndrome in macaques. AIDS. Res. Hum. Retroviruses 13(8), 635–645. Joag, S. V., Liu, Z. Q., Stephens, E. B., Smith, M. S., Kumar, A., Li, Z., Wang, C., Sheffer, D., Jia, F., Foresman, L., Adany, I., Lifson, J., McClure, H. M., and Narayan, O. (1998). Oral immunization of macaques with attenuated vaccine virus induces protection against vaginally transmitted AIDS. J. Virol. 72(11), 9069–9078. Johnson, R. P. (1999). Live attenuated AIDS vaccines: Hazards and hopes. Nature Med. 5(2), 154–155. Johnson, R. P., and Desrosiers, R. C. (1998). Protective immunity induced by live attenuated simian immunodeficiency virus. Curr. Opin. Immunol. 10(4), 436–443. Karlsson, G. B., Halloran, M., Li, J., Park, I. W., Gomila, R., Reimann, K. A., Axthelm, M. K., Iliff, S. A., Letvin, N. L., and Sodroski, J. (1997). Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD41 lymphocyte depletion in rhesus monkeys. J. Virol. 71(6), 4218–4225. Karlsson, G. B., Halloran, M., Schenten, D., Lee, J., Racz, P., TennerRacz, K., Manola, J., Gelman, R., Etemad-Moghadam, B., Desjardins, E., Wyatt, R., Gerard, N. P., Marcon, L., Margolin, D., Fanton, J., Axthelm, M. K., Letvin, N. L., and Sodroski, J. (1998). The envelope glycoprotein ectodomains determine the efficiency of CD4 1 T lymphocyte depletion in simian-human immunodeficiency virus-infected macaques. J. Exp. Med. 188(6), 1159–1171. Kestler, H., Kodama, T., Ringler, D., Marthas, M., Pedersen, N., Lackner, A., Regier, D., Sehgal, P., Daniel, M., and King, N. (1990). Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus. Science 248(4959), 1109–1112. Kestler, H. W. D., Ringler, D. J., Mori, K., Panicali, D. L., Sehgal, P. K., Daniel, M. D., and Desrosiers, R. C. (1991). Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65(4), 651–662. Khabbaz, R. F., Heneine, W., George, J. R., Parekh, B., Rowe, T., Woods, T., Switzer, W. M., McClure, H. M., Murphey-Corb, M., and Folks, T. M. (1994). Brief report: Infection of a laboratory worker with simian immunodeficiency virus. N. Engl. J. Med. 330(3), 172–177. Khabbaz, R. F., Rowe, T., Murphey-Corb, M., Heneine, W. M., Schable, C. A., George, J. R., Pau, C. P., Parekh, B. S., Lairmore, M. D., and

262

UI ET AL.

