Episode 293 EBOLA Update

Episode 293/294: EBOLA and vaccine development

Dear colleagues,

While SARS-CoV-2 is still evolving and merits to be followed up in a next Episode, I was intrigued this week by a paper in Nature Briefing on a candidate mRNA vaccine for EBOLA (see Ep 294-16).  Before discussing it, I would like to it into the context of the disease and existing vaccines.  As always, I will try to tell the story, based on illustrative  self-explanatory (?) pictures, tables and graphs.  Of course, I’m aware that in my audience, there are colleagues, who have more knowledge and experience with various aspects of EBOLA.  If you (Peter, Guido, Kevin, Erika…) think that I skip some important aspects or make questionable interpretations, please let me know: I like to learn….

The first part (Ep 293) is more general. Tomorrow I will send Ep 294 on EBOLA vaccines

Ep 293 General background  (Ep 293-1-5)

1. Phylogeny

2. Morphology and genetic composition

3. Viral cycle

The genome of Ebola virus (EBOV) is depicted, in the 3ʹ‑to‑5ʹ orientation, to indicate that the genomic RNA is negative sense. The proteins encoded are nucleoprotein (NP), viral protein 35 (VP35), VP40, glycoprotein (GP), soluble GP (sGP), VP30, VP24 and large protein (L). Note that GP and sGP are encoded by a single gene in EBOV owing to transcriptional editing. Marburg virus (MARV) does not encode sGP. Filoviruses have unusually long non-coding regions at the 5ʹ and 3ʹ ends of their mRNAs. The genome regions corresponding to these non-protein-coding sequences are not drawn to scale. b | The general life cycle of a filovirus is displayed. First, the filovirus attaches to the cell membrane by its surface GP. The virus is then taken up by a process known as macropinocytosis37,153–155. Upon acidification of the endosome, the cellular proteases cathepsin B and cathepsin L cleave GP32. This allows GP and the host protein Niemann–Pick C1 (NPC1) to interact, which is a prerequisite for the fusion of viral and endosomal membranes33,34. Host endosomal calcium channels called two-pore channels play a crucial part in the endosomal trafficking of incoming viral particles to the site of fusion156. GP mediates fusion of the viral and the endosomal membrane, releasing the viral ribonucleocapsid into the cytoplasm, where the negative-strand RNA genome undergoes transcription and replication157. Production of 5ʹ‑capped, 3ʹ‑polyadenylated mRNAs from individual viral genes occurs, and genome replication follows, in which the genomic RNA template is copied into a full-length complement. The full-length complement serves as a template for the synthesis of additional negative-sense genomes. The RNA synthesis reactions require NP, VP35 and L. For initiation of transcription, EBOV also requires VP30. Translation of viral proteins occurs, and new viral particles are assembled at the plasma membrane. VP40 functions as the viral matrix protein and directs budding of particles from the cell surface158–160. GP, a type I transmembrane protein, is incorporated into the budding particles, as are viral nucleocapsids containing the viral genome161

4. Conceptualized pathogenesis

• General

Ebola virus particles enter the body through dermal injuries (microscopic or macroscopic wounds) or via direct contact via mucosal membranes. Primary targets of infection are macrophages and dendritic cells.

Infected macrophages and dendritic cells migrate to regional lymph nodes while producing progeny virions.

Through suppression of intrinsic, innate and adaptive immune responses, systemic distribution of progeny virions and infection of secondary target cells occur in almost all organs.

Key organ-specific interactions occur in the gastrointestinal tract, liver and spleen, with corresponding markers of organ injury or dysfunction that correlate with human disease outcome.

 The question marks indicate speculated manifestations. RIG-I, antiviral innate immune response receptor RIG-I.

• Interference with type 1 and type 2 IFN

EBOV VP35 suppresses IFN-ß  production via multiple inhibitory effects

Schematic of the interferon (IFN) production pathway and mechanisms of filoviral evasion. The image depicts how Ebola virus (EBOV) viral protein 35 (eVP35) and Marburg virus (MARV) VP35 (mVP35) antagonize signalling pathways that lead to the expression of type I IFNs. RIG‑I‑like receptors (RLRs), which include RIG‑I and melanoma differentiation-associated protein 5 (MDA5), detect RNA products of viral replication, such as cytoplasmic double-stranded RNAs (dsRNAs) or RNAs with 5ʹ triphosphates. Activation of RLRs is facilitated by the protein PACT (a protein activator of the IFN-induced antiviral kinase protein kinase R (PKR)). Upon activation, RLRs signal through the mitochondrial antiviral-signalling protein (MAVS) to activate kinases IκB kinase-ε (IKKε) and TBK1. These kinases phosphorylate IFN regulatory factor 3 (IRF3) or IRF7, which then accumulates in the nucleus and promotes expression of type I IFNs. VP35 can bind to dsRNAs and to PACT, preventing RLR activation. In addition, VP35 interacts with and acts as a decoy substrate for IKKε and TBK1

EBOV VP24 disrupts both type I (IFN-α andIFN-β) and type II IFN signaling by inhibiting the dimerization of phosphorylated STAT proteins

Schematic of the interferon (IFN) response pathway and mechanisms of filoviral evasion. Type I IFNs bind to the extracellular domains of the heterodimeric type I IFN receptor (IFNAR), which is composed of the subunits IFNAR1 and IFNAR2. This activates receptor-associated tyrosine kinases Janus kinase 1 (JAK1) and TYK2. JAK1 and TYK2 phosphorylate signal transducer and activator of transcription 1 (STAT1) and STAT2, which leads to the formation of STAT1–STAT1 homodimers or STAT1–STAT2 heterodimers. Tyrosine-phosphorylated STAT (pSTAT) dimers associate with karyopherin α1 (KPNA1), KPNA5 and KPNA6, and are transported into the nucleus, where they bind to IFN-sensitive response elements (ISREs). Binding of pSTAT dimers to ISREs induces the expression of IFN-stimulated genes (ISGs), which include the genes that encode the antiviral kinase protein kinase R (PKR) and major histocompatibility complex I (MHC) class I molecules. Marburg virus (MARV) VP40 (mVP40) inhibits the IFN-induced activation of JAK1. Ebola virus (EBOV) VP24 (eVP24) binds to KPNA1, KPNA5 and KPNA6 to block KPNA–STAT1 interactions. Both mechanisms inhibit ISG expression.

5. Clinical course

• Acute phase

The time course of the clinical manifestations (top), laboratory findings (middle) and viraemia and immune responses (bottom) in patients with Ebola virus disease (EVD).

The coloured lines in the top and middle panels do not have defined start and end points as these may vary. aIncubation periods of 2–21 days have been reported.

Renal dysfunction is common and not well-characterized in patients with EVD; it is probably a multifactorial combination of hypovolaemia (related to gastrointestinal fluid losses, decreased fluid input, fever, hypoalbuminaemia and sepsis pathophysiology), intrinsic renal injury (acute tubular necrosis related to myoglobin pigment injury secondary to rhabdomyolysis or direct viral infection of tubular epithelial cells) or cytokine-mediated nephrotoxicity.

Whereas respiratory symptoms and signs may reflect respiratory compensation for a primary metabolic acidosis, primary causes of hypoxaemic respiratory failure include acute lung injury (related to systemic inflammatory response syndrome and/or sepsis or Ebola virus (EBOV)-related cytokinaemia), pulmonary oedema (in the setting of capillary leak or direct infection) and viral pneumonia. Respiratory muscle fatigue may also contribute to ventilatory respiratory failure.

Haemorrhagic manifestations include oozing from venepuncture sites, haemoptysis (coughing up blood), haematemesis (vomiting blood), melaena (dark stools as a result of bleeding) and vaginal bleeding.

Neurological manifestations include meningoencephalitis and cerebrovascular accidents (such as strokes).

ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CPK , creatine phosphokinase; Hb, haemoglobin; HCT, haematocrit; PLT, platelet; PMN, polymorphonuclear leukocyte; PT, prothrombin time; PTT, partial thromboplastin time; WBC, white blood cell count.

• Clinical sequelae in survivors of EVD

EBOV, Ebola virus; PTSD, post-traumatic stress disorder.

1. In the PREVAIL III clinical trial, a prospective, controlled study assessing symptoms in survivors that had a >10% increase in prevalence compared with control close contacts, this symptom had an increased odds ratio (P < 0.0001) compared with close contact controls.
2. In the PREVAIL III clinical trial, in which symptoms in survivors were compared with symptoms in control close contacts (regardless of any increase in their prevalence in survivors), this symptom had an increased odds ratio (P < 0.01) compared with control close contacts.
3. Data from uncontrolled cohorts, case series or case reports.
4. Most common abnormalities in neurological examinations are abnormal oculomotor examination, abnormal reflexes, tremor and abnormal sensory examination.
5. Most common abnormalities include irregular heart rate, cardiac murmur, decreased breath sounds, rales (crackling lung sounds) and wheezes.
6. Most common abnormalities include abdominal tenderness, mass or distension.
7. Most common abnormalities include muscle tenderness and decreased range of motion.

6. Correlates of fatal disease, survival and sequelae

A very nice review is Ep 293-6:  Stéphanie Longet Front Immunol 2021

Ep 293-7: Stéphanie Reynard JCI Insight : Transcriptome and soluble factors in fatalities vs survivors:

• Massive upregulation of chemokines, cytokines (including anti-inflammatory ones such as IL-10) and cytotoxic molecules in fatal cases vs survivors =  “cyto-chemokine storm)
• Exception is soluble CD40L (immune-suppressive) and IL-38 (anti-inflammatory) upregulated in survivors during recovery

Some pro-inflammatory cytokines

Some chemokines

Some anti-inflammatory cytokines

Oposite for sCD40L and IL-38

The most predictive set, predictive of outcome includes: 2 chemokines MIP1α and Fractaline; 2 cytokines IL-6 and IFN-α2; one anti-inflamm IL-1RA

Ep 293-8: Ruth Thom Lancet Infect Dis 2019: longitudinal antibody and T cell responses in survivors and contacts of the West-African outbreak in 2014-16

A continuous high titre of neutralising antibodies and increased T cell response in survivors: might support the concept of long-term protective immunity in survivors.

Antibody and T cell responses in some contacts adds evidence to the existence of sub-clinical Ebola virus infection.

Ep 293-9: Francesca Colavita Viruses 2019 Inflammatory and Humoral Immune Response during

Ebola Virus Infection in Survivor and Fatal Cases

1. A dysregulated inflammatory response in fatal patients as compared to survivors, mainly consisting of the upregulation of inflammatory mediators, whose extent directly correlated with viremia levels.

While most cytokines and chemokines are upregulated during acute and chronic phase in fatal cases vs survivors, the immune suppressive soluble CD40L (consistent with Ep 293-7), but also the chemokine CX3CL1 (= Fractalkine) and PPBP (which is a potent neutrophils chemoattractant and activator involved in platelet activation, but also a protein involved in the host defense against bacterial and fungal infections

2. An earlier and more robust EBOV antibody response was observed in survivor patients.

Ep 293-10: Paula Ruibal Nature 2019: T cell immune suppression/exhaustion as a hallmark of fatal EBOLA

Fatal EVD was characterized by a high percentage of CD4+ and CD8+ T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load.

Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation.

Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response

Ep 293-11: Stéphanie Lavergne JID 2021 Increased EBOV-specific T cel responses in surivors with versus without sequelae

…suggesting EBOV induced pathogenesis of sequelae and even a possible auto-immune component…

7. Latency in humans as a source of resurgence?

Ep 293-12  New outbreak 7 years after the declaration of the first epidemic of Ebola virus disease in Guinea,—between 14 February and 19 June 2021—near the epicentre of the previous epidemic. 

The 2021 lineage shows considerably lower divergence than would be expected during sustained human-to-human transmission, which suggests a persistent infection with reduced replication or a period of latency.

8. History of epidemics

From Ep 293-2

From Ep 293-5

Note: Deaths in 2010-2019 is 13,648 (not 136,489)

9. Ongoing epidemics in Uganda and DRC

Uganda: Ep 293-13 and -14

This outbreak is caused by the Sudan virus: 4 of the 5 outbreaks in Uganda were caused by this rather rare EBOV. 

The largest was in 2000 with 425 infections and 224 deaths.

Vaccine development was oriented towards the more “common” Zaire strain.   

Overview by CDC (Ep 293-13)

Latest information from WHO (Ep 293-14)

DRC

1. April 2022: Outbreak in Equateur Province

On April 23, 2022, the Ministry of Health in the Democratic Republic of the Congo (DRC) declared an outbreak of Ebola virus disease (EVD) in Mbandaka health zone, Equateur Province. This is the 14th EVD outbreak in DRC and marks the third in a series of outbreaks in Equateur province since 2018 – one which started in the spring of 2018 and the other which was declared in the summer of 2020. Sequencing data from the first confirmed case in this outbreak indicates that this is a new spillover event from an animal to a person and is not directly linked to previous outbreaks.

The first patient identified in this outbreak sought treatment at several healthcare facilities and was taken to the Wangata Ebola Treatment Center after signs of hemorrhage (bleeding) began. Results from an initial test conducted in Mbandaka were positive for EVD. The DRC’s national laboratory in Kinshasa—the Institut National de la Recherche Biomédicale (INRB)— confirmed the positive test result through polymerase chain reaction (PCR).

2. On August 22, 2022, the Ministry of Health (MOH) in the Democratic Republic of the Congo (DRC) announced an outbreak of Ebola virus disease (EVD) in North Kivu Province. This announcement came after a fatal case was discovered in Beni Health Zone, one of the epicenters of DRC’s 10th outbreak in 2018-2020. A specimen was collected and tested positive at the Beni Referral Hospital laboratory and later confirmed at the DRC’s national laboratory in Goma – the Institut National de la Recherche Biomédicale (INRB).

Sequencing results from the INRB lab in Goma showed a link between this case and the 2018-2020 EVD outbreak in the same region  (= another argument for latency in humans?). While an investigation to identify how the individual became infected is ongoing, the sequencing results suggest a relapse of EVD, or infection by a survivor experiencing a relapse or who had a persistent EVD infection.

10. Full history of all outbreaks till early 2022: see https://www.cdc.gov/vhf/ebola/history/chronology.html    (Ep 293-15)

A quick calculation:

1. EBOV Zaire strain: in Central Africa (DRC and Congo) 15 outbreaks 4925 cases
2. EBOV Zaire strain in West-Afrika: 9 outbreaks with 28,869 cases
3. Sudan virus in Sudan and Uganda:   7 outbreaks with 778 cases
4. Bundibugyo virus 2 outbreaks: DRC with 38 cases and Uganda with 131 cases: Case fatality = 33 %
5. Reston: sporadic and non-fatal.