HIV Research: iPSC Models

• Cellular manipulation methods enable in vitro disease modeling for high-throughput screening under physiologically relevant conditions with a species-accurate genetic background
• Advancements in induced pluripotent stem cell (iPSC) technology allow for the generation of infinite amounts of multiple cell types from a single genetic source without repeated biopsies
• iPSC tech is leveraged to uncover mechanisms of pathogenesis, target latent reservoirs, and screen for optimal drug dosages to limit adverse drug reactions in HIV context
• iPSC models hold promise for geographical regions disproportionately affected by HIV, as Africa's genetic diversity impacts disease progression and treatment
• Researchers aim to fully harness iPSC tech to eradicate HIV

HIV Key Tissues

• Cells of the immune system

• Neurological space

• Liver

• Human Immunodeficiency Virus (HIV) causes Acquired Immunodeficiency Syndrome (AIDS), affecting over 37 million people globally, with no curative treatment or vaccine

• HIV pathogenesis causes depletion of specific immune cell subsets and collapse of the host immune system

• Individuals become highly susceptible to opportunistic infections and other diseases as a result

• HIV/AIDS related mortalities have claimed over 36 million lives since the start of the epidemic, with Africa disproportionately affected

• Antiretroviral treatment (ART) uses combinations of antiretrovirals (ARVs) against at least two distinct targets

• ART aims to restricts active HIV replication but cannot specifically target and eliminate viral reservoirs

• Latently infected quiescent cells and HIV infectable cell populations in poorly accessible anatomical compartments are hard to treat

• A single HIV infected cell can catalyse deregulation of the human immune system in the absence of treatment

• Diagnosis of HIV infection requires life-long treatment

• Broader access to combined antiretroviral therapy (CART) has greatly improved clinical prognosis for HIV/AIDS

• Therapies present socio-economic and health challenges that are particularly relevant in the African context

• Economic implications are evident in resource-challenged regions like sub-Saharan Africa where South Africa alone spent ~$2.5 billion USD on ARVs between 2012 and 2018

• Chronic ARV therapy is linked to adverse drug events which further impact treatment

• Adverse drug events linked to CART can be compounded by the richness of genetic diversity within the African continent

• Diverse inter- and intra-population pharmacokinetics of ARVs, such as nevirapine, advocate for the use of genotyping in clinical dose optimisation

• Improved cellular models which can facilitate the identification of novel treatment strategies, recapitulate HIV pathogenesis, and predict potential adverse drug events still have an important role to play as global research efforts strive towards the eradication of HIV

• Shinya Yamanaka published a method to reprogram differentiated cells back to a pluripotent state in 2006 / 2007 (Nobel Prize in 2012)

• Induced pluripotent stem cell (iPSC) technology has provided opportunities in the realm of creating models of disease-in-a-dish

• Pluripotency provides the potential to guide differentiation into any cell type

iPSC Technology

• Strategic utilization enables investigations through generation of various HIV relevant cell lineages using a singular source of cellular material
• Retains the combinatorial advantage over transformed and primary cell lines by retaining genomic integrity and being an infinite and easily accessible source of cellular material
• Cells are permissive to genome engineering

Immune System

• The human immune response to pathogenic challenge relies on the interplay between innate and adaptive cellular responses mediated by specialised cell types
• Innate immune cells such as neutrophils, macrophages, dendritic cells, and natural killer (NK) cells play a role in the rapid detection, restriction, and clearance of pathogens
• Adaptive immunity relies on antigen presenting cells and T lymphocytes to clear infections and establish long term, pathogen-specific immunological memory
• Cluster of differentiation 4 positive (CD4+) T lymphocyte populations are the most well characterised target cells of HIV
• Rapid depletion in response to both productive and non-productive HIV infection is the predominant driver of immunodeficiency and progression to AIDS
• Macrophages represent an equally compelling target for HIV research
• Macrophages are among the first cell types infected following exposure to HIV and are critical effectors of contact-mediated inter-cellular HIV transmission and various other modes of viral pathogenesis
• Macrophages are able to sustain in vivo viral replication independently of T lymphocytes and are critical effectors of contact-mediated inter-cellular HIV transmission and various other modes of viral pathogenesis
• Studies that have used immortalised cell lines to identify therapeutic targets have demonstrated poor concordance when compared to primary cell types and in vivo models
• Atypical cellular physiology inherent to oncogenic lines
• Gene-editable and readily-scalable iPSCs represent a tool for the evaluation of specific functional elements on HIV replication dynamics in a macrophage model that offers improved physiological relevance over immortalised lines

