REVIEW Gene-Mutated HIV-1/SIV Chimeric Viruses As AIDS Live Attenuated Vaccines for Potential Human

Gene-mutated HIV-1/SIV chimeric viruses as AIDS live attenuated vaccines for potential human use M Hayami1, T Igarashi1, T Kuwata1,MUi1, T Haga1, Y Ami2, K Shinohara2 and M Honda2

1Institute for Virus Research, Kyoto University, Kyoto; and 2AIDS Center, Division of Experimental Animal Research, National Institute of Infectious Diseases, Japan

To develop an AIDS vaccine for human use as well as a suitable T cells against HIV-1 Env without any clinical symptoms; (2) animal model for AIDS research, we constructed a series of SIV Gag crossreacts with HIV-1 Gag, which can be another HIV-1/SIVmac chimeric viruses (SHIVs). We successfully gener- ated a SHIV (designated as NM-3rN) having the HIV-1 env gene, neutralizing epitope besides Env; (3) SIV is genetically close which enabled the evaluation of the efficacy of HIV-1 Env-tar- to HIV-2 which is less pathogenic than HIV-1, and accidental geted vaccines in macaque monkeys instead of chimpanzees. SIV infection in humans resulted in silent infection, suggesting Two NM-3rN derivatives (NM-3 and NM-3n) induced long-term that SIV is not pathogenic in humans; (4) these SHIVs can anti-virus immunities without manifesting the disease. The infect human lymphocytes, suggesting that the infection monkeys vaccinated with NM-3 or NM-3n became resistant to immunity by SHIVs may be induced in humans and protect a challenge inoculation with NM-3rN. Serum from a monkey vaccinated with NM-3 neutralized not only the parental HIV-1 humans from the HIV-1 infection. For the purpose of making (NL432), but also an antigenically different HIV-1 (MN). In vivo a vaccine, therefore, some genes essential for pathogenicity experiments confirmed the heterologous protection against an were mutated to attenuate these chimeric viruses. We SHIV having the HIV-1 (MN) env. In addition to specific immun- observed that the monkeys infected with these chimeric ity including neutralizing antibodies and cytotoxic T lympho- viruses were protected against a challenge infection. In this cyte activity, nonspecific immunity such as natural killer report, we present recent progress in our studies of SHIVs, and activity is associated with this protection. These data suggest that the live vaccine has the ability to protect individuals discuss their applications to the development of an AIDS against various types of HIVs. These SHIVs should contribute vaccine. to the development of future anti-HIV-1 live vaccines in humans. Keywords: HIV-1; SIV; chimeric virus; AIDS; live vaccine Strategy for constructing chimeric viruses First, each gene of HIV-1 and SIV was investigated by constructing various mutant viruses and determining their viral Introduction replication capacity and their exchangeability between the two viruses.6,7 As a result, the strategy for construction of The lack of a suitable animal model for evaluating vaccine chimeric viruses between HIV-1 and SIV was determined as candidates of human immunodeficiency virus type 1 (HIV-1) follows: (1) vpr and nef can be sacrificed; (2) tat and TAR (in is a difficult problem in developing the vaccine, because few LTR) can be from different parental viruses, but rev and RRE animal species are susceptible to HIV-1 infection. Pig-tailed (in env), which interact, should have the same origin; (3) gag macaques1 and rabbits2 were reported to be infected with and env can be from different origins, but gag and pol should HIV-1, but they could not be used practically. At present, be from the same origin. chimpanzees are the only animals used for evaluation of HIV- Based on this strategy, we constructed chimeric viruses 1 vaccines, although some human trials have been conduc- between HIV-1 and SIVmac. SIVmac is known to induce an ted.3 Chimpanzees, however, rarely show any AIDS-like dis- AIDS-like disease in macaque monkeys. At first, chimeric ease, and their use is extremely limited for experiments viruses were constructed by replacing the 5′-half containing because they are an endangered species. We therefore tried gag and pol, and the 3′-half containing env, tat and rev to generate an HIV-1 that is infectious to, and able to induce, between HIV-1 and SIVmac, and their replicative abilities an AIDS-like disease in macaque monkeys, by constructing were investigated in human or monkey peripheral blood HIV-1 chimeric viruses based on SIVmac (SHIVs). As a result, mononuclear cells (PBMC). As a result, the chimeric viruses we succeeded in constructing chimeric viruses, which include that had the env of HIV-1 and the gag and pol of SIVmac the env gene of HIV-1 and which could infect macaque mon- could replicate in monkey PBMC, but the other viruses, which keys, a species that is available for experimental use.4,5 These had the env of SIVmac and the gag and pol of HIV-1, could chimeric viruses express the HIV-1 Env protein, and enable not4 (Figure 1). This result shows that the gag and pol genes us to use macaque monkeys instead of chimpanzees for evalu- of SIVmac determine the tropism to monkey PBMC and that ation of vaccine candidates. This is done by vaccinating the the env gene has no relation to this cell tropism. This fact was monkeys with a vaccine candidate and then challenging them unexpected because Env, whose function is to bind to cells, with the chimeric viruses. was generally supposed to determine the cell tropism. In addition, SHIVs may be used as anti-HIV-1 live attenuated vaccines for humans for the following reasons: (1) SHIV- infected monkeys produced neutralizing antibodies and killer Experimental infection of monkeys with HIV-1/SIVmac chimeric viruses

