Latent reservoirs of human immunodeficiency virus (HIV) present significant challenges for eradicating HIV from infected persons, particularly reservoirs in the brain established during acute infection. A simian immunodeficiency virus (SIV)/macaque model of HIV dementia was used to show that viral DNA levels in the brain remained at constant levels from acute through asymptomatic infection, despite significant down-regulation of viral RNA in the brain after the acute phase of infection. Viral replication in the brain coincided with activation of macrophages and microglia in the central nervous system; down-regulation of viral replication coincided with increased infiltration of cytotoxic lymphocytes and reduced activation of macrophages and microglia in the brain. Comparison of viral genotypes in the central nervous system and peripheral blood mononuclear cells suggests that recrudescence of viral replication in brain occurs by reactivation of latent viral DNA. Latent virus in the brain must be considered in therapeutic strategies to eliminate HIV from infected persons.

Whole inactivated viral particles have been successfully used as vaccines for some viruses, but procedures historically used for inactivation can denature virion proteins. Results have been inconsistent, with enhancement of disease rather than protection seen in some notable instances following vaccination. We used the compound 2,2′-dithiodipyridine (aldrithiol-2; AT-2) to covalently modify the essential zinc fingers in the nucleocapsid (NC) protein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) virions, thereby inactivating infectivity. The inactivated virus was not detectably infectious in vitro (up to 5 log units of inactivation). However, in contrast to virions inactivated by conventional methods such as heat or formalin treatment, viral and host cell-derived proteins on virion surfaces retained conformational and functional integrity. Thus, immunoprecipitation of AT-2-treated virions was comparable to precipitation of matched untreated virus, even when using antibodies to conformational determinants on gp120. AT-2 inactivated virions bound to CD4+ target cells and mediated virus-induced, CD4-dependent “fusion from without” comparably to native virions. However, viral entry assays demonstrated that the viral life cycle of AT-2-treated virions was arrested before initiation of reverse transcription. The major histocompatibility complex (MHC) class II molecules on the surface of AT-2-treated virions produced from MHC class II-expressing cells retained the ability to support class II-dependent, superantigen-triggered proliferative responses by resting T lymphocytes. These findings indicate that inactivation via this method results in elimination of infectivity with preservation of conformational and functional integrity of virion surface proteins, including both virally encoded determinants and proteins derived from the host cells in which the virus was produced. Such inactivated virions should provide a promising candidate vaccine antigen and a useful reagent for experimentally probing the postulated involvement of virion surface proteins in indirect mechanisms of HIV-1 pathogenesis.

Internally controlled RT-PCR methods (QC-RT-PCR) for quantification of SIV RNA are effective, but are relatively cumbersome, expensive, and time and labor intensive. For greater throughput and efficiency, we have developed a method for quantification of plasma SIV RNA levels by real-time RT-PCR using the Applied Biosystems Prism 7700 sequence detection system. This assay format allows real-time kinetic analysis of PCR product generation, providing a broad linear dynamic range and ensuring that quantification is based on analysis during the exponential phase of amplification, regardless of the input template copy number. Simultaneous amplification and analysis eliminates any requirement for handling amplified products, increasing throughput and eliminating a potential source of assay contamination. The assay we have developed for quantification of SIV RNA has a nominal threshold sensitivity of 300 copy Eq/ml of plasma, although as little as 10 copy Eq/reaction of SIV RNA template can be detected. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 25% (coefficient of variation), based on studies of extraction and analysis of replicate aliquots of the same plasma specimens. The combination of sensitivity, precision, and broad dynamic range allows reliable quantification of viral load even during dynamic phases of SIV infection, such as through the onset and resolution of primary infection, or during treatment with antiretroviral agents. The primer-probe combinations we have developed allow quantification of SIV isolates most commonly used for experimental studies. Availability of this assay should greatly facilitate studies of basic pathogenesis and evaluation of therapeutic and prophylactic approaches in the SIV-infected macaque.