Myeloid Cell Types

• Derivation can support HIV replication, including macrophages, from iPSCs have been well established and functionally characterised
• Macrophage models closely recapitulate the physiology of primary macrophages through their transcriptomic profiles; inflammatory responses; polarisation potential and response to microbial challenge including that of HIV
• Higaki and colleagues (2018) demonstrated the utility of iPSC-derived macrophages for modelling transcriptional gene silencing-based HIV therapeutic approaches by modifying iPSCs
• Macrophage populations expressing shRNA expression confers an HIV-protective phenotype through increased methylation of the HIV promoter
• HIV susceptibility may be impacted by the choice of macrophage differentiation strategy
• The expression of infectious virions per cell in macrophage models with different cell culture media compositions may influence energy utilisation and subsequent viral replication dynamics
• HIV-refractive macrophages have been engineered through lentiviral transduction of iPSC lines to express an shRNA targeting HIV dependency factor CCR5 along with a chimeric version of the HIV restriction factor Trim5-a
• iPSC-derived macrophages represent a resource for large-scale experimental evaluations like functional genomics and drug screens
• Application is evidenced by the recent utilization of iPSC-derived macrophages in a genome-wide CRISPR-Cas9 based screen to resolve the functional pathways mediating inflammatory responses in macrophages
• Combining outcomes from orthogonal functional screening approaches specifically targeted to uncover HIV-host interactions in iPSC-derived macrophages may offer insight towards the delineation of HIV-host dependencies and more specifically, the characterisation of novel HIV-host interactions
• CRISPR edited iPSC-derived macrophages with a knockout of the IFN-stimulated gene, ubiquitin-specific proteinase 18 (USP18), revealed deficient HIV-1 replication
• Successful use of iPSC-derived macrophages in modelling Mycobacterium tuberculosis (Mtb) infection provides an ideal and sustainable platform for the interrogation of HIV/Mtb co-infection dynamics
• Advancements in the tissue specification of macrophage models and new differentiation strategies present an opportunity for the targeted evaluation of cell populations that have been proposed to play a role in HIV pathogenesis but are not readily accessible
• Rare ARV-naive subsets of the human population are able to naturally restrict HIV
• Several genetic and functional phenotypes have been linked to viremic controllers ranging from specific human leukocyte antigen (HLA) variants and improved cytotoxic immune responses to functional mutations in host factors required for viral entry

Polymorphisms

• Various chemokine coreceptors (CCR5, CCR2, CXCR4) protect against HIV
• The most well characterised is a 32 base deletion in the gene encoding the chemokine coreceptor CCR5 (CCR5 Δ32)
• Most common in individuals of eastern European descent and results in a non-functional protein product
• CCR5 Δ32 homozygosity confers a high degree of immunity against macrophage-tropic viral variants which also dominate transmission events
• Protective homozygous phenotype has been further shown to be transferable via stem cell-mediated transplantation resulting in the long-term control of HIV within a previously infected and naturally progressing individual
• The prevalence of viremic controllers in the Democratic Republic of Congo is potentially 4-fold greater than the global average
• Viremic controllers show great promise for the development of curative treatments
• It also necessitates the strategic utilisation of scientific methods to capitalize on these valuable and rare resources
• Derivation of patient-specific iPSCs from individuals harbouring protective phenotypes against HIV have been demonstrated
• This approach could therefore be readily applied to establish African-relevant, patient-derived, iPSC repositories that are representative of phenotypes of interest
• Sub-Saharan Africa retains unique genetic diversity
• Since iPSC technology produces a clonal cell line which retains the full genetic background it enables improved stratification of patients into specific drug response groups
• Can be used to develop a regional precision medicine drug development pipeline without the crippling cost of extensive clinical trials
• Extensive prior art has demonstrated that iPSC-derived macrophages can recapitulate HIV infection, as well as be generated in high-throughput
• Africanised drug screening on cohorts of specific groups would be technically feasible

Engineering and Genome Editing

• In the absence of patient derived cellular material, the combination of iPSCs and genome engineering would provide a complementary approach to functionally validate the putative contribution of individual genetic perturbations to viremic control phenotypes
• Conferring resistance against HIV in iPSC-derived macrophages via both gene knockout of CCR5 and the introduction of the CCR5 Δ32 genotype into a wild-type iPSC line
• Adoptive cell transfer of murine iPSC-derived, viral antigen-specific cytotoxic T lymphocytes resulted in the suppression of HIV replication in peritoneal macrophages
• Enrichment of HIV-targeted memory T cell populations
• Co-culture of iPSC-derived NK cells with an HIV infected reporter T cell line resulted in a 90% restriction of HIV replication
• HIV exposed primary CD4+ T cells revealed enhanced contact-mediated cytotoxicity, enhanced cytokine production and antibody-dependent cellular toxicity as potential mechanisms for NK-mediated restriction of HIV
• Co-culture models that better mimic in vivo conditions represent another important tool for the improved modelling of HIV replication and the preclinical validation of putative therapeutic strategies
• IPSCs can provide a universal cell source for the derivation of multiple HIV-relevant immune cell types
• Various combinations of co-culture models would provide a cell-based system to unravel complex cellular interactions
• The use of such an approach would be incredibly powerful specifically in the characterisation of viremic control as it relates to intercellular interactions against the backdrop of a specific genetic perturbation
• Scalable and efficient production of mature iPSC-derived CD4+ T lymphocytes has historically proven to be more challenging
• Recent advances towards more robust differentiation methods will likely invigorate their utilisation in future research efforts
• In addition, iPSC-derived immune cells also hold potential in terms of their capacity to support adoptive cell immunotherapies
• Modifications such as the introduction of pathogen-specific chimeric antigen receptors (CARs) or the expression of viral restriction factors can be readily implemented at the iPSC stage

Neurological Space:

• HIV infection and the clinical manifestation of AIDS was initially considered primarily due to the susceptibility of the cells of the immune system
• Early infection of the brain via the blood brain barrier creates reservoirs of HIV leading to latency.
• HIV neuropathy in aging populations after decades of ARV treatment are prevalent, and further exacerbated by substance abuse
• The increasing appreciation for the neurological space within the context of HIV infection and disease progression necessitates advanced cellular models of the brain

Advanced Cellular Models

• iPSC derived microglia
• iPSC derived 3D cerebral organoids
• Co-culture models of the two combined with endothelial cells to mimic the blood brain barrier
• Microglia are a key element of HIV research
• Significant evidence from post-mortem studies has indicated their reservoir status and activation promote neuronal damage leading to HIV-induced neuropathology
• IPSC-derived microglia have been generated by several groups
• Simplified method generates iPSC-MG that cluster closely with human foetal and adult microglia with respect to gene expression
• In comparison to alternative in vitro microglial models, and in the context of HIV infection, these more closely mimic characteristics of primary human microglia
• This includes the expression of HIV-1 restriction factors
• Understanding of HIV-induced microglial activation in the CNS, revealing that this activation is driven by the cytoplasmic export of the HIV intron-containing RNA
• Co-culture microglial and neuronal iPSC-derived models have been shown to mimic the cross talk between neurons and microglia in the modulation of HIV expression.
• Such established systems will go a long way towards providing high-throughput drug screens which would seek to alter the cyclical neuronal damage induced activation of HIV infected microglia and the subsequent neurotoxicity which leads to HAND
• Systems include iPSC-derived neurons and astrocytes
• Each separately generated and combined with infected, uninfected, or infected and ART treated iPSC-MG provide highly modular opportunities for assessing cell-type specific effects of infection and treatment on different cells of the neurological space
• Advantages of in vitro systems mimic the 3D space
• This takes into account cell to cell contact and signalling, chromatin organisation, and the extra-cellular matrix
• Apprecation for mechano-transduction
• conversion of mechanical stimuli to biological signalling
• Cerebral organoid can be used to study many of neurological aspects of HIV neuropathology
• Validated such a model in which infected microglia
• Increased levels of TNFa and Il1β after incorporation into a 3D cerebral organoid

Trade Offs

• Tradeoff of increased complexity in multi-cellular organoids confers a lack of uniformity, which is necessary in reproducible drug screens
• No one model is necessarily better
• The research question remains paramount and dictates the 'best' model
• Continue to add new drugs, with the aim to lower costs and reduce ADRs
• Long-term CART improves clinical prognosis, ADRs can be associated with intrinsic drug toxicity
• ADR is associated with genetic polymorphisms: may also impact circulating drug concentrations and reactive metabolites

ADR Reporting

• Reporting of ADRs in pharmacovigilance databases show a small reporting contribution from African members in ARVs
• ARV-associated ADRs are not exclusive to a specific organ system or pathway
• The liver is a common cause of grade four ADRs
• CART regimens result in drug-induced toxicity in 9-30% of patients,
• Drug toxicity necessitates monitoring of liver enzymes
• Co-infections, steatohepatitis, and other metabolic disorders modulate the liver
• Genotyping is clinically relevant for the pharmacokinetics of the ARV, efavirenz (EFV)
• Efavirenz: strong predictor of systemic exposure and subsequently the potential for serious ADRs
• Remians the alternative first-line regimen
• The cornerstone of first-line regimens
• remains a concerning example of both intrinsic liver injury and pharmacogenetics impacting efficacy and toxicity in Africa.
• Cellular models, such as immortalised HepG2 cells, have been used to investigate retrospectively
• Numerous in vitro strategies exist to evaluate the risk of liver injury , and these are continuing to evolve
• ARVs are not represented in all drug-induced toxicity screensHistorically, uncovering the burden of ARV-induced ARV has mostly been evidenced by clinical outcomes
• Co-culture models, primary and/or immortalised cells have been used to investigate the progression of hepatic fibrogenesis in HIV-HCV co-infections
• Can provide population-specific context for safety and toxicity profiles, and explore complex aspects of injury in patients with HIV

Hepatocyte Like Cells

• Can be successfully differentiated -Becoming increasingly competent in their mimicry of primary human hepatocytes
• Translational relevance of iPSC-HLCs is further being functionally improved by scalable 3D cell culture technologies
• Improves human physiological processes, these platforms will become increasingly valuable in their predictivity, mechanistic relevance, and translatability
• High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids
• Differentiated to hepatocyte- and Kupffer-like cell fates.
• Could be utilised singularly or as a component of more complex models
• Addresses aspects of drug toxicity, liver injury, and the functional consequences of African relevant genetic variants for xenobiotic metabolism in HIV

Future

• Outlined several highlights of HIV relevant models
• Noted to recognise that a number of non-IPSC models have been used with great effect
• Non-human primates and humanised mice
• Tissue explants and both primary and immortalised cell lines across various lineages
• Potential cell populations are produced across various differentiation protocols
• Careful consideration of differentiation approaches