5,8 Correspondence: M Hayami, Institute for Virus Research, Kyoto Uni- In the series of chimeric viruses constructed, NM-3 and versity, Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan NM-3n,9 which could replicate in monkey PBMC, were HIV-1/SIVmac chimeric viruses M Hayami et al S43

Figure 1 Replication and pathogenicity of HIV-1, SIVmac and HIV-1/SIVmac chimeric viruses in vitro and in vivo. inoculated to cynomolgus monkeys intravenously (i.v.). Their (CTL) were detected at 3 years post-inoculation. In the other abilities to infect monkeys were examined by virus recovery, monkey, proviral DNA was detected in PBMC only in the the polymerase chain reaction (PCR) and antibody response early period after inoculation, and anti-Env antibodies were (Table 1). NM-3 has LTR, gag, pol, vif and vpx of SIVmac and detected for a while. Thus, this monkey was considered to be env, tat, rev and vpu of HIV-1. NM-3n, which has a similar infected transiently.5 These results showed that NM-3 infec- structure but contains the intact nef gene was constructed tion induced both humoral and cellular anti-virus immunities based on the report by Kestler et al10 who demonstrated that in monkeys, although its infectivity was not so efficient. the nef gene was required for the induction of AIDS-like Two monkeys (a 3-year-old male and a 3-year-old female) 3.5 disease in monkeys infected with SIVmac. were inoculated with 10 TCID50 of NM-3n. Viruses were Of two monkeys (8- and 11-year-old males) inoculated with occasionally re-isolated from one of these monkeys (male), 4 10 TCID50 of NM-3, one developed a persistent viremic state, and proviruses were also detected in its PBMC by PCR in the and anti-Env and anti-Gag antibodies were raised as expected early period after inoculation. Antibodies which neutralized from the structure of this chimeric virus. These antibodies had NM-3n and the parental HIV-1 were detected persistently in virus neutralizing activity, which neutralized NM-3 and the monkeys infected with NM-3n. HIV-1 Env-specific killer T parental HIV-1. HIV-1 Env-specific cytotoxic T lymphocytes cells were observed at 2 years post-inoculation in the monkey

Table 1 Summary of experimental infection of NM-3 and NM-3n

Animal Experimental infection with NM-3 (weeks)

0 2 6 10 21 28 40 94 100 116 129 138 149 virus recovery MF1 −++−−−−++++++ MF2 −−−−−−−−−−−−− antibodies (Western) anti-HIV-1 Env MF1 −−−++++++++++ MF2 −−++++−ND ND ND ND ND ND anti-SIVmac Gag MF1 −−+++++++++++ MF2 −−−−−−−ND ND ND ND ND ND

Experimental infection with NM-3n (weeks)

0 2 6 10 12 14 20 30 37 42 52 63 73 88 virus recovery MF3080 −−−−−−−−−−−−−− MF3096 −−−−−−−−+−−+−− PCR (env) MF3080 −++−−−−−ND −−−−ND MF3096 −++−+++−ND −−−−ND antibodies (Western) anti-HIV-1 Env MF3080 − ND ND + ND +++++++++ MF3096 − ND ND − ND +++++++++ anti-SIVmac Gag MF3080 − ND ND + ND +++++++++ MF3096 − ND ND − ND −−−++++++

