Perspectives in Immunotherapy: meeting report from Immunotherapy Bridge (Naples, November 30th-December 1st, 2022).

The discovery and development of novel treatments that harness the patient's immune system and prevent immune escape has dramatically improved outcomes for patients across cancer types. However, not all patients respond to immunotherapy, acquired resistance remains a challenge, and responses are poor in certain tumors which are considered to be immunologically cold. This has led to the need for new immunotherapy-based approaches, including adoptive cell transfer (ACT), therapeutic vaccines, and novel immune checkpoint inhibitors. These new approaches are focused on patients with an inadequate response to current treatments, with emerging evidence of improved responses in various cancers with new immunotherapy agents, often in combinations with existing agents. The use of cell therapies, drivers of immune response, and trends in immunotherapy were the focus of the Immunotherapy Bridge (November 30th-December 1st, 2022), organized by the Fondazione Melanoma Onlus, Naples, Italy, in collaboration with the Society for Immunotherapy of Cancer.

Influence of HIV-1 Genomic RNA on the Formation of Gag Biomolecular Condensates.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4+ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

Influence of HIV-1 genomic RNA on the formation of Gag biomolecular condensates.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) and the HIV-1 pr55 Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yield self-assembling BMCs having HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4 + T cell nuclear lysates led to the formation of larger BMCs as compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggests that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

Scaffolding viral protein NC nucleates phase separation of the HIV-1 biomolecular condensate.

Membraneless biomolecular condensates (BMCs) contribute to the replication of a growing number of viruses but remain to be functionally characterized. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) proteins phase separated into condensates regulating virus assembly. Here we discover that intrinsically disordered human immunodeficiency virus-type 1 (HIV-1) core proteins condense with the viral genomic RNA (vRNA) to assemble as BMCs attaining a geometry characteristic of viral reverse transcription complexes. We explore the predisposition, mechanisms, and pharmacologic sensitivity of HIV-1 core BMCs in living cells. HIV-1 vRNA-interacting NC condensates were found to be scaffolds onto which client capsid, reverse transcriptase, and integrase condensates assemble. HIV-1 core BMCs exhibit fundamental characteristics of BMCs and are drug-sensitive. Lastly, protease-mediated maturation of Gag and Gag-Pol precursor proteins yield abundant and visible BMCs in cells. This study redefines HIV-1 core components as fluid BMCs and advances our understanding of the nature of viral cores during ingress.

Hallmarks of Resistance to Immune-Checkpoint Inhibitors.

Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, biopharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known biological processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.

The double-stranded RNA-binding protein, Staufen1, is an IRES-transacting factor regulating HIV-1 cap-independent translation initiation.

Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5'untranslated region (5'UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.

Society for Immunotherapy of Cancer clinical and biomarkers data sharing resource document: Volume II-practical challenges.

The development of strongly predictive validated biomarkers is essential for the field of immuno-oncology (IO) to advance. The highly complex, multifactorial data sets required to develop these biomarkers necessitate effective, responsible data-sharing efforts in order to maximize the scientific knowledge and utility gained from their collection. While the sharing of clinical- and safety-related trial data has already been streamlined to a large extent, the sharing of biomarker-aimed clinical trial derived data and data sets has been met with a number of hurdles that have impaired the progression of biomarkers from hypothesis to clinical use. These hurdles include technical challenges associated with the infrastructure, technology, workforce, and sustainability required for clinical biomarker data sharing. To provide guidance and assist in the navigation of these challenges, the Society for Immunotherapy of Cancer (SITC) Biomarkers Committee convened to outline the challenges that researchers currently face, both at the conceptual level (Volume I) and at the technical level (Volume II). The committee also suggests possible solutions to these problems in the form of professional standards and harmonized requirements for data sharing, assisting in continued progress toward effective, clinically relevant biomarkers in the IO setting.

Society for Immunotherapy of Cancer clinical and biomarkers data sharing resource document: Volume I-conceptual challenges.