Curran, J. W. (1992). Simian immunodeficiency virus needlestick accident in a laboratory worker. Lancet 340(8814), 271–273. Klinger, J. M., Himathongkham, S., Legg, H., Luciw, P. A., and Barnett, S. W. (1998). Infection of baboons with a simian immunodeficiency virus/HIV-1 chimeric virus constructed with an HIV-1 Thai subtype E envelope. AIDS 12(8), 849–857. Kuwata, T., Igarashi, T., Ido, E., Jin, M., Mizuno, A., Chen, J., and Hayami, M. (1995). Construction of human immunodeficiency virus 1/simian immunodeficiency virus strain mac chimeric viruses having vpr and/or nef of different parental origins and their in vitro and in vivo replication. J. Gen. Virol. 76(Pt. 9), 2181–2191. Kuwata, T., Shioda, T., Igarashi, T., Ido, E., Ibuki, K., Enose, Y., StahlHennig, C., Hunsmann, G., Miura, T., and Hayami, M. (1996). Chimeric viruses between SIVmac and various HIV-1 isolates have biological properties that are similar to those of the parental HIV-1. AIDS 10(12), 1331–1337. Lang, S. M., Weeger, M., Stahl-Hennig, C., Coulibaly, C., Hunsmann, G., Muller, J., Muller-Hermelink, H., Fuchs, D., Wachter, H., and Daniel, M. M. (1993). Importance of vpr for infection of rhesus monkeys with simian immunodeficiency virus. J. Virol. 67(2), 902–912. Lekutis, C., and Letvin, N. L. (1997). HIV-1 envelope-specific CD4 1 T helper cells from simian/human immunodeficiency virus-infected rhesus monkeys recognize epitopes restricted by MHC class II DRB1*0406 and DRB*W201 molecules. J. Immunol. 159(4), 2049– 2057. Letvin, N. L. (1998). Progress in the development of an HIV-1 vaccine. Science 280(5371), 1875–1880. Letvin, N. L., Li, J., Halloran, M., Cranage, M. P., Rud, E. W., and Sodroski, J. (1995). Prior infection with a nonpathogenic chimeric simian-human immunodeficiency virus does not efficiently protect macaques against challenge with simian immunodeficiency virus. J. Virol. 69(7), 4569–4571. Li, J., Lord, C. I., Haseltine, W., Letvin, N. L., and Sodroski, J. (1992). Infection of cynomolgus monkeys with a chimeric HIV-1/SIVmac virus that expresses the HIV-1 envelope glycoproteins. J. Acquired Immune Defic. Syndr. 5(7), 639–646. Lohman, B. L., McChesney, M. B., Miller, C. J., McGowan, E., Joye, S. M., Van Rompay, K. K., Reay, E., Antipa, L., Pedersen, N. C., and Marthas, M. L. (1994). A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge. J. Virol. 68(11), 7021–7029. Luciw, P. A., Pratt-Lowe, E., Shaw, K. E., Levy, J. A., and Cheng-Mayer, C. (1995). Persistent infection of rhesus macaques with T-cell-line-tropic and macrophage-tropic clones of simian/human immunodeficiency viruses (SHIV). Proc. Natl. Acad. Sci. USA 92(16), 7490–7494. Marthas, M. L., Sutjipto, S., Higgins, J., Lohman, B., Torten, J., Luciw, P. A., Marx, P. A., and Pedersen, N. C. (1990). Immunization with a live, attenuated simian immunodeficiency virus (SIV) prevents early disease but not infection in rhesus macaques challenged with pathogenic SIV. J. Virol. 64(8), 3694–3700. Mellado, M., Rodriguez-Frade, J. M., Vila-Coro, A. J., de Ana, A. M., and Martinez, A. C. (1999). Chemokine control of HIV-1 infection. Nature 400(6746), 723–724. Miller, C. J., McChesney, M. B., Lu, X., Dailey, P. J., Chutkowski, C., Lu, D., Brosio, P., Roberts, B., and Lu, Y. (1997). Rhesus macaques previously infected with simian/human immunodeficiency virus are protected from vaginal challenge with pathogenic SIVmac239. J. Virol. 71(3), 1911–1921. Montefiori, D. C., Reimann, K. A., Wyand, M. S., Manson, K., Lewis, M. G., Collman, R. G., Sodroski, J. G., Bolognesi, D. P., and Letvin, N. L. (1998). Neutralizing antibodies in sera from macaques infected with chimeric simian-human immunodeficiency virus containing the envelope glycoproteins of either a laboratory-adapted variant or a primary isolate of human immunodeficiency virus type 1. J. Virol. 72(4), 3427–3431. Nara, P. L., Robey, W. G., Arthur, L. O., Asher, D. M., Wolff, A. V., Gibbs, C. J., Jr., Gajdusek, D. C., and Fischinger, P. J. (1987). Persistent

