HIV-1 controllers exhibit an enhanced antiretroviral innate state characterised by overexpression of p21 and MCPIP1 and silencing of ERVK-6 RNA expression.

BACKGROUND: Human immunodeficiency virus (HIV)-1 infection can activate the expression of human endogenous retroviruses (HERVs), particularly HERV-K (HML-2). HIV controllers (HICs) are rare people living with HIV (PLWHs) who naturally control HIV-1 replication and overexpress some cellular restriction factors that negatively regulate the LTR-driven transcription of HIV-1 proviruses. OBJECTIVES: To understand the ability of HICs to control the expression of endogenous retroviruses. METHODS: We measured endogenous retrovirus type K6 (ERVK-6) RNA expression in peripheral blood mononuclear cells (PBMCs) of HICs (n = 23), antiretroviral (ART)-suppressed subjects (n = 8), and HIV-1-negative (NEG) individuals (n = 10) and correlated the transcript expression of ERVK-6 with multiple HIV-1 cellular restriction factors. FINDINGS: Our study revealed that ERVK-6 RNA expression in PBMCs from HICs was significantly downregulated compared with that in both the ART and NEG control groups. Moreover, we detected that ERVK-6 RNA levels in PBMCs across all groups were negatively correlated with the expression levels of p21 and MCPIP1, two cellular restriction factors that limit the activation of macrophages and T cells by downregulating the activity of NF-kB. MAIN CONCLUSIONS: These findings support the hypothesis that HICs activate innate antiviral mechanisms that may simultaneously downregulate the transcription of both exogenous (HIV-1) and endogenous (ERVK-6) retroviruses. Future studies with larger cohorts should be performed to confirm this hypothesis and to explore the role of p21 and MCPIP1 in regulating HERV-K expression in physiological and pathological conditions.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy.

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.

A Long-Running Arms Race between APOBEC1 Genes and Retroviruses in Tetrapods.

Activation-induced cytidine deaminase/apolipoprotein B mRNA editing catalytic polypeptide-like (AID/APOBEC) proteins are cytosine deaminases implicated in diverse biological functions. APOBEC1 (A1) proteins have long been thought to regulate lipid metabolism, whereas the evolutionary significance of A1 proteins in antiviral defense remains largely obscure. Endogenous retroviruses (ERVs) document past retroviral infections and are ubiquitous within the vertebrate genomes. Here, we identify the A1 gene repertoire, characterize the A1-mediated mutation footprints in ERVs, and interrogate the evolutionary arms race between A1 genes and ERVs across vertebrate species. We find that A1 genes are widely present in tetrapods, recurrently amplified and lost in certain lineages, suggesting that A1 genes might have originated during the early evolution of tetrapods. A1-mediated mutation footprints can be detected in ERVs across tetrapods. Moreover, A1 genes appear to have experienced episodic positive selection in many tetrapod lineages. Taken together, we propose that a long-running arms race between A1 genes and retroviruses might have persisted throughout the evolutionary course of tetrapods. IMPORTANCE APOBEC3 (A3) genes have been thought to function in defense against retroviruses, whereas the evolutionary significance of A1 proteins in antiviral defense remains largely obscure. In this study, we identify the A1 gene repertoire, characterize the A1-mediated mutation footprints in endogenous retroviruses (ERVs), and explore the evolutionary arms race between A1 genes and ERVs across vertebrate species. We found A1 proteins originated during the early evolution of tetrapods, and detected the footprints of A1-induced hypermutations in retroviral fossils. A1 genes appear to have experienced pervasive positive selection in tetrapods. Our study indicates a long-running arms race between A1 genes and retroviruses taking place throughout the evolutionary course of tetrapods.

Endogenous Retroviruses Augment Amphibian (Xenopus laevis) Tadpole Antiviral Protection.

The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.

A Systematic Review of Expression and Immunogenicity of Human Endogenous Retroviral Proteins in Cancer and Discussion of Therapeutic Approaches.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections that have become fixed in the human genome. While HERV genes are typically silenced in healthy somatic cells, there are numerous reports of HERV transcription and translation across a wide spectrum of cancers, while T and B cell responses against HERV proteins have been detected in cancer patients. This review systematically categorizes the published evidence on the expression of and adaptive immune response against specific HERVs in distinct cancer types. A systematic literature search was performed using Medical Search Headings (MeSH) in the PubMed/Medline database. Papers were included if they described the translational activity of HERVs. We present multiple tables that pair the protein expression of specific HERVs and cancer types with information on the quality of the evidence. We find that HERV-K is the most investigated HERV. HERV-W (syncytin-1) is the second-most investigated, while other HERVs have received less attention. From a therapeutic perspective, HERV-K and HERV-E are the only HERVs with experimental demonstration of effective targeted therapies, but unspecific approaches using antiviral and demethylating agents in combination with chemo- and immunotherapies have also been investigated.

