Human immunodeficiency virus (HIV) is a retrovirus isolated in 1983 and is the causative agent of acquired immunodeficiency syndrome (AIDS) (Barré-Sinoussi et al.
1983). Entry of HIV into target cells occurs through a multistep process that involves four different steps: attachment, co-receptor binding, fusion and entry (Wilen et al.
2012). Attachment is mediated by viral glycoprotein gp120 and CD4 cell receptor on the surface of the cells and is followed by co-receptor binding. These co-receptors are specific chemokine receptors: the CC-chemokine receptor 5 (CCR5), used by HIV R5 strains, and CXC-chemokine receptor 4 (CXCR4), used by HIV X4 strains (Berkowitz et al.
1998). Following attachment, the viral envelope protein fuses with the host cell membrane and the viral core containing the HIV RNA, viral proteins and enzymes can finally enter the host cell (Berger et al.
1999). After the initial infection, the virus can cross the blood-brain barrier (BBB), gaining access to the central nervous system (CNS), where it can establish viral reservoirs, eventually leading to neuropathogenesis (Zayyad and Spudich
2015). This can happen as early as 3 to 5 days after initial infection (Koenig et al.
1986; Whitney et al.
2014). The major cellular reservoirs in the brain are microglia and perivascular macrophages. Several studies have provided evidence of susceptibility and productive infection of HIV in these cells (Watkins et al.
1990; Peudenier et al.
1991; Ioannidis et al.
1995; McCarthy et al.
1998; Albright et al.
2000; Joseph et al.
2015), whereas HIV infection in astrocytes remains a controversial topic. Studies have shown HIV infection in astrocytes with expression of early viral proteins, but markers of HIV replication were not detected in these cells, leading to the belief that HIV infection in astrocytes is not productive (Brack-Werner
1999). Astrocytes are the most abundant cells in the CNS and are considered HIV reservoirs, playing an important role in HIV induced neuropathogenesis (Valdebenito et al.
2021; Wahl and Al-Harthi
2023). Neuronal cells are not infected by HIV, but viral proteins and neurotoxicity from glial activation may cause neuronal damage, leading to neuronal dysfunction and cell death (Kovalevich and Langford
2012). Furthermore, viral proteins such as Tat, Vpr and gp120 can contribute to neuronal dysfunction, either directly or indirectly through microglia stimulation resulting in the release of proinflammatory cytokines such as IL-1β and TNF-α (Gelbard et al.
1993; Yeung et al.
1995; Nicolini et al.
2008). Another consequence of HIV invasion in the brain is the induction of an inflammatory response, which may also contribute to the development of HIV-associated neurocognitive disorders (HAND) (Navia et al.
1986; Brown
2015; Hong and Banks
2015; Saylor et al.
2016). HAND includes a range of neurocognitive impairments, with a classification based on severity: asymptomatic neurocognitive impairment (ANI), mild neurocognitive disorder (MND), and HIV associated dementia (HAD). Diagnosis of HAND involves neuropsychological testing and assessments of functional status (Antinori et al.
2007). Severity of HAND has changed since the beginning of the HIV/AIDS epidemic. In the beginning, HAD, the most severe form of HAND, was diagnosed in 20–30% of HIV patients (González-Scarano and Martín-García
2005). With the start of combination antiretroviral therapy (cART), HAD frequency has significantly decreased, although 50% of people with HIV (PWH) present neurological disorders (Clifford and Ances
2013; Saylor et al.
2016). This percentage did not change in the post-cART era, but now the majority of HAND cases are diagnosed as MND or ANI (McArthur et al.
1993; Heaton et al.
2010). The introduction of cART also had an impact on the neuropathology in HAND. In the pre-cART era, neuronal loss and HIV encephalitis were considered to have major roles in HIV neuropathogenesis. However, with the advent of cART, these dysfunctions became less common, and they are no longer considered enough to account for neurological dysfunction. Consequently, the lack of clear neuropathological changes strictly related to HIV infection in patients receiving cART indicates that the underlying mechanism of HAND is more likely related to functional changes in neurons (Gelman
2015; Saylor et al.
2016).
Gaining insight into the neuropathology of HIV is crucial for comprehending the mechanisms underlying cognitive impairment in PWH. Research on HIV neuropathology in humans has primarily relied on the examination and analysis of brain tissues obtained post-mortem. A significant obstacle in investigating HIV neuropathogenesis is the scarcity of suitable in vitro culture models that can recapitulate HAND pathology, considering that multiple types of cells within the CNS may contribute to the pathology. HIV infection cellular models in the CNS usually include cell lines and primary cells. Studies using these 2D culture models have enabled the identification and characterization of cellular processes associated with neuronal toxicity. In particular, viral proteins such as gp120, Tat, Nef and Vpr have shown to be toxic when exposed to neuronal cultures (Brenneman et al.
1988; Adamson et al.
1996; Piller et al.
1998; New et al.
1998; Kaul and Lipton
1999; Kaul et al.
2001; Chen et al.
2005; Mattson et al.
2005; Agrawal et al.
2007; Shah et al.
2013; Sami Saribas et al.
2017; Fields et al.
2017; Dong et al.
2019). To capture the nature of cell-to-cell interactions, it is necessary to employ a primary culture system that combines neurons and glial cells, resembling the composition of cells typically found in the intact brain. Various systems have been developed to investigate the impact of soluble factors released by microglia. Majority of the studies on neuroHIV were performed using immortalized microglial cell lines, macrophages derived from peripheral blood monocytes or primary human microglia that were isolated from tissues (Garcia-Mesa et al.
2017; Rawat and Spector
2017; Rai et al.
2020). Furthermore, the use of animal models, such as nonhuman primates (NHPs) and genetically modified rodent models, has played a significant role in advancing our knowledge of specific aspects related to HIV pathology. While these animal models have provided valuable insights, limitations remain in comprehending CNS infection in humans (Mallard and Williams
2018). Research on HIV neuropathology in humans has been constrained to the collection and examination of brain tissues post-mortem (Wiley et al.
1986; Koenig et al.
1986; Everall et al.
1991; Masliah et al.
2000). Thus, it is important to develop a 3D model of human origins to investigate HIV neuropathology.
Human induced pluripotent stem cells (hiPSCs) allow for easy generation of primary human neural cell types in culture through differentiation. As a result, they have been used to generate complex 3D cell systems, such as brain organoids, which contain multiple cell types. iPSC derived 3D-brain organoids were first generated by Lancaster et al. (Lancaster et al.
2013), which they named “cerebral organoids” (COs). These COs displayed functional neurons and glial cell populations, discrete brain regions, and proper dorsal cortical organization. The establishment of the CO model was a major breakthrough, allowing the human brain to be modeled in vitro with proper organization and cellular connections. The human CO model can be especially helpful in studying human neurotropic viral infections, such as HIV, which has been difficult to study due to limitations of the previously used 2D in vitro and in vivo models. In vitro models used lack a multicellular composition, which is crucial for studying HIV effects as HIV infects microglial cells, which in turn possess neurotoxic effects on neurons that are unable to be infected by HIV (Kovalevich and Langford
2012).
Here, we first developed and characterized hiPSCs from human dermal fibroblasts obtained from healthy individuals. We then developed and characterized a 3D model of human Cerebral Organoids (hCOs) containing the major cell types present in the CNS, including astrocytes, neurons, oligodendrocytes, and microglia. The hCOs are further characterized for the expression of HIV receptor and co-receptors. Finally, we demonstrated the susceptibility of hCOs to HIV infection, in the absence or presence of cART regimens by a series of biochemical, histological, and virological studies. We were able for the first time to show the efficacy of cART treatment in suppressing HIV replication in a human 3D CO model. The findings from our study provide a unique platform to enhance our understanding of the neuropathological aspects of HIV infection in the brain.