The sharing of clinical trial data and biomarker data sets among the scientific community, whether the data originates from pharmaceutical companies or academic institutions, is of critical importance to enable the development of new and improved cancer immunotherapy modalities. Through data sharing, a better understanding of current therapies in terms of their efficacy, safety and biomarker data profiles can be achieved. However, the sharing of these data sets involves a number of stakeholder groups including patients, researchers, private industry, scientific journals and professional societies. Each of these stakeholder groups has differing interests in the use and sharing of clinical trial and biomarker data, and the conflicts caused by these differing interests represent significant obstacles to effective, widespread sharing of data. Thus, the Society for Immunotherapy of Cancer (SITC) Biomarkers Committee convened to identify the current barriers to biomarker data sharing in immuno-oncology (IO) and to help in establishing professional standards for the responsible sharing of clinical trial data. The conclusions of the committee are described in two position papers: Volume I-conceptual challenges and Volume II-practical challenges, the first of which is presented in this manuscript. Additionally, the committee suggests actions by key stakeholders in the field (including organizations and professional societies) as the best path forward, encouraging the cultural shift needed to ensure responsible data sharing in the IO research setting.

Zinc and Copper Ions Differentially Regulate Prion-Like Phase Separation Dynamics of Pan-Virus Nucleocapsid Biomolecular Condensates.

Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming elucidated. BMCs are now appreciated as required for several steps of gene regulation, while their deregulation promotes pathological aggregates, such as stress granules (SGs) and insoluble irreversible plaques that are hallmarks of neurodegenerative diseases. Treatment of BMC-related diseases will greatly benefit from identification of therapeutics preventing pathological aggregates while sparing BMCs required for cellular functions. Numerous viruses that block SG assembly also utilize or engineer BMCs for their replication. While BMC formation first depends on prion-like disordered protein domains (PrLDs), metal ion-controlled RNA-binding domains (RBDs) also orchestrate their formation. Virus replication and viral genomic RNA (vRNA) packaging dynamics involving nucleocapsid (NC) proteins and their orthologs rely on Zinc (Zn) availability, while virus morphology and infectivity are negatively influenced by excess Copper (Cu). While virus infections modify physiological metal homeostasis towards an increased copper to zinc ratio (Cu/Zn), how and why they do this remains elusive. Following our recent finding that pan-retroviruses employ Zn for NC-mediated LLPS for virus assembly, we present a pan-virus bioinformatics and literature meta-analysis study identifying metal-based mechanisms linking virus-induced BMCs to neurodegenerative disease processes. We discover that conserved degree and placement of PrLDs juxtaposing metal-regulated RBDs are associated with disease-causing prion-like proteins and are common features of viral proteins responsible for virus capsid assembly and structure. Virus infections both modulate gene expression of metalloproteins and interfere with metal homeostasis, representing an additional virus strategy impeding physiological and cellular antiviral responses. Our analyses reveal that metal-coordinated virus NC protein PrLDs initiate LLPS that nucleate pan-virus assembly and contribute to their persistence as cell-free infectious aerosol droplets. Virus aerosol droplets and insoluble neurological disease aggregates should be eliminated by physiological or environmental metals that outcompete PrLD-bound metals. While environmental metals can control virus spreading via aerosol droplets, therapeutic interference with metals or metalloproteins represent additional attractive avenues against pan-virus infection and virus-exacerbated neurological diseases.

Pan-retroviral Nucleocapsid-Mediated Phase Separation Regulates Genomic RNA Positioning and Trafficking.

The duality of liquid-liquid phase separation (LLPS) of cellular components into membraneless organelles defines the nucleation of both normal and disease processes including stress granule (SG) assembly. From mounting evidence of LLPS utility by viruses, we discover that HIV-1 nucleocapsid (NC) protein condenses into zinc-finger (ZnF)-dependent LLPSs that are dynamically influenced by cytosolic factors. ZnF-dependent and Zinc (Zn2+)-chelation-sensitive NC-LLPS are formed in live cells. NC-Zn2+ ejection reverses the HIV-1 blockade on SG assembly, inhibits NC-SG assembly, disrupts NC/Gag-genomic RNA (vRNA) ribonucleoprotein complexes, and causes nuclear sequestration of NC and the vRNA, inhibiting Gag expression and virus release. NC ZnF mutagenesis eliminates the HIV-1 blockade of SG assembly and repositions vRNA to SGs. We find that NC-mediated, Zn2+-coordinated phase separation is conserved among diverse retrovirus subfamilies, illustrating that this exquisitely evolved Zn2+-dependent feature of virus replication represents a critical target for pan-antiretroviral therapies.

A Bifunctional Nucleoside Probe for the Inhibition of the Human Immunodeficiency Virus-Type 1 Reverse Transcriptase.

Nucleoside analogs have proven effective for the inhibition of viral polymerases and are the foundation of many antiviral therapies. In this work, the antiretroviral potential of 6-azauracil analogs was assessed using activity-based protein profiling techniques and functional assays. Probes based on the 6-azauracil scaffold were examined and found to bind to HCV polymerase and HIV-1 reverse transcriptase through covalent modification of residues near the active site. The modified sites on the HIV-1 RT were examined using a mass spectrometry approach, and it was discovered that the azauracil moieties modified the enzyme in proximity to its active site. However, these scaffolds gave little or no inhibition of enzyme activity. Instead, a bifunctional inhibitor was prepared using click chemistry to link the 6-azauracil moiety to azidothymidine (AzT) and the corresponding triphosphate (AzTTP). These bifunctional inhibitors were found to have potent inhibitory function through a mode of action that includes both alkylation and chain termination. An in vitro assay demonstrated that the bifunctional inhibitor was 23-fold more effective in inhibiting HIV-1 RT activity than the parent AzTTP. The bifunctional inhibitor was also tested in HIV-1 permissive T cells where it decreased Gag expression similarly to the front-line drug Efavirenz with no evidence of cytotoxicity. This new bifunctional scaffold represents an interesting tool for inhibiting HIV-1 by covalently anchoring a chain-terminating nucleoside analog in the active site of the reverse transcriptase, preventing its removal and abolishing enzymatic activity, and represents a novel mode of action for inhibiting polymerases including reverse transcriptases.

Viral Infections Exacerbate FUS-ALS Phenotypes in iPSC-Derived Spinal Neurons in a Virus Species-Specific Manner.

Amyotrophic lateral sclerosis (ALS) arises from an interplay of genetic mutations and environmental factors. ssRNA viruses are possible ALS risk factors, but testing their interaction with mutations such as in FUS, which encodes an RNA-binding protein, has been difficult due to the lack of a human disease model. Here, we use isogenic induced pluripotent stem cell (iPSC)-derived spinal neurons (SNs) to investigate the interaction between ssRNA viruses and mutant FUS. We find that rabies virus (RABV) spreads ALS phenotypes, including the formation of stress granules (SGs) with aberrant composition due to increased levels of FUS protein, as well as neurodegeneration and reduced restriction activity by FUS mutations. Consistent with this, iPSC-derived SNs harboring mutant FUS are more sensitive to human immunodeficiency virus (HIV-1) and Zika viruses (ZIKV). We demonstrate that RABV and HIV-1 exacerbate cytoplasmic mislocalization of FUS. Our results demonstrate that viral infections worsen ALS pathology in SNs with genetic risk factors, suggesting a novel role for viruses in modulating patient phenotypes.

Correction to: Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop.

Following publication of the original article [1], the author reported that an author name, Roberta Zappasodi, was missed in the authorship list.

Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop.

