Background: HIV-associated neurocognitive impairments (NCIs) affect ~50% of individuals with HIV, leading to cognitive, motor, and behavioral declines primarily driven by brain inflammation. Activation of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor involved in regulating lipid and glucose metabolism, has demonstrated anti-inflammatory effects in neurodegenerative diseases. However, its efficacy in addressing HIV-associated neurological complications, as well as potential sex differences in its effects, remains unclear. This study hypothesized that the selective, novel, PPARγ agonist, INT131, could mitigate HIV-induced brain inflammation and neurocognitive deficits in male and female mice using the ecotropic HIV-1 (EcoHIV) mouse model.

Methods: Male and female mice were IP-injected with saline or EcoHIV (4×10⁶ pg/ml of p24) and evaluated 6 weeks post-infection. Experimental groups included: (i) non-infected mice, (ii) non-infected mice treated with INT131 (20 mg/kg/day administered IP via osmotic pumps for 28 days), (iii) EcoHIV-infected mice, and (iv) EcoHIV-infected mice treated with INT131. Molecular analyses were conducted to quantify viral genes, inflammatory cytokines/chemokines, oxidative stress markers, and blood-brain barrier (BBB) tight junction proteins using qPCR and IHC in brain and spleen tissues. Behavioral assessments performed during INT131 treatment evaluated locomotion, learning, memory, and anxiety.

Results: In both male and female mouse brain and spleen tissues, INT131 treatment significantly reduced the expression of viral genes (vif, tat), inflammatory cytokines/chemokines (Tnf-α, Il-1β, Ifn-γ, and Ccl2), oxidative stress marker (Nos2) as well as expression of the viral p24 protein. Furthermore, INT131 restored the expression of BBB tight junction proteins (Cldn5, Ocln, and Tjp1). Behavioral deficits, including impairments in locomotion, learning, memory, and anxiety, were reversed following INT131 treatment in both sexes.

Conclusion: These findings suggest that PPARγ could serve as a novel molecular target for the treatment of HIV-associated brain inflammation, BBB dysfunction, and NCIs. (Supported by the Canadian Institutes of Health Research.)

Emily Y. Ying, Mitchell J. Mumby, Jimmy D. Dikeakos

Department of Microbiology & Immunology, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada

The Human Immunodeficiency Virus Type 1 (HIV-1) accessory protein Nef downregulates cell surface molecules such as CD4, MHC-I, CD28, and the restriction factor Serine Incorporator 5 (SERINC5) to promote HIV-1 pathogenesis and infectivity in vivo. Although these receptor downregulation activities mediated by Nef contribute to disease progression from acute HIV-1 infection to Acquired Immunodeficiency Syndrome (AIDS), it remains unclear how differential surface molecule downregulations by Nef influence HIV-1 virulence.

A recently identified hypervirulent subtype B (VB) strain of HIV-1 circulating in the Netherlands accelerates CD4+ T cell loss and progression to AIDS compared to other non-VB subtype B HIV-1 strains. Given the critical role of Nef in HIV-1 pathogenesis, we hypothesized that the VB Nef protein downregulates specific receptors, such as SERINC5, more efficiently than non-VB Nef proteins.

To address this, we compared the abilities of VB Nef and non-VB Nef to downregulate CD4 and SERINC5. In CD4+ HeLa cells coexpressing Nef and SERINC5, flow cytometry revealed that both VB and non-VB Nef proteins downregulate CD4 to similar extents. However, a polymorphic VB Variant harboring F103 mutation displayed enhanced SERINC5 downregulation. This finding is further supported by infection of Sup-T1 cells with pseudotyped viral vectors, where CD4 downregulation remained comparable across strains. Luciferase assay in infected TZM-bl cells also confirmed this enhanced SERINC5 downregulation by the F103 VB variant.
Our findings highlight the effects of specific polymorphisms on the ability of Nef to downregulate surface CD4 and SERINC5. Future work characterizing additional Nef-mediated receptor downregulations capabilities will provide further insights into the hypervirulent strain.

The Human Immunodeficiency Virus (HIV) compromises the immune system by depleting mainly CD4+ T cells, which can be categorized into different subsets based on their differentiation states and functions. HIV disrupts the intricate balance between these subsets, which is suggested to cause people living with HIV (PLWH) heightened susceptibility to opportunistic infections by pathogens such as Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) and leading cause of death among PLWH. Little is known about the adaptive immune responses during co-infection in specifically the lungs. Using humanized mice (hu-mice), we aim to address this research gap by characterizing the effects of HIV-1 on the different lung CD4+ T cell subsets and investigating how these effects may correlate with progression of TB. In the lungs of HIV-infected hu-mice, we observed significant depletion of CD4+ effector (TEFF) and effector memory (TEM) cells, whereas the relative proportion of naïve CD4+ T cells shifted towards increase. On the other hand, in Mtb-infected hu-mice, there was a significant increase and a trend towards increase in the lung CD4+ TEFF and TEM cells respectively, likely indicative of the activation and proliferation of these subsets in response to infection. At 2- and 4-weeks post-Mtb-infection, we also observed gradual increase in the proportion of CCR5-expressing CD4+ T cells, which have been reported to help recruit immune cells to the lungs for defense. In contrast, the CCR5-expressing CD4+ T cells were significantly depleted in the lungs of HIV-infected mice. These data show certain subsets that may be more involved in the defense against TB are preferentially depleted by HIV, providing a potential mechanism for how the backdrop of HIV infection promotes TB. In upcoming HIV/Mtb co-infection studies, we will examine how HIV’s effects on these subsets may associate with TB disease severity, providing further insight into their interactions.