infection of chimpanzees with human immunodeficiency virus: serological responses and properties of reisolated viruses. J. Virol. 61(10), 3173–3180. Novembre, F. J., Saucier, M., Anderson, D. C., Klumpp, S. A., O’Neil, S. P., Brown, C. R. II, Hart, C. E., Guenthner, P. C., Swenson, R. B., and McClure, H. M. (1997). Development of AIDS in a chimpanzee infected with human immunodeficiency virus type 1. J. Virol. 71(5), 4086–4091. Otsyula, M. G., Miller, C. J., Tarantal, A. F., Marthas, M. L., Greene, T. P., Collins, J. R., van Rompay, K. K., and McChesney, M. B. (1996). Fetal or neonatal infection with attenuated simian immunodeficiency virus results in protective immunity against oral challenge with pathogenic SIVmac251. Virology 222(1), 275–278. Pantaleo, G., Graziosi, C., Demarest, J. F., Butini, L., Montroni, M., Fox, C. H., Orenstein, J. M., Kotler, D. P., and Fauci, A. S. (1993). HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature 362(6418), 355–358. Pantaleo, G., Graziosi, C., Demarest, J. F., Cohen, O. J., Vaccarezza, M., Gantt, K., Muro-Cacho, C., and Fauci, A. S. (1994). Role of lymphoid organs in the pathogenesis of human immunodeficiency virus (HIV) infection. Immunol. Rev. 140, 105–130. Pantaleo, G., Menzo, S., Vaccarezza, M., Graziosi, C., Cohen, O. J., Demarest, J. F., Montefiori, D., Orenstein, J. M., Fox, C., Schrager, L. K., Margolick, J. B., Buchbinder, S., Giorgi, J. V., and Fauci, A. S. (1995). Studies in subjects with long-term nonprogressive human immunodeficiency virus infection. N. Engl. J. Med. 332(4), 209–216. Petry, H., Dittmer, U., Stahl-Hennig, C., Coulibaly, C., Makoschey, B., Fuchs, D., Wachter, H., Tolle, T., Morys-Wortmann, C., Kaup, F. J., Jurkiewicz, E., Luke, W., and Hunsmann, G. (1995). Reactivation of human immunodeficiency virus type 2 in macaques after simian immunodeficiency virus SIVmac superinfection. J. Virol. 69(3), 1564– 1574. Putkonen, P., Walther, L., Zhang, Y. J., Li, S. L., Nilsson, C., Albert, J., Biberfeld, P., Thorstensson, R., and Biberfeld, G. (1995). Long-term protection against SIV-induced disease in macaques vaccinated with a live attenuated HIV-2 vaccine. Nature Med. 1(9), 914–918. Quesada-Rolander, M., Makitalo, B., Thorstensson, R., Zhang, Y. J., Castanos-Velez, E., Biberfeld, G., and Putkonen, P. (1996). Protection against mucosal SIVsm challenge in macaques infected with a chimeric SIV that expresses HIV type 1 envelope. AIDS Res. Hum. Retroviruses 12(11), 993–999. Reimann, K. A., Li, J. T., Veazey, R., Halloran, M., Park, I. W., Karlsson, G. B., Sodroski, J., and Letvin, N. L. (1996a). A chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys. J. Virol. 70(10), 6922–6928. Reimann, K. A., Li, J. T., Voss, G., Lekutis, C., Tenner-Racz, K., Racz, P., Lin, W., Montefiori, D. C., Lee-Parritz, D. E., Lu, Y., Collman, R. G., Sodroski, J., and Letvin, N. L. (1996b). An env gene derived from a primary human immunodeficiency virus type 1 isolate confers high in vivo replicative capacity to a chimeric simian/human immunodeficiency virus in rhesus monkeys. J. Virol. 70(5), 3198–3206. Robinson, H. L., Montefiori, D. C., Johnson, R. P., Manson, K. H., Kalish, M. L., Lifson, J. D., Rizvi, T. A., Lu, S., Hu, S. L., Mazzara, G. P., Panicali, D. L., Herndon, J. G., Glickman, R., Candido, M. A., Lydy, S. L., Wyand, M. S., and McClure, H. M. (1999). Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations. Nature Med. 5(5), 526–534. Sakuragi, S., Shibata, R., Mukai, R., Komatsu, T., Fukasawa, M., Sakai, H., Sakuragi, J., Kawamura, M., Ibuki, K., and Hayami, M. (1992). Infection of macaque monkeys with a chimeric human and simian immunodeficiency virus. J. Gen. Virol. 73(Pt. 11), 2983–2987. Shibata, R., Kawamura, M., Sakai, H., Hayami, M., Ishimoto, A., and Adachi, A. (1991). Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells. J. Virol. 65(7), 3514–3520.