Tumor immunology has changed the landscape of cancer treatment. Yet, not all patients benefit as cancer immune responsiveness (CIR) remains a limitation in a considerable proportion of cases. The multifactorial determinants of CIR include the genetic makeup of the patient, the genomic instability central to cancer development, the evolutionary emergence of cancer phenotypes under the influence of immune editing, and external modifiers such as demographics, environment, treatment potency, co-morbidities and cancer-independent alterations including immune homeostasis and polymorphisms in the major and minor histocompatibility molecules, cytokines, and chemokines. Based on the premise that cancer is fundamentally a disorder of the genes arising within a cell biologic process, whose deviations from normality determine the rules of engagement with the host's response, the Society for Immunotherapy of Cancer (SITC) convened a task force of experts from various disciplines including, immunology, oncology, biophysics, structural biology, molecular and cellular biology, genetics, and bioinformatics to address the complexity of CIR from a holistic view. The task force was launched by a workshop held in San Francisco on May 14-15, 2018 aimed at two preeminent goals: 1) to identify the fundamental questions related to CIR and 2) to create an interactive community of experts that could guide scientific and research priorities by forming a logical progression supported by multiple perspectives to uncover mechanisms of CIR. This workshop was a first step toward a second meeting where the focus would be to address the actionability of some of the questions identified by working groups. In this event, five working groups aimed at defining a path to test hypotheses according to their relevance to human cancer and identifying experimental models closest to human biology, which include: 1) Germline-Genetic, 2) Somatic-Genetic and 3) Genomic-Transcriptional contributions to CIR, 4) Determinant(s) of Immunogenic Cell Death that modulate CIR, and 5) Experimental Models that best represent CIR and its conversion to an immune responsive state. This manuscript summarizes the contributions from each group and should be considered as a first milestone in the path toward a more contemporary understanding of CIR. We appreciate that this effort is far from comprehensive and that other relevant aspects related to CIR such as the microbiome, the individual's recombined T cell and B cell receptors, and the metabolic status of cancer and immune cells were not fully included. These and other important factors will be included in future activities of the taskforce. The taskforce will focus on prioritization and specific actionable approach to answer the identified questions and implementing the collaborations in the follow-up workshop, which will be held in Houston on September 4-5, 2019.

Immune-enrichment of non-small cell lung cancer baseline biopsies for multiplex profiling define prognostic immune checkpoint combinations for patient stratification.

BACKGROUND: Permanence of front-line management of lung cancer by immunotherapies requires predictive companion diagnostics identifying immune-checkpoints at baseline, challenged by the size and heterogeneity of biopsy specimens. METHODS: An innovative, tumor heterogeneity reducing, immune-enriched tissue microarray was constructed from baseline biopsies, and multiplex immunofluorescence was used to profile 25 immune-checkpoints and immune-antigens. RESULTS: Multiple immune-checkpoints were ranked, correlated with antigen presenting and cytotoxic effector lymphocyte activity, and were reduced with advancing disease. Immune-checkpoint combinations on TILs were associated with a marked survival advantage. Conserved combinations validated on more than 11,000 lung, breast, gastric and ovarian cancer patients demonstrate the feasibility of pan-cancer companion diagnostics. CONCLUSIONS: In this hypothesis-generating study, deepening our understanding of immune-checkpoint biology, comprehensive protein-protein interaction and pathway mapping revealed that redundant immune-checkpoint interactors associate with positive outcomes, providing new avenues for the deciphering of molecular mechanisms behind effects of immunotherapeutic agents targeting immune-checkpoints analyzed.

Failed immune responses across multiple pathologies share pan-tumor and circulating lymphocytic targets.

Rationale Tumor infiltrating lymphocytes are widely associated with positive outcomes, yet carry key indicators of a systemic failed immune response against unresolved cancer. Cancer immunotherapies can reverse their tolerance phenotypes, while preserving tumor-reactivity and neoantigen-specificity shared with circulating immune cells. Objectives We performed comprehensive transcriptomic analyses to identify gene signatures common to circulating and tumor infiltrating lymphocytes in the context of clear cell renal cell carcinoma. Modulated genes also associated with disease outcome were validated in other cancer types. Findings Using bioinformatics, we identified practical diagnostic markers and actionable targets of the failed immune response. On circulating lymphocytes, three genes, LEF1, FASLG, and MMP9, could efficiently stratify patients from healthy control donors. From their associations with resistance to cancer immunotherapies and microbial infections, we uncovered not only pan-cancer, but pan-pathology failed immune response profiles. A prominent lymphocytic matrix metallopeptidase cell migration pathway, is central to a panoply of diseases and tumor immunogenicity, correlates with multi-cancer recurrence, and identifies a feasible, non-invasive approach to pan-pathology diagnoses. Conclusions The non-invasive differently expressed genes we have identified warrant future investigation towards the development of their potential in precision diagnostics and precision pan-disease immunotherapeutics.

