18 Feb 2023 Episode 316: Under which circumstances could type I or type III IFN be a useful treatment?

Episode 316: Under which circumstances could type I or type III IFN be a useful treatment?  

Dear colleagues,

This question was triggered by a recent publication in NEJM on positive results of subcutaneous type III IFN in a first phase 3 trial in COVID patients (see Ep 316-12).  Many trials with type I IFN have been published with variable results (Ep 316-3, -7, -)8, -9). 

We know that both types of IFN have a largely overlapping mechanism of action and there is clear evidence that IFN is crucial for our defense against COVID.  

I will elaborate on these elements in this episode, starting with a (long) introduction about the role of interferons in SARS-CoV-2 infection (Par 1), followed by a summary of clinical trials (Par 2) and an update on the genetic background (Par 3).

Finally, I will address the question whether there are potentially therapeutically relevant differences between type I and type III IFN.

Par 1 Introduction on type I, II and III interferons and COVID

1. 1. Definition

Ep 316-1: https://www.thermofisher.com/be/en/home/life-science/cell-analysis/cell-analysis-learning-center/immunology-at-work/interferons-overview.html

Type I IFNs bind to a specific cell surface receptor known as IFN-α/β (IFNAR1, IFNAR2) and function as a warning system for uninfected cells against viral infection. In humans, Type I IFNs are the largest IFN family and include IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω; they are produced by many cell types, including plasmacytoid dendritic cells and fibroblasts.

Two major functions of Type I IFN (Fig 1)

• inactivation of eukaryotic translation initiation factor 2a (eIF-2a), thereby inhibiting the synthesis of viral protein
• activation of RNase L, which cleaves any ssRNA within the cytoplasm, further inhibiting viral replication.

IFN-α has been used to treat hairy cell leukemia, while IFN-β has been used to slow the progression of multiple sclerosis.

Type II IFNs (IFN-γ in humans) bind to the IFN-γ receptor complex (IFNGR1, IFNGR2) and are involved in immune and inflammatory responses; they are produced by activated T cells and natural killer (NK) cells.

When Type II IFNs are released by T helper cells, type 1 (Th1 cells), leukocytes are recruited and activated to the sites of infection, leading to killing of intracellular bacteria and increased inflammation

Type III IFNs include IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4 and are implicated in inhibition of viral infections similar to Type I IFNs. IFN-λ1, IFN-λ2, and IFN-λ3 were originally named IL-29, IL-28a, and IL-28b, resp.; IFN-λ4 is the newest Type III IFN discovered. Type III IFNs bind to the receptors IFRL1 and IL-10R2, which are distinct from Type I receptors. Although not as well understood as Type I and II, Type III IFNs have been associated with the JAK-STAT pathway and are synthesized when the host detects pathogen-associated molecular patterns (PAMPs), similar to Type I IFNs.

Abbreviations: IFN, interferon; IFNAR1, interferon alpha receptor 1; IFNAR2, interferon alpha receptor 2; IFNGR1, interferon gamma receptor 1; IFNGR2, interferon gamma receptor 2; IFNLR1, interferon lambda receptor 1; IL-10R2, interleukin-10 receptor 2.

1. 2. Intracellular signaling

Ep 316-2: Kim and Shin Experimental & Molecular Medicine (2021)

The receptors and downstream signaling pathways of type I, type II, and type III interferons (IFNs).

Type I and type III IFNs bind to the heterodimeric receptor complexes IFNAR1/IFNAR2 and IFNLR1/IL-10Rβ, respectively. Upon IFN binding, the receptor-associated kinases JAK1 and TYK2 phosphorylate STAT1 and STAT2. Together with IRF9, phosphorylated STAT1 and STAT2 form a trimeric complex called IFN-stimulated gene factor 3 (ISGF3). ISGF3 subsequently enters the nucleus and binds IFN-stimulated response elements (ISREs) to promote the transcription of hundreds of IFN-stimulated genes (ISGs).

Type II IFN binds to the receptor complex composed of IFNGR1 and IFNGR2 and promotes the phosphorylation of STAT1 via JAK1 and JAK2. Phosphorylated STAT1 forms homodimers, which bind gamma-activated sequences (GASs) in the nucleus and induce proinflammatory gene expression. Unlike type III IFNs, type I IFNs can also signal via STAT1 homodimers and promote proinflammatory gene expression.

Thus, IFN type I and II share a similar intracellular pathway, which is clearly distinct from IFN type II  

Ep 316-3: Jorge Quarleri  Cytokine and Growth Factor Research Revies 2021: Similar effects of type I and type III action

Type I and III interferons response following SARS-CoV-2 sensing during controlled (left and central cell) and uncontrolled (right cell) infection

Schematic representing the canonical pathway of pathogen recognition receptor (PRR) sensing of intracellular pathogens. Pathogen-associated molecular patterns (PAMPs) -such as viral RNA- are recognized by pathogen recognition receptors –PRRs- (RIG-1, MDA5, and LGP2) lead to activation and phosphorylation of IRF3/7 and IRF1, their nuclear translocation, and drive the expression of both types I and III interferons.

