A Targeted Mass Spectrometry Assay for Detection of HIV Gag Protein Following Induction of Latent Viral Reservoirs

During early infection, HIV-1 establishes a reservoir of latently infected cells that persist during antiretroviral therapy. These reservoirs are considered the primary obstacle to eradicating HIV-1 from patients, and multiple strategies are being investigated to eliminate latently infected cells. Measuring the reservoir size using an affordable and scalable assay is critical as these approaches move into clinical trials: the current “gold-standard” viral outgrowth assay is costly, labor-intensive, and requires large numbers of cells. Here, we assessed whether selective reaction monitoring-mass spectrometry (SRM-MS) is sufficiently sensitive to detect latent HIV reservoirs following reactivation of virus. The Gag structural proteins were the most abundant viral proteins in purified virus and infected cells, and tractable peptides for monitoring Gag levels were identified. We then optimized a Gag immunoprecipitation procedure that permitted sampling of more than 10⁷ CD4+ T cells, a requirement for detecting exceedingly rare latently infected cells. Gag peptides were detectable in both cell lysates and supernatants in CD4+ T cells infected in vitro at frequencies as low as ∼1 in 10⁶ cells and in cells from HIV-infected patients on suppressive antiretroviral therapy with undetectable viral loads. To our knowledge, this represents the first detection of reactivated latent HIV reservoirs from patients without signal amplification. Together, these results indicate that SRM-MS is a viable method for measuring latent HIV-1 reservoirs in patient samples with distinct advantages over current assays

A Targeted Mass Spectrometry Assay for Detection of HIV Gag Protein Following Induction of Latent Viral Reservoirs

During early infection, HIV-1 establishes a reservoir of latently infected cells that persist during antiretroviral therapy. These reservoirs are considered the primary obstacle to eradicating HIV-1 from patients, and multiple strategies are being investigated to eliminate latently infected cells. Measuring the reservoir size using an affordable and scalable assay is critical as these approaches move into clinical trials: the current “gold-standard” viral outgrowth assay is costly, labor-intensive, and requires large numbers of cells. Here, we assessed whether selective reaction monitoring-mass spectrometry (SRM-MS) is sufficiently sensitive to detect latent HIV reservoirs following reactivation of virus. The Gag structural proteins were the most abundant viral proteins in purified virus and infected cells, and tractable peptides for monitoring Gag levels were identified. We then optimized a Gag immunoprecipitation procedure that permitted sampling of more than 10⁷ CD4+ T cells, a requirement for detecting exceedingly rare latently infected cells. Gag peptides were detectable in both cell lysates and supernatants in CD4+ T cells infected in vitro at frequencies as low as ∼1 in 10⁶ cells and in cells from HIV-infected patients on suppressive antiretroviral therapy with undetectable viral loads. To our knowledge, this represents the first detection of reactivated latent HIV reservoirs from patients without signal amplification. Together, these results indicate that SRM-MS is a viable method for measuring latent HIV-1 reservoirs in patient samples with distinct advantages over current assays

A Targeted Mass Spectrometry Assay for Detection of HIV Gag Protein Following Induction of Latent Viral Reservoirs

During early infection, HIV-1 establishes a reservoir of latently infected cells that persist during antiretroviral therapy. These reservoirs are considered the primary obstacle to eradicating HIV-1 from patients, and multiple strategies are being investigated to eliminate latently infected cells. Measuring the reservoir size using an affordable and scalable assay is critical as these approaches move into clinical trials: the current “gold-standard” viral outgrowth assay is costly, labor-intensive, and requires large numbers of cells. Here, we assessed whether selective reaction monitoring-mass spectrometry (SRM-MS) is sufficiently sensitive to detect latent HIV reservoirs following reactivation of virus. The Gag structural proteins were the most abundant viral proteins in purified virus and infected cells, and tractable peptides for monitoring Gag levels were identified. We then optimized a Gag immunoprecipitation procedure that permitted sampling of more than 10⁷ CD4+ T cells, a requirement for detecting exceedingly rare latently infected cells. Gag peptides were detectable in both cell lysates and supernatants in CD4+ T cells infected in vitro at frequencies as low as ∼1 in 10⁶ cells and in cells from HIV-infected patients on suppressive antiretroviral therapy with undetectable viral loads. To our knowledge, this represents the first detection of reactivated latent HIV reservoirs from patients without signal amplification. Together, these results indicate that SRM-MS is a viable method for measuring latent HIV-1 reservoirs in patient samples with distinct advantages over current assays