HIV-1 requires Staufen1 to dissociate stress granules and to produce infectious viral particles.

The human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) has two major fates during viral replication: to serve as the template for the major structural and enzymatic proteins, or to be encapsidated and packaged into assembling virions to serve as the genomic vRNA in budding viruses. The dynamic balance between vRNA translation and encapsidation is mediated by numerous host proteins, including Staufen1. During HIV-1 infection, HIV-1 recruits Staufen1 to assemble a distinct ribonucleoprotein complex promoting vRNA encapsidation and viral assembly. Staufen1 also rescues vRNA translation and gene expression during conditions of cellular stress. In this work, we utilized novel Staufen1-/- gene-edited cells to further characterize the contribution of Staufen1 in HIV-1 replication. We observed a marked deficiency in the ability of HIV-1 to dissociate stress granules (SGs) in Staufen1-deficient cells and remarkably, the vRNA repositioned to SGs. These phenotypes were rescued by Staufen1 expression in trans or in cis, but not by a dsRBD-binding mutant, Staufen1F135A. The mistrafficking of the vRNA in these Staufen1-/- cells was also accompanied by a dramatic decrease in viral production and infectivity. This work provides novel insight into the mechanisms by which HIV-1 uses Staufen1 to ensure optimal vRNA translation and trafficking, supporting an integral role for Staufen1 in the HIV-1 life cycle, positioning it as an attractive target for next-generation antiretroviral agents.

Spatially distinct tumor immune microenvironments stratify triple-negative breast cancers.

Understanding the tumor immune microenvironment (TIME) promises to be key for optimal cancer therapy, especially in triple-negative breast cancer (TNBC). Integrating spatial resolution of immune cells with laser capture microdissection gene expression profiles, we defined distinct TIME stratification in TNBC, with implications for current therapies including immune checkpoint blockade. TNBCs with an immunoreactive microenvironment exhibited tumoral infiltration of granzyme B+CD8+ T cells (GzmB+CD8+ T cells), a type 1 IFN signature, and elevated expression of multiple immune inhibitory molecules including indoleamine 2,3-dioxygenase (IDO) and programmed cell death ligand 1 (PD-L1), and resulted in good outcomes. An "immune-cold" microenvironment with an absence of tumoral CD8+ T cells was defined by elevated expression of the immunosuppressive marker B7-H4, signatures of fibrotic stroma, and poor outcomes. A distinct poor-outcome immunomodulatory microenvironment, hitherto poorly characterized, exhibited stromal restriction of CD8+ T cells, stromal expression of PD-L1, and enrichment for signatures of cholesterol biosynthesis. Metasignatures defining these TIME subtypes allowed us to stratify TNBCs, predict outcomes, and identify potential therapeutic targets for TNBC.

T Lymphocytes as Measurable Targets of Protection and Vaccination Against Viral Disorders.

Continuous epidemiological surveillance of existing and emerging viruses and their associated disorders is gaining importance in light of their abilities to cause unpredictable outbreaks as a result of increased travel and vaccination choices by steadily growing and aging populations. Close surveillance of outbreaks and herd immunity are also at the forefront, even in industrialized countries, where previously eradicated viruses are now at risk of re-emergence due to instances of strain recombination, contractions in viral vector geographies, and from their potential use as agents of bioterrorism. There is a great need for the rational design of current and future vaccines targeting viruses, with a strong focus on vaccine targeting of adaptive immune effector memory T cells as the gold standard of immunity conferring long-lived protection against a wide variety of pathogens and malignancies. Here, we review viruses that have historically caused large outbreaks and severe lethal disorders, including respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic fevers. To observe trends in vaccinology against these viral disorders, we describe viral genetic, replication, transmission, and tropism, host-immune evasion strategies, and the epidemiology and health risks of their associated syndromes. We focus on immunity generated against both natural infection and vaccination, where a steady shift in conferred vaccination immunogenicity is observed from quantifying activated and proliferating, long-lived effector memory T cell subsets, as the prominent biomarkers of long-term immunity against viruses and their associated disorders causing high morbidity and mortality rates.