Antiviral Responses in Cancer: Boosting Antitumor Immunity Through Activation of Interferon Pathway in the Tumor Microenvironment.

In recent years, it became apparent that cancers either associated with viral infections or aberrantly expressing endogenous retroviral elements (EREs) are more immunogenic, exhibiting an intense intra-tumor immune cell infiltration characterized by a robust cytolytic apparatus. On the other hand, epigenetic regulation of EREs is crucial to maintain steady-state conditions and cell homeostasis. In line with this, epigenetic disruptions within steady-state cells can lead to cancer development and trigger the release of EREs into the cytoplasmic compartment. As such, detection of viral molecules by intracellular innate immune sensors leads to the production of type I and type III interferons that act to induce an antiviral state, thus restraining viral replication. This knowledge has recently gained momentum due to the possibility of triggering intratumoral activation of interferon responses, which could be used as an adjuvant to elicit strong anti-tumor immune responses that ultimately lead to a cascade of cytokine production. Accordingly, several therapeutic approaches are currently being tested using this rationale to improve responses to cancer immunotherapies. In this review, we discuss the immune mechanisms operating in viral infections, show evidence that exogenous viruses and endogenous retroviruses in cancer may enhance tumor immunogenicity, dissect the epigenetic control of EREs, and point to interferon pathway activation in the tumor milieu as a promising molecular predictive marker and immunotherapy target. Finally, we briefly discuss current strategies to modulate these responses within tumor tissues, including the clinical use of innate immune receptor agonists and DNA demethylating agents.

Systems Biology to Understand and Regulate Human Retroviral Proinflammatory Response.

The majority of human genome are non-coding genes. Recent research have revealed that about half of these genome sequences make up of transposable elements (TEs). A branch of these belong to the endogenous retroviruses (ERVs), which are germline viral infection that occurred over millions of years ago. They are generally harmless as evolutionary mutations have made them unable to produce viral agents and are mostly epigenetically silenced. Nevertheless, ERVs are able to express by still unknown mechanisms and recent evidences have shown links between ERVs and major proinflammatory diseases and cancers. The major challenge is to elucidate a detailed mechanistic understanding between them, so that novel therapeutic approaches can be explored. Here, we provide a brief overview of TEs, human ERVs and their links to microbiome, innate immune response, proinflammatory diseases and cancer. Finally, we recommend the employment of systems biology approaches for future HERV research.

TDP-43 and HERV-K Envelope-Specific Immunogenic Epitopes Are Recognized in ALS Patients.

The human endogenous retrovirus-K (HERV-K) and TAR DNA-binding protein 43 (TDP-43) have been associated with the pathophysiology of amyotrophic lateral sclerosis (ALS). Given these findings, we investigated the humoral response against HERV-K envelope surface (env-su) glycoprotein antigens and TDP-43 in the plasma of ALS patients and healthy controls (HCs). The measured levels of Abs against the different epitopes' fragments were significantly elevated in ALS patients, both in long-survivor (LS) and newly diagnosed (ND) patients, compared to HCs. We observed a positive correlation between HERV-K and TDP-43 antibodies (Abs) levels, which seemed to strengthen with disease progression, that was not found in HCs. The TDP-43 and HERV-K epitopes identified in this study are highly immunogenic and recognized by the humoral response of ALS patients. Increased circulating levels of Abs directed against specific HERV-K- and TDP-43-derived epitopes could serve as possible biomarkers.

Infections and Multiple Sclerosis: From the World to Sardinia, From Sardinia to the World.

Multiple Sclerosis (MS) is an inflammatory disease of the central nervous system. Sardinia, an Italian island, is one of the areas with the highest global prevalence of MS. Genetic factors have been widely explored to explain this greater prevalence among some populations; the genetic makeup of the Sardinians appears to make them more likely to develop autoimmune diseases. A strong association between MS and some infections have been reported globally. The most robust evidence indicating the role of infections is MS development concerns the Epstein-Barr virus (EBV). Anti-EBV antibodies in patients once infected by EBV are associated with the development of MS years later. These features have also been noted in Sardinian patients with MS. Many groups have found an increased expression of the Human endogenous retroviruses (HERV) family in patients with MS. A role in pathogenesis, prognosis, and prediction of treatment response has been proposed for HERV. A European multi-centre study has shown that their presence was variable among populations, ranging from 59% to 100% of patients, with higher HERV expression noted in Sardinian patients with MS. The mycobacterium avium subspecies paratuberculosis (MAP) DNA and antibodies against MAP2694 protein were found to be associated with MS in Sardinian patients. More recently, this association has also been reported in Japanese patients with MS. In this study, we analysed the role of infectious factors in Sardinian patients with MS and compared it with the findings reported in other populations.

