CD4 tropism is conserved among all primate lentiviruses and likely contributes to viral pathogenesis by targeting cells that are critical for the adaptive anti-viral immune responses. Although CD4-independent variants of HIV and SIV have been described that can utilize coreceptors CCR5 or CXCR4 in the absence of CD4, these viruses typically retain their CD4 binding sites and can still interact with CD4. In this thesis, I present the characterization and evaluation, both in vitro and in vivo, of a novel CD4-independent variant of SIV lacking a CD4 binding site. I first describe the derivation of iMac239, a CD4-independent variant of SIVmac239. Like other CD4-independent variants, we found that a mutation in the V1/V2 loops of Env was required for CD4-independent entry, and that acquisition of CD4-independence resulted in an increase in neutralization sensitivity. While iMac239 was CD4-independent, its CD4-binding site was intact, thus we removed the Aspartic Acid residue at position 385 (analogous to D-368 in HIV-1) to ablate CD4 binding. We found that this novel variant, iMac239-ΔD385, exhibited replication kinetics similar to that of the parental iMac239 strain, and was insensitive to neutralization by soluble CD4. Both CD4-independent strains exhibited an expansion of cellular tropism in vitro with infection of CD4- CD8+ T cells in stimulated rhesus PBMCs. Next, I present our evaluation of iMac239-ΔD385 pathogenesis and immunogenicity in four rhesus macaques. iMac239-ΔD385 replicated to a high acute viral peak, but was quickly controlled to undetectable levels by the host immune response. iMac239-ΔD385 infection elicited high and sustained neutralizing antibody titers and polyfunctional T cell responses. Strikingly, we observed an alteration in the distribution of infected cells in the lymph node and expansion in the types of cells infected. We tested iMac239-ΔD385 as a live attenuated vaccine against a pathogenic SIVsmE660, and while the number of animals in the study is too small to determine significance we observed a trend toward improved outcomes post challenge, potentially due to a synergistic interaction between iMac239-ΔD385 vaccination and Trim5α alleles.