PROTECTIVE EFFECT OF GENE-DELETED SHIVs Shibata, R., Seimon, C., Cho, M. W., Arthur, L. O., Nigida, S. M., Jr., Matthews, T., Sawyer, L. A., Schultz, A., Murthy, K. K., Israel, Z., Javadian, A., Frost, P., Kennedy, R. C., Lane, H. C., and Martin, M. A. (1996). Resistance of previously infected chimpanzees to successive challenges with a heterologous intraclade B strain of human immunodeficiency virus type 1. J. Virol. 70(7), 4361–4369. Shibata, R., Seimon, C., Czajak, S. C., Desrosiers, R. C., and Martin, M. A. (1997). Live, attenuated simian immunodeficiency virus vaccines elicit potent resistance against a challenge with a human immunodeficiency virus type 1 chimeric virus. J. Virol. 71(11), 8141– 8148. Stahl-Hennig, C., Dittmer, U., Nisslein, T., Petry, H., Jurkiewicz, E., Fuchs, D., Wachter, H., Matz-Rensing, K., Kuhn, E. M., Kaup, F. J., Rud, E. W., and Hunsmann, G. (1996). Rapid development of vaccine protection in macaques by live-attenuated simian immunodeficiency virus. J. Gen. Virol. 77(Pt. 12), 2969–2981. Steger, K. K., Dykhuizen, M., Mitchen, J. L., Hinds, P. W., Preuninger, B. L., Wallace, M., Thomson, J., Montefiori, D. C., Lu, Y., and Pauza, C. D. (1998). CD41-T-cell and CD201-B-cell changes predict rapid disease progression after simian-human immunodeficiency virus infection in macaques. J. Virol. 72(2), 1600–1605. Stephens, E. B., Joag, S. V., Atkinson, B., Sahni, M., Li, Z., Foresman, L., Adany, I., and Narayan, O. (1997). Infected macaques that controlled replication of SIVmac or nonpathogenic SHIV developed sterilizing resistance against pathogenic SHIV(KU-1). Virology 234(2), 328–339. Suryanarayana, K., Wiltrout, T. A., Vasquez, G. M., Hirsch, V. M., and

263

Lifson, J. D. (1998). Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction. AIDS. Res. Hum. Retroviruses 14(2), 183–189. Ten Haaft, P., Verstrepen, B., Uberla, K., Rosenwirth, B., and Heeney, J. (1998). A pathogenic threshold of virus load defined in simian immunodeficiency virus- or simian-human immunodeficiency virus-infected macaques. J. Virol. 72(12), 10281–10285. Van Rompay, K. K., Otsyula, M. G., Tarara, R. P., Canfield, D. R., Berardi, C. J., McChesney, M. B., and Marthas, M. L. (1996). Vaccination of pregnant macaques protects newborns against mucosal simian immunodeficiency virus infection. J. Infect. Dis. 173(6), 1327–1335. Voss, G., and Letvin, N. L. (1996). Definition of human immunodeficiency virus type 1 gp120 and gp41 cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I alleles in simian-human immunodeficiency virus-infected rhesus monkeys. J. Virol. 70(10), 7335–7340. Watson, A., Ranchalis, J., Travis, B., McClure, J., Sutton, W., Johnson, P. R., Hu, S. L., and Haigwood, N. L. (1997). Plasma viremia in macaques infected with simian immunodeficiency virus: plasma viral load early in infection predicts survival. J. Virol. 71(1), 284–290. Yamamoto, H., Miller, M. D., Tsubota, H., Watkins, D. I., Mazzara, G. P., Stallard, V., Panicali, D. L., Aldovini, A., Young, R. A., and Letvin, N. L. (1990). Studies of cloned simian immunodeficiency virus-specific T lymphocytes: Gag-specific cytotoxic T lymphocytes exhibit a restricted epitope specificity. J. Immunol. 144(9), 3385–3391.