Antiretroviral therapy (ART) successfully reduces mortality rates in people living with HIV (PLWH) by suppressing Human Immunodeficiency Virus-1 (HIV) replication to undetectable levels. However, ART is not curative due to the establishment of the HIV reservoir that continues to persist despite prolonged ART suppression. It has been established that long-lived memory T cells represent the most important HIV reservoir population which is predominantly composed of CD4+ T cell clones that are established early after infection and increase under ART treatment. While factors such as integration site effects have been proposed to induce clonal expansion of HIV-infected T cells, our data points to cognate dendritic cell (DC):T cell interactions as the main driver of clonal expansion of latent T cell subsets through T cell receptor (TCR)-dependent signaling and cytokines that support a pro-survival state in these cells. Depending on the nature of the TCR stimulus, latent T cell proliferation can occur in the absence of virus reactivation. However, a remaining gap in knowledge is how antigen stimulation regulates opposing biological processes of (i) proliferation leading to clonal expansion or (ii) viral production leading to cell death. Here, we utilized a dual-fluorescent HIV latency reporter and antigen-specific human CD4+ T cell clones to modulate TCR signaling strengths using a panel of altered peptide ligands and directly examined the relationship between TCR signals and proliferative responses by latent T cells. Our preliminary data suggests that low avidity antigen stimulation drives proliferation without viral reactivation in latent T cell subsets while high avidity stimulation promotes virus reactivation and cell death. Our data argue that a critical balance between stimulatory and inhibitory pathways dictate which T cell subsets clonally expand under ART suppression. These studies have implications on stimulatory signals that can be therapeutically targeted to reduce the HIV reservoir size in PLWH.

Human immunodeficiency virus type I (HIV-1) arose following cross-species transmission of simian immunodeficiency virus (SIV) infecting chimpanzees (SIVcpz). While SIV infections of its natural primate hosts are typically non-pathogenic, SIVcpz and HIV-1 infection is highly pathogenic in chimpanzees and humans, respectively. SIV/HIV pathogenesis is strongly influenced by the accessory protein, Nef. Indeed, Nef isolates from non-pathogenic SIV strains robustly downregulate the T cell receptor component CD3ζ, which leads to lower levels of T cell activation. Conversely, Nef isolates derived from pathogenic infections fail to downregulate CD3ζ, leading to increased chronic immune activation. We hypothesize that the increased pathogenicity of SIVcpz/HIV-1 resulted from Nef losing its ability to downregulate CD3ζ, thereby leading to increased chronic T cell activation. Nef sequences generated from a reconstructed time-scaled phylogeny were used to measure CD3ζ downregulation and investigate how this Nef function changed over time. We generated human CD8α-CD3ζ fusion constructs encoding the CD3ζ cytoplasmic tail from diverse primate hosts, using the detection of cell surface huCD8α as a measure of CD3ζ downregulation by the reconstructed ancestral Nef proteins. Within the SIV/HIV lineage, only the reconstructed ancestor of all primate lentiviruses robustly downregulated CD3ζ, while Nefs derived from the intermediate nodes in the SIVcpz/HIV-1 lineage lost this function altogether. This demonstrates that Nefs derived from the SIVcpz/HIV-1 lineage cannot downregulate CD3ζ, which could therefore contribute to the elevated levels of chronic T cell activation consistent with pathogenic infections. Moreover, we observed Nef-mediated CD3ζ downregulation was largely species-independent as the ancestor of all primate lentiviruses downregulated CD3ζ from various primate species, however the exact magnitude may vary due to virus-host co-evolution. These results support the use of ancestral sequence reconstruction to characterize the deep evolutionary history of HIV-1. Our findings highlight the importance of Nef modulation of T cell activation and its contribution to pathogenicity.

Background: Children who are born HIV-exposed but uninfected (HEU) are the fastest growing HIV-affected population, numbering at over 16 million children globally. There is a large gap in our understanding of how in utero exposure to HIV and antiretrovirals influence the development and long-term health of these children. A mouse HIV pregnancy model would greatly facilitate such research.
EcoHIV is an HIV virus that has been modified to express the envelope protein from the murine leukemia virus to allow infection in mouse immune cells. Here we present a new EcoHIV infection model in a mouse pregnancy.

Methods: 7-week-old C57BL/6 mice were infected with 2.5x106 pg EcoHIV virus or mock infected and were mated 1 week post infection. Pregnant dams were euthanized on gestational day (GD) 14.5 or 18.5 (N=5/group). Tissue and blood were collected. HIV infection was detected through HIV gag expression by qPCR. mRNA expression of neuroinflammatory markers were assessed in fetal brains (N=24/group). Mann-Whitney test was used for statistical comparisons.

Results: Injection with EcoHIV resulted in 100% maternal infection, confirmed by HIV gag detection in spleen tissue of all dams. Infection was detected in 4.7% of EcoHIV exposed fetuses. EcoHIV infection was associated with significantly lower fetal weights, lower placenta efficiency, and lower viability rates. mRNA levels of the inflammatory marker TNFa were increased, and mRNA levels of synapsin 1 and synaptophysin, both markers of neuronal health, were decreased in brains of EcoHIV exposed fetuses compared to controls.

Conclusions: The EcoHIV pregnancy model represents the first mouse pregnancy model that includes HIV. In utero EcoHIV exposure was associated with fetal growth restriction and placental insufficiency even in the absence of fetal infection, paralleling clinical findings. Neuroinflammation and neuronal toxicity in EcoHIV exposed uninfected fetuses, may represent a mechanism contributing to neurodevelopmental deficits seen in children who are HEU.