Enhanced Infectivity of an R5-Tropic Simian/Human Immunodeficiency Virus Carrying Human Immunodeficiency Virus Type 1 Subtype C Envelope after Serial Passages in Pig-Tailed Macaques (Macaca nemestrina)

Subtype C viruses have become the most prevalent human immunodeficiency virus type 1 (HIV-1) genotype globally (49). UNAIDS has estimated that there are now eight million subtype C infections worldwide, mainly in sub-Saharan Africa and Asia. In these respective geographic areas, subtype C is more common than any other subtype, and it now accounts for about 40% of all new HIV-1 infections in the world. In one recent study in two cities in southern China, 22 of 23 infected patients were found to carry subtype C viruses (Z. Chen, Y. Cao, L. Zhang, and D. Ho, unpublished data). Despite mounting efforts, it remains unclear why this subtype has gained dominance so quickly and whether means can be developed to slow down its spread. To address these questions effectively, a relevant animal model to study HIV-1 subtype C would be very useful. One of the current animal models for AIDS research consists of Asian macaques experimentally infected with simian immunodeficiency virus (SIV) (13, 14). Indeed, several molecular clones of SIV are pathogenic in vivo, causing a fatal AIDS-like disease in macaques (25, 26). For this reason, the model has been widely used to evaluate various vaccine strategies and to study AIDS pathogenesis (4, 12, 14, 19, 29, 36, 39). Nevertheless, because the env genes of SIV and HIV-1 show significant sequence diversity (28), the SIV/macaque model is of limited utility for in vivo analyses of the phenotypic and immunological properties of HIV-1 envelope. Some groups have attempted to adapt HIV-1 in macaques (2, 3, 6, 16). These efforts, however, were largely unsuccessful. The value of the macaque model has increased since the development of a chimeric simian/human immunodeficiency virus (SHIV) (31, 34, 44). Traditionally, SHIV is a chimeric lentivirus that uses pathogenic SIVmac239 as a genetic background, except that its tat, rev, and env genes are replaced by the corresponding regions of HIV-1 (23, 32, 34, 44). Since SHIV retains the ability to infect macaques, it provides a unique in vivo model for studying the pathogenic properties of HIV-1 envelope and for examining the efficacy of HIV-1 vaccines based on envelope glycoproteins. Several SHIV strains have been constructed, and their pathogenicity in nonhuman primates has been evaluated. Most current SHIV constructs utilize envelope genes derived from HIV-1 subtype B strains, either from lab-adapted, syncytium-inducing (SI), T-tropic viruses (HIV-1HXB2 and HIV-1NL43) or from primary, non-syncytium-inducing (NSI), M-tropic (HIV-1162), SI T-tropic (HIV-133), and dual-tropic (HIV89.6 and HIV-1DH12) isolates (23, 32, 34, 44). Since these chimeras retain biological properties of corresponding parental HIV-1 env, they have been used to reveal envelope-determined differences in the replication capacity of the SHIVs in vivo and in the induction of various virus-specific immune responses. These SHIV/macaque models have allowed researchers to explore the significance of HIV-1 env variation, as well as to evaluate vaccines based on HIV-1 Env antigens. In addition to SHIVs based on subtype B, one has been successfully developed for subtype E (27). However, there has been no SHIV for subtype C. In this study, approaches similar to those used for constructing subtype B and E SHIVs were adopted to make a subtype C envelope-based SHIV. We focused on primary, NSI HIV-1 subtype C viruses, as they have been demonstrated to use CCR5 for entry (52). This subtype was selected because of its emerging dominance in the epidemic, and the particular NSI, R5-tropic phenotype was selected because it represents the dominant type of HIV-1 strains transmitted sexually (54, 55). Moreover, it has been demonstrated recently that R5-tropic viruses cause distinct pathogenic effects in comparison to X4-tropic ones (20, 50). Here, we report that a replication-competent SHIVCHN19 was generated by using HIV-1 subtype C envelope in the background of SHIV33. SHIVCHN19 was found to be different from SHIV162 in that the new virus did not infect rhesus peripheral blood mononuclear cells (PBMC) despite CD8+ T-cell depletion. The virus was, however, replication competent in CD4+ T lymphocytes of pig-tailed macaques. To test its in vivo growth capacity, SHIVCHN19 was inoculated into two pig-tailed and two rhesus macaques. We found that SHIVCHN19 replicated preferentially in pig-tailed macaques. To determine whether in vivo adaptation would enhance the infectivity of SHIVCHN19, serial passages were carried out in three groups of two pig-tailed macaques each, via intravenous blood-bone marrow transfusion. In comparison to two passage 1 (P1) pig-tailed macaques, the passages were successful as shown by (i) the increasingly elevated levels of plasma viremia in animals from later passages, (ii) the shortened doubling time of plasma virus during acute infection with each passage, (iii) faster seroconversion in P2 to P4 animals, (iv) higher levels of sustained viral load in animals from later passages, (v) the enhanced viral infectivity in rhesus PBMC, and (vi) profound CD4+ T-cell depletion in the jejunal lamina propria of P4 animals. Importantly, the serial passages did not change the viral phenotype as determined by the persistence of the R5 tropism of SHIVCHN19 isolated from the two P4 animals. Our data indicate the establishment of the first R5-tropic SHIV/macaque model for HIV-1 subtype C vaccine and pathogenesis studies.