ND, not done. HIV-1/SIVmac chimeric viruses M Hayami et al S44 from which the virus was re-isolated at 63 weeks post-inocu- keys died of an AIDS-like syndrome, from which we are now lation (w.p.i.).9 These results showed that NM-3n, as well as trying to clone the pathogenic chimeric virus. NM-3, infected monkeys and induced both humoral and cellular anti-virus immunities. Viruses re-isolated from the monkeys infected with NM-3 Development of vaccines using chimeric viruses having or NM-3n basically maintained their chimeric structure, HIV-1 Env which had Env of HIV-1 and Gag of SIVmac. Anti-HIV-1 vaccines including inactivated viral particles, recombinant viral proteins and synthetic peptides, are cur- Construction of a chimeric virus containing vpr and rently on trial in various laboratories. They are so-called ‘inac- nef, which produces a more efficient infection tivated-vaccines’ which cannot replicate, and are not thought to be very efficient. In the history of vaccines against viral diseases, such as polio-myelitis and measles, live-attenuated NM-3 and NM-3n, whose vpr genes were not intact (Figure 2), vaccines have been proven to be the most effective by were able to infect the monkeys, but did not induce an AIDS- inducing long-lasting and strong humoral, cellular and other like disease. Lang et al11 reported that the vpr gene was asso- protective immunities. Therefore, attempts should be made to ciated with induction of AIDS-like disease. In an attempt to develop a live attenuated vaccine. For the development of a generate a pathogenic SHIV, the vpr of HIV-1 and the nef of live vaccine, it is necessary to select an attenuated strain. SIVmac were introduced to the chimeric virus.12 Accordingly, Although high-pathogenic or low- (non-) pathogenic HIV-1 this chimeric virus has LTR, gag, pol, vif, vpx and nef of strains have been described, these reports were based only on SIVmac and env, tat, rev, vpu and vpr of HIV-1. As this virus, the observations of in vitro cultures or on indirect evidence. termed NM-3rN, could replicate in monkey PBMC more The pathogenicities of these strains in individuals were not efficiently than NM-3 and NM-3n, 105 TCID of NM-3rN was 50 directly confirmed. Live attenuated vaccines so far established inoculated intravenously into a total of six monkeys including in other viral infections have been developed by repeated cynomolgus, rhesus and pig-tailed monkeys. Viruses were passage in cells of a different animal species. In HIV-1 vaccine continuously re-isolated from 2 w.p.i. in all the monkeys development, however, this method was considered to be infected with NM-3rN. It was shown that this virus could dangerous because a pathogenic revertant could appear due infect monkeys much more efficiently than NM-3 and NM- to the high mutation rate of HIV-1. In a study of SIV, it was 3n.12 reported that the nef gene was required to induce disease in These results indicate that NM-3rN is useful as a challeng- monkeys.10 Daniel et al15 showed that monkeys infected with ing virus for the evaluation of vaccine candidates, because a nef-deleted non-pathogenic SIVmac were protected from a NM-3rN has HIV-1 Env, and candidates of AIDS vaccines pathogenic SIVmac. They showed a new approach to attenu- under development are directed to an induction of an anti- ate viruses by deleting genes associated with the onset of dis- Env immunity. Thus, the evaluation of vaccines has become ease. In our chimeric viruses, the nef and vpr of NM-3 were possible in macaque monkeys instead of chimpanzees by not intact, and the vpr of NM-3n was not intact. These viruses using NM-3rN as a challenging virus. HIV-1NL432 was used induced neutralizing antibodies and CTL without any clinical to construct NM-3rN, and its V3 of Env, a primary neutralizing manifestations for 2–3 years. epitope, is of the prototypic IIIb type. We also constructed We performed a protection experiment to examine the chimeric viruses from HIV-1SF13 and HIV-1HAN2, repli- ability of immunities induced by NM-3 or NM-3n (Table 2). cation-competent in macaque monkeys.13 The V3 of these The viral and immunological status varied widely among the chimeric viruses is an MN type which is endemic in America monkeys ‘vaccinated’ with NM-3 or NM-3n. Seven ‘vacci- and Europe. The evaluation of vaccines against an MN-type nated’ monkeys and four normal monkeys as a control were virus has become possible by these chimeric viruses. challenged with NM-3rN. Virus re-isolation was carried out from PBMC of these monkeys collected at 2, 6 and 10 w.p.i. Infectious viruses were recovered from all the naive monkeys Onset of AIDS-like disease by an in vivo passaged chimeric at all times, but were not recovered from three of the seven virus vaccinated monkeys after the challenge. In the four vaccinated monkeys from which viruses were recovered, cell-asso- Although monkeys infected with NM-3rN have shown no ciated virus loads were lower than those in the naive controls clinical signs associated with AIDS up to the present (2 years by about 10- to 100-fold. Attempts to recover virus from post-inoculation), virus loads in some monkeys have lymph nodes and spleen were made 10–20 weeks post-chal- increased. A variant of NM-3rN (R43–56) recovered at 56 lenge and the samples were examined for virus using PCR. w.p.i. from one monkey persistently infected with NM-3rN, Viruses were not recovered from three of the seven vaccinated improved its replication ability in monkey PBMC compared monkeys, but they were recovered from the other four. The with the original NM-3rN. When R43–56 was inoculated into differentiating PCR proved that all the viruses detected or three monkeys, cell-associated virus loads were about 10–50 recovered from PBMC, lymph nodes and spleens in vacci- times higher than that of the original NM-3rN-inoculated nated monkeys were NM-3 or NM-3n, not the challenging monkeys. In one of the monkeys, the number of CD4+ lym- virus, NM-3rN. These results indicate that all the monkeys phocytes in PBMC declined to the moribund stage. The mon- vaccinated with NM-3 or NM-3n were protected from key lost weight (Figure 3) and diarrhea was observed at the last challenge inoculation with NM-3rN.16 stage. At autopsy, systemic lymphoadenopathy and follicular Both cell-mediated and humoral immunities were supposed depletion with an expanded paracortex in the lymphnodes to contribute to this protection because both immunities were and spleen was observed. The monkey was also found to have detected in most of the vaccinated monkeys. In two monkeys, acute hepatitis which was probably caused by an opportun- however, no neutralizing antibodies was detected, and in one istic viral infection.14 The above findings suggest that the mon- monkey, no CTL activity was found. Nevertheless, all the HIV-1/SIVmac chimeric viruses M Hayami et al S45

Figure 2 Genomic structure of HIV-1, SIVmac and HIV-1/SIVmac chimeric viruses. (᭿) Sequences derived from HIV-1NL432; (ᮀ) from SIVmac239; ( ) from HIV-1MN; ( ) chimeric region of HIV-1NL432 and SIVmac239. HIV-1/SIVmac chimeric viruses M Hayami et al S46 NM-3 neutralized not only NL432, the immunizing chimeric virus and parental HIV-1, but also the antigenically different HIV-1 MN. The neutralizing titer is shown in Table 3. It is based on the highest serum dilution of the culture supernatants that caused 50% or more reduction of RT activity compared with the RT activity of culture supernatants treated with a normal monkey serum. The main neutralizing epitope of the V3 region in NL432 is of the IIIb type, which is different from the MN type. Thus, the vaccinated monkey serum showed a broad spectrum of neutralization. An in vivo experiment also confirmed the heterologous protection by a live vaccine: two monkeys vaccinated with the chimeric virus having HIV-1 Env of the IIIb type were challenged by the chimera having an Env of MN type. No virus was recovered and no PCR product was obtained from the vaccinated monkeys (Table 4). To ensure the safety of a live vaccine, we also constructed a chimeric virus which was completely defective with respect to vpx, vpr and nef, and we are now examining its abilities to infect monkeys and to induce immunities. In addition, we have constructed and examined chimeric viruses using non- pathogenic SIVagm as a parental virus, which originated from an African green monkey.17 Figure 3 Changes of peripheral CD4 and CD8 positive lympho- cyte subsets and body weight in a monkey infected with R43–56. Conclusions monkeys were resistant to the challenge inoculation. It is note- worthy that natural killer (NK) cell activity was raised in all To date, many attempts have been made to develop anti-HIV- of these monkeys by vaccination.16 Therefore, these vaccine 1 vaccines. At present, however, there is some pessimism viruses seem to induce an infectious immunity including not regarding the development of vaccines due to the failure of only specific neutralization and CTL activity, but also a non- several vaccine trials in humans. There are difficult problems specific defense mechanism such as NK activity. This could that must be overcome in order to develop anti-HIV-1 vac- be a big advantage of a live-attenuated vaccine, especially cines because of particular properties of HIV-1, such as a because of the rapid mutation and recombination of HIV-1. broad antigenicity. Our data suggest that a live vaccine could There is additional evidence that the SHIV live vaccine is protect individuals from infection of various types of HIV. effective in protecting individuals from infection by various Another difficulty is that vaccine candidates must be evalu- types of HIV-1. The serum of one monkey vaccinated with ated directly in humans, which can take years. Chimeric

Table 2 Monkeys vaccinated with NM-3 or NM-3n were protected from the challenge inoculation with NM-3rN

Monkey Vaccinated Viral and immunological Cell-associated virus loads after virus status challengec (weeks)

Virus recovery Antibody Killer cell 2 6 10 (just before activity b challenge)Env Gag Neutralizing (Env) titera

MF1 NM-3 +(+) + + ++ ++ +(NM-3) ++(NM-3) − MF12d NM-3 +(−) −+−± −−− MF15d NM-3 +(−) −+−++−−− MF11d NM-3 +(+) ++++ −+(NM-3) − MF13d NM-3 +(+) ++++ −+(NM-3) − MF3080 NM-3n −(−) +++− −−− MF3096 NM-3n +(−) ++++ −−+(NM-3n) MF47 none / −−−− +++++++ MM50 none / −−−− +++++++++ MF3445 none / −−−− +++++ MF3446 none / −−−− ++++++

aThe highest serum dilution of the culture supernatants causing 50% or more reduction of RT activity of that treated with a normal monkey serum was determined. ++, Ͼ100×; +; 20–100×; −, Ͻ20×. bTarget and effector cells were mixed at various ratio (100:1–12.5:1) and target cell destruction was evaluated by 51Cr release in the supernatant. The representative value at a ratio of 100:1 was shown by ++, Ͼ25%; +, 15–25%; ±, 10–15%; −, Ͻ10%. cThe serially three-fold diluted PBMC were cocultivated with human T lymphoid M8166 cells. The cell-associated virus load was determined by the minimal number of PBMC required for virus recovery. +++, Ͻ104 cells; ++,104–105 cells; +, Ͼ105 cells, −, not recovered. dInfection by blood transfusion from MF1. 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J Virol will make rapid progress once an evaluation system based on 1993; 67: 902–912. challenges with chimeric viruses has been established. 12 Kuwata T, Igarashi T, Ido E, Jin M, Mizuno A, Jiangli C, Hayami Among the variety of candidates examined as anti-HIV-1 M. Construction of human immunodeficiency virus 1/simian immunodeficiency strain mac chimeric viruses having vpr and/or vaccines, a promising approach may be the use of live attenu- nef of different parental origins and their in vitro and in vivo repli- ated vaccines in view of the history of vaccines against viral cation. J Gen Virol 1995; 76: 2181–2191. diseases. However, it is difficult to use attenuated strains of 13 Kuwata T, Shioda T, Igarashi T, Ido E, Ibuki K, Enose Y, Stahl- HIV-1 as live vaccines. The mutation rate of HIV-1 is so high Hennig C, Hunsmann G, Miura T, Hayami M. Chimeric viruses that it is hard to prevent the appearance of virulent virus. As between SIVmac and various HIV-1 isolates have biological our trial to attenuate our chimeric viruses by deleting genes properties that are similar to those of the parental HIV-1. AIDS 1996; 10: 1331–1337. is limited in the case of monkeys, these chimeric viruses may 14 Nagamachi D, Igarashi T, Shimada T, Kuwata T, Ui M, Ami Y, not be directly used in humans. Even in that case, this Enose Y, Ido E, Fukumoto M, Hayami M. Rapid and progressive approach provides useful information on the pathogenicity of CD4+ decline in a monkey infected with an SIV+HIV-1 chimeric HIV infection and AIDS, and eventually will contribute to the virus. J Vet Med Sci 1998; 60: 361–363. development of future vaccines. Further efforts should be 15 Daniel MD, Kirchhoff F, Czajak SC, Sehgal PK, Desrosiers RC. made to ensure the safety and efficacy of live-attenuated Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 1992; 258: 1938–1941. AIDS vaccine. 16 Igarashi T, Ami Y, Yamamoto H, Shibata R, Kuwata T, Mukai R, Shinohara K, Komatsu T, Adachi A, Hayami M. Protection of monkeys vaccinated with vpr- and/or nef-defective simian immuno- Acknowledgments deficiency virus strain mac/human immunodeficiency virus type 1 chimeric viruses: a potential candidate live-attenuated human This work was supported in part by a grant-in-aid for AIDS AIDS vaccine. J Gen Virol 1997; 78: 985–989. 17 Jin M, Ido E, Kuwata T, Igarashi T, Cichutek K, Kurth R, Miura T, research from the Organization for Drug ADR Relief, R&D Enose Y, Chen J, Hayami M. Replication and cytopathogenicity Promotion and Product Review, Japan. of human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus agm3 chimeric viruses in human and monkey cells: the 5′ half of the HIV-1 genome is responsible for virus cytopathogenicity. J Gen Virol 1996; 77: 2427–2431.