Antibodies against human endogenous retrovirus K102 envelope activate neutrophils in systemic lupus erythematosus.

Neutrophil activation and the formation of neutrophil extracellular traps (NETs) are hallmarks of innate immune activation in systemic lupus erythematosus (SLE). Here we report that the expression of an endogenous retrovirus (ERV) locus ERV-K102, encoding an envelope protein, was significantly elevated in SLE patient blood and correlated with autoantibody levels and higher interferon status. Induction of ERV-K102 in SLE negatively correlated with the expression of epigenetic silencing factors. Anti-ERV-K102 IgG levels in SLE plasma correlated with higher interferon stimulated gene expression, and further promoted enhanced neutrophil phagocytosis of ERV-K102 envelope protein through immune complex formation. Finally, phagocytosis of ERV-K102 immune complexes resulted in the formation of NETs consisting of DNA, neutrophil elastase, and citrullinated histone H3. Together, we identified an immunostimulatory ERV-K envelope protein that in an immune complex with SLE IgG is capable of activating neutrophils.

Immunotherapy-induced antibodies to endogenous retroviral envelope glycoprotein confer tumor protection in mice.

Following curative immunotherapy of B16F10 tumors, ~60% of mice develop a strong antibody response against cell-surface tumor antigens. Their antisera confer prophylactic protection against intravenous challenge with B16F10 cells, and also cross-react with syngeneic and allogeneic tumor cell lines MC38, EL.4, 4T1, and CT26. We identified the envelope glycoprotein (env) of a murine endogenous retrovirus (ERV) as the antigen accounting for the majority of this humoral response. A systemically administered anti-env monoclonal antibody cloned from such a response protects against tumor challenge, and prophylactic vaccination against the env protein protects a majority of naive mice from tumor establishment following subcutaneous inoculation with B16F10 cells. These results suggest the potential for effective prophylactic vaccination against analogous HERV-K env expressed in numerous human cancers.

Activation of endogenous retroviruses during brain development causes an inflammatory response.

Endogenous retroviruses (ERVs) make up a large fraction of mammalian genomes and are thought to contribute to human disease, including brain disorders. In the brain, aberrant activation of ERVs is a potential trigger for an inflammatory response, but mechanistic insight into this phenomenon remains lacking. Using CRISPR/Cas9-based gene disruption of the epigenetic co-repressor protein Trim28, we found a dynamic H3K9me3-dependent regulation of ERVs in proliferating neural progenitor cells (NPCs), but not in adult neurons. In vivo deletion of Trim28 in cortical NPCs during mouse brain development resulted in viable offspring expressing high levels of ERVs in excitatory neurons in the adult brain. Neuronal ERV expression was linked to activated microglia and the presence of ERV-derived proteins in aggregate-like structures. This study demonstrates that brain development is a critical period for the silencing of ERVs and provides causal in vivo evidence demonstrating that transcriptional activation of ERV in neurons results in an inflammatory response.

Viral Infections and Systemic Lupus Erythematosus: New Players in an Old Story.

A causal link between viral infections and autoimmunity has been studied for a long time and the role of some viruses in the induction or exacerbation of systemic lupus erythematosus (SLE) in genetically predisposed patients has been proved. The strength of the association between different viral agents and SLE is variable. Epstein-Barr virus (EBV), parvovirus B19 (B19V), and human endogenous retroviruses (HERVs) are involved in SLE pathogenesis, whereas other viruses such as Cytomegalovirus (CMV) probably play a less prominent role. However, the mechanisms of viral-host interactions and the impact of viruses on disease course have yet to be elucidated. In addition to classical mechanisms of viral-triggered autoimmunity, such as molecular mimicry and epitope spreading, there has been a growing appreciation of the role of direct activation of innate response by viral nucleic acids and epigenetic modulation of interferon-related immune response. The latter is especially important for HERVs, which may represent the molecular link between environmental triggers and critical immune genes. Virus-specific proteins modulating interaction with the host immune system have been characterized especially for Epstein-Barr virus and explain immune evasion, persistent infection and self-reactive B-cell "immortalization". Knowledge has also been expanding on key viral proteins of B19-V and CMV and their possible association with specific phenotypes such as antiphospholipid syndrome. This progress may pave the way to new therapeutic perspectives, including the use of known or new antiviral drugs, postviral immune response modulation and innate immunity inhibition. We herein describe the state-of-the-art knowledge on the role of viral infections in SLE, with a focus on their mechanisms of action and potential therapeutic targets.

Endogenous Retroviruses Provide Protection Against Vaginal HSV-2 Disease.

Endogenous retroviruses (ERVs) are genomic sequences that originated from retroviruses and are present in most eukaryotic genomes. Both beneficial and detrimental functions are attributed to ERVs, but whether ERVs contribute to antiviral immunity is not well understood. Here, we used herpes simplex virus type 2 (HSV-2) infection as a model and found that Toll-like receptor 7 (Tlr7-/-) deficient mice that have high systemic levels of infectious ERVs are protected from intravaginal HSV-2 infection and disease, compared to wildtype C57BL/6 mice. We deleted the endogenous ecotropic murine leukemia virus (Emv2) locus on the Tlr7-/- background (Emv2-/-Tlr7-/-) and found that Emv2-/-Tlr7-/- mice lose protection against HSV-2 infection. Intravaginal application of purified ERVs from Tlr7-/- mice prior to HSV-2 infection delays disease in both wildtype and highly susceptible interferon-alpha receptor-deficient (Ifnar1-/-) mice. However, intravaginal ERV treatment did not protect Emv2-/-Tlr7-/- mice from HSV-2 disease, suggesting that the protective mechanism mediated by exogenous ERV treatment may differ from that of constitutively and systemically expressed ERVs in Tlr7-/- mice. We did not observe enhanced type I interferon (IFN-I) signaling in the vaginal tissues from Tlr7-/- mice, and instead found enrichment in genes associated with extracellular matrix organization. Together, our results revealed that constitutive and/or systemic expression of ERVs protect mice against vaginal HSV-2 infection and delay disease.

Opportunities for Antigen Discovery in Metastatic Breast Cancer.

Immune checkpoint inhibitor-based immunotherapy (ICI) of breast cancer is currently efficacious in a fraction of triple negative breast cancers (TNBC) as these cancers generally carry high tumor mutation burden (TMB) and show increased tumor infiltration by CD8+ T cells. However, most estrogen receptor positive breast cancers (ERBC) have low TMB and/or are infiltrated with immunosuppressive regulatory T cells (Tregs) and thus fail to induce a significant anti-tumor immune response. Our understanding of the immune underpinning of the anti-tumor effects of CDK4/6 inhibitor (CDKi) treatment coupled with new knowledge about the mechanisms of tolerance to self-antigens suggests a way forward, specifically via characterizing and exploiting the repertoire of tumor antigens expressed by metastatic ERBC. These treatment-associated tumor antigens (TATA) may include the conventional tumor neoantigens (TNA) encoded by single nucleotide mutations, TNA encoded by tumor specific aberrant RNA transcription, splicing and DNA replication induced frameshift (FS) events as well as the shared tumor antigens. The latter may include the conventional tumor associated antigens (TAA), cancer-testis antigens (CTA) and antigens encoded by the endogenous retroviral (ERV) like sequences and repetitive DNA sequences induced by ET and CDKi treatment. An approach to identifying these antigens is outlined as this will support the development of a multi-antigen-based immunotherapy strategy for improved targeting of metastatic disease with potential for minimal autoimmune toxicity against normal tissues.

Koalas vaccinated against Koala retrovirus respond by producing increased levels of interferon-gamma.

Koala retrovirus (KoRV) is believed to be in an active state of endogenization into the koala genome. KoRV is present as both an endogenous and exogenous infection in all koalas in northern Australia. KoRV has been linked to koala pathologies including neoplasia and increased susceptibility to Chlamydia. A KoRV vaccine recently trialled in 10 northern koalas improved antibody response and reduced viral load. This communication reports the expression of key immune genes underlining the innate and adaptive immune response to vaccination in these northern koalas. The results showed that prior to vaccination, IL-8 was expressed at the highest levels, with at least 200-fold greater expression compared to other cytokines, while CD8 mRNA expression was significantly higher than CD4 mRNA expression level. Interferon-γ was up-regulated at both 4- and 8-weeks post-vaccination while IL-8 was down-regulated at 8-weeks post-vaccination.

The role of endogenous retroviruses-K in human cancer.

It is known that human endogenous retroviruses (HERVs) constitute almost 8% of the human genome. Although the expression of HERVs from the human genome is tightly regulated, different exogenous and endogenous factors could trigger HERV activation. Aberrant expression of different HERVs may potentially cause a variety of diseases such as neurological and autoimmune diseases as well as cancer. It is suggested that HERV-K can induce cancer through different mechanisms that are discussed. The interplay between some tumor viruses and HERV-K seems to be a key player in progression of viral-associated cancers because elevated levels of Rec and Np9 proteins are observed in several cancers. The frequent over expression of HERV proteins and some specific antibodies in cancer cells could be considered as suitable prognostic and therapeutic biomarkers in diagnosis and treatment of cancers. The expression of HERV proteins in cancers and development of immune responses against them may also be used as targets for cancer immunotherapy. Further studies are warranted to evaluate the role of HERVs in cancer formation and use of different HERV proteins in developing new diagnostic and therapeutic approaches for cancer treatments.

Human Endogenous Retrovirus K in Cancer: A Potential Biomarker and Immunotherapeutic Target.

In diseases where epigenetic mechanisms are changed, such as cancer, many genes show altered gene expression and inhibited genes become activated. Human endogenous retrovirus type K (HERV-K) expression is usually inhibited in normal cells from healthy adults. In tumor cells, however, HERV-K mRNA expression has been frequently documented to increase. Importantly, HERV-K-derived proteins can act as tumor-specific antigens, a class of neoantigens, and induce immune responses in different types of cancer. In this review, we describe the function of the HERV-K HML-2 subtype in carcinogenesis as biomarkers, and their potential as targets for cancer immunotherapy.

Cancer Associated Endogenous Retroviruses: Ideal Immune Targets for Adenovirus-Based Immunotherapy.

Cancer is a major challenge in our societies, according to the World Health Organization (WHO) about 1/6 deaths were cancer related in 2018 and it is considered the second leading cause of death globally. Immunotherapies have changed the paradigm of oncologic treatment for several cancers where the field had fallen short in providing competent therapies. Despite the improvement, broadly acting and highly effective therapies capable of eliminating or preventing human cancers with insufficient mutated antigens are still missing. Adenoviral vector-based vaccines are a successful tool in the treatment of various diseases including cancer; however, their success has been limited. In this review we discuss the potential of adenovirus as therapeutic tools and the current developments to use them against cancer. More specifically, we examine how to use them to target endogenous retroviruses (ERVs). ERVs, comprising 8% of the human genome, have been detected in several cancers, while they remain silent in healthy tissues. Their low immunogenicity together with their immunosuppressive capacity aid cancer to escape immunosurveillance. In that regard, virus-like-vaccine (VLV) technology, combining adenoviral vectors and virus-like-particles (VLPs), can be ideal to target ERVs and elicit B-cell responses, as well as CD8+ and CD4+ T-cells responses.

The paradigm of immunosenescence in atherosclerosis-cardiovascular disease (ASCVD).

The new immunosenescence paradigm (2015) was an attempt to explain a mechanism by which macrophages could be immunosuppressed and dysfunctional, yet paradoxically release proinflammatory factors in an unregulated manner. This mechanism was linked to the loss of dehydroepiandrosterone (DHEA) with aging and thus explained how immunosenescence could be causally related to the risk of stress and/or age-associated chronic diseases. At the center of this paradigm was lipid body negative (LB-) foamy macrophage (CD14+CD16+) which produced human endogenous retrovirus K102 (HERV-K102) particles. HERV-K102 may be a protector foamy virus of humans, and its induction may generate trained innate immunity, a special type of autoimmunity, in response to intracellular pathogens, their constituents, toxins, and/or tumors. Overwhelming evidence now suggests that the proinflammatory foamy macrophages driving ASCVD are LB-. Moreover, the monocyte/macrophage phenotype implicated in atherosclerosis-cardiovascular disease (ASCVD) appears to be the CD14+CD16+ intermediate phenotype. These and other observations directly challenge the cholesterol hypothesis. For the prevention and treatment of ASCVD, it is important to address the putative cause of ASCVD -- immunosenescence, rather than the signs or symptoms such as inflammation or elevated cholesterol. Therefore, strategies to reverse or prevent immunosenescence, which improve or maintain an optimal cortisol/DHEA ratio such as isoflavonoids, would be expected to alleviate not only ASCVD but the risk of many other age-associated chronic diseases. Here, the new immunosenescence paradigm will be appraised for its suitability to explain ASCVD risks.