Both interferons are translated and secreted from the infected cell in an autocrine or paracrine manner. Upon engagement of IFN-I to the IFNAR and of IFN-III to the IFNLR receptor, signal transductions start, leading to the expression of interferon-stimulated genes (ISG). These opposite scenarios compare the IFN-I/III optimal response controlling SARS-CoV-2 infection (left and central cell), and the counteracted and/or deficient IFN-I/III response (right cell) associated with high virus replication, immune-mediated complications (including down-regulation of ISGs, together with exacerbated NF-κB activation), and lung immunopathology (“cytokine storm”, accumulation of monocytes, vascular damage, and abnormal T cell response).

IFN signaling through the Jak-STAT pathway

Type I (left panel) and type III (right panel) IFNs bind to distinct receptors but activate the same downstream signaling events, and induce almost identical sets of genes mainly through the activation of IFN-stimulated gene factor 3 (ISG3) and STAT1 homodimers.

Ep 316-4: Fatemeh Sodeifian : Induction and effects of type 1 and type 3 IFN

In the first step, infectious viral particles (PAMPs) were sensed through pattern recognition receptors PRRs.

• SARS‐CoV‐2 is detected through endosomal PRRs including TLR 3, 7, 8, and 9 and/or through cytoplasmic PRRs such as MDA5 and RIG‐1. Moreover, the virus can be sensed through TLR4, which is localized in the cell membrane.
• Following TLRs activation, NF‐'kB's transcription factor induces inflammatory cytokines, whereas activated MDA5 and RIG‐1 recruit IRF3 and IRF7 for interferon production.

When interferon binds its receptor, IFRAR1/2,an IFN‐induced signaling pathway is initiated, resulting in the recruitment of JAK‐1 and TYK. JAK‐1 and TYK activation trigger the phosphorylation of the signal transducer and activator of transcription (STAT)1 and 2, respectively.

Subsequently, an IFN‐stimulated gene factor 3 (ISGF3) complex is formed, containing STAT1, STAT2, and IRF‐9, which is translocated to the nucleus and increases the expression of IFN‐stimulated genes (ISGs).

1. 3. Immunological effects of type I and III IFN

ISGs translation and posttranslational modification trigger antiviral action interferons, including

• Inhibition of mRNA production, RNA degradation, RNA editing, and the initiation of T cell response, and NO synthesis.

Abbreviations IFN, interferon; IRF, IFN regulatory factor; MDA, melanoma differentiation‐associated protein; mRNA, messenger RNA; PRRs, pattern recognition receptors; RIG, retinoic acid‐inducible gene‐1‐like receptor; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; TLR, Toll‐like receptor

Ep 316-5: Hossein Khorramdelazad Eur J Pharmacol 2022 IFN and COVID pathogenesis

Effects of type 1 IFN on innate immunity

Effect of type 1 IFN on adaptive immunity

Note: this simplified scheme already indicates that the effects of type 1 IFN on the adaptive immunity can be both stimulatory and inhibitory, depending on timing and quantity.

This is further illustrated  in the next slides showing a delicate balance between type 1 Interferon and inflammation

• Type 1 IFN = essential for protection against severe disease: see deleterious genetic defects of inborn errors and auto-Ab.
• Age effect: children are “primed” for rapid type 1 response versus elderly are too slow
• Low type -1 IFN levels  associated with high inflammatory markers
• In the lungs of severe COVID there is accumulation of pro-inflammatory cells (neutrophils, all monocyte subsets, cDC and NK cells), but decrease of type-1 IFN producing pDC

Ep 316-2: Kim and Shin

Hypothesis of how delayed but exaggerated type I IFN responses are involved in hyperinflammation and contribute to the severe progression of COVID-19

After respiratory epithelial cells are infected (a), SARS-CoV-2 proteins block type I and III interferon (IFN) responses (b) (See Ep 316-6  for more details). The viral load increases (c) and uninfected innate immune cells, such as monocytes, macrophages, and dendritic cells, are stimulated by viral components via Toll-like receptors and produce type I and III IFNs (d). Type I and III IFNs further induce the accumulation and activation of monocytes and macrophages, leading to the production of large amounts of IFNs and proinflammatory cytokines (e). Type I IFNs also enhance TNF-mediated inflammation by disrupting TNF-induced tolerance to TLR stimulation in monocytes and macrophages.

1. 4.  How SARS-CoV-2 interferes with type 1 IFN

Ep 316-6: Sa Ribeiro PLoS Pathogens July 2020

SARS-CoV-2 genomic organization and encoded proteins

In blue:  ORF1a/1b encode a polyprotein, which is proteolytically processed into Nsp1–16

In green: Structural proteins, including S, E, M, and N proteins

In orange:  Accessory proteins encoded at the 30 end of the viral genome comprise ORF3a, 3b, 6, 7a,7b, 8, 9b, 9c, and 10.

Untranslated extremities of the genome (50-UTR and 30-UTR) are also represented.

In red are depicted SARS-CoV-2 proteins that interfere with IFN induction pathway as well as their known or hypothetic target].

E, envelope; IFN, interferon; M, membrane; MAVS, mitochondrial antiviral-signaling protein; N, nucleocapsid; Nrdp1, neuregulin receptor degradation protein-1; