Modifications to rhesus macaque TCR constant regions improve TCR cell surface expression.

T cell immunotherapy success is dependent on effective levels of antigen receptor expressed at the surface of engineered cells. Efforts to optimize surface expression in T cell receptor (TCR)-based therapeutic approaches include optimization of cellular engineering methods and coding sequences, and reducing the likelihood of exogenous TCR α and β chains mispairing with the endogenous TCR chains. Approaches to promote correct human TCR chain pairing include constant region mutations to create an additional disulfide bond between the two chains, full murinization of the constant region of the TCR α and β sequences, and a minimal set of murine mutations to the TCR α and β constant regions.

Broad coverage of neutralization-resistant SIV strains by second-generation SIV-specific antibodies targeting the region involved in binding CD4.

Both SIV and SHIV are powerful tools for evaluating antibody-mediated prevention and treatment of HIV-1. However, owing to a lack of rhesus-derived SIV broadly neutralizing antibodies (bnAbs), testing of bnAbs for HIV-1 prevention or treatment has thus far been performed exclusively in the SHIV NHP model using bnAbs from HIV-1-infected individuals. Here we describe the isolation and characterization of multiple rhesus-derived SIV bnAbs capable of neutralizing most isolates of SIV.

Antibody-mediated depletion of viral reservoirs is limited in SIV-infected macaques treated early with antiretroviral therapy.

The effectiveness of virus-specific strategies, including administered HIV-specific mAbs, to target cells that persistently harbor latent, rebound-competent HIV genomes during combination antiretroviral therapy (cART) has been limited by inefficient induction of viral protein expression. To examine antibody-mediated viral reservoir targeting without a need for viral induction, we used an anti-CD4 mAb to deplete both infected and uninfected CD4+ T cells. Ten rhesus macaques infected with barcoded SIVmac239M received cART for 93 weeks starting 4 days after infection.

Validation of the Viral Barcoding of Simian Immunodeficiency Virus SIVmac239 and the Development of New Barcoded SIV and Subtype B and C Simian-Human Immunodeficiency Viruses.

Genetically barcoded viral populations are powerful tools for evaluating the overall viral population structure as well as assessing the dynamics and evolution of individual lineages over time. Barcoded viruses are generated by inserting a small, genetically unique tag into the viral genome, which is retained in progeny virus. We recently reported barcoding the well-characterized molecular clone simian immunodeficiency virus (SIV) SIVmac239, resulting in a synthetic swarm (SIVmac239M) containing approximately 10,000 distinct viral clonotypes for which all genetic differences were within a 34-base barcode that could be tracked using next-generation deep sequencing.

Preferential Small Intestine Homing and Persistence of CD8 T Cells in Rhesus Macaques Achieved by Molecularly Engineered Expression of CCR9 and Reduced Manipulation.

doptive ell ransfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques.

Clonal expansion of SIV-infected cells in macaques on antiretroviral therapy is similar to that of HIV-infected cells in humans.

Clonal expansion of HIV infected cells plays an important role in the formation and persistence of the reservoir that allows the virus to persist, in DNA form, despite effective antiretroviral therapy. We used integration site analysis to ask if there is a similar clonal expansion of SIV infected cells in macaques. We show that the distribution of HIV and SIV integration sites in vitro is similar and that both viruses preferentially integrate in many of the same genes.

Rational design and in vivo selection of SHIVs encoding transmitted/founder subtype C HIV-1 envelopes.

Chimeric Simian-Human Immunodeficiency Viruses (SHIVs) are an important tool for evaluating anti-HIV Env interventions in nonhuman primate (NHP) models. However, most unadapted SHIVs do not replicate well in vivo limiting their utility. Furthermore, adaptation in vivo often negatively impacts fundamental properties of the Env, including neutralization profiles.

Short Communication: Ultrasensitive Immunoassay for Assessing Residual Simian-Tropic HIV in Nonhuman Primate Models of AIDS.

Persistence of replication-competent viral reservoirs during infection remains a barrier to HIV cure, despite the ability of combination antiretroviral therapy (cART) to effectively suppress viral replication. Simian-tropic HIV (stHIV) is a minimally chimeric HIV-1 that is comprised of 94% HIV-1 sequence, contains HIV-1 drug and immunologic targets, and is capable of replicating to high levels and causing authentic HIV-like pathogenesis leading to clinical AIDS in pigtail macaques. Suppression of stHIV replication by cART provides a model for study of viral reservoirs and HIV-specific intervention strategies targeting them.

Ultrasensitive Immunoassay for Simian Immunodeficiency Virus p27.

Although effective for suppressing viral replication, combination antiretroviral treatment (cART) does not represent definitive therapy for HIV infection due to persistence of replication-competent viral reservoirs. The advent of effective cART regimens for simian immunodeficiency virus (SIV)-infected nonhuman primates (NHP) has enabled the development of relevant models for studying viral reservoirs and intervention strategies targeting them. Viral reservoir measurements are crucial for such studies but are problematic.

A single gp120 residue can affect HIV-1 tropism in macaques.

Species-dependent variation in proteins that aid or limit virus replication determines the ability of lentiviruses to jump between host species. Identifying and overcoming these differences facilitates the development of animal models for HIV-1, including models based on chimeric SIVs that express HIV-1 envelope (Env) glycoproteins, (SHIVs) and simian-tropic HIV-1 (stHIV) strains. Here, we demonstrate that the inherently poor ability of most HIV-1 Env proteins to use macaque CD4 as a receptor is improved during adaptation by virus passage in macaques.

Derivation and Characterization of a CD4-Independent, Non-CD4-Tropic Simian Immunodeficiency Virus.

Unlabelled: CD4 tropism is conserved among all primate lentiviruses and likely contributes to viral pathogenesis by targeting cells that are critical for adaptive antiviral immune responses. Although CD4-independent variants of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have been described that can utilize the coreceptor CCR5 or CXCR4 in the absence of CD4, these viruses typically retain their CD4 binding sites and still can interact with CD4. We describe the derivation of a novel CD4-independent variant of pathogenic SIVmac239, termed iMac239, that was used to derive an infectious R5-tropic SIV lacking a CD4 binding site.

Quantification of entry phenotypes of macrophage-tropic HIV-1 across a wide range of CD4 densities.

Defining a macrophage-tropic phenotype for HIV-1 to assess a role in pathogenesis is complicated by the fact that HIV-1 isolates vary continuously in their ability to enter monocyte-derived macrophages (MDMs) in vitro, and MDMs vary in their ability to support HIV-1 entry. To overcome these limitations, we identified consistent differences in entry phenotypes between five paired blood-derived, T cell-tropic HIV-1 env genes, four of which are CCR5-using (R5) and one of which is CXCR4-using (X4), and cerebrospinal fluid (CSF)-derived, R5 macrophage-tropic env genes. We performed entry assays using the CD4- and CCR5-inducible Affinofile cell line, expressing a range of CD4 levels that approximates the range from MDMs to CD4(+) T cells.

Circadian regulation of ATP release in astrocytes.

Circadian clocks sustain daily oscillations in gene expression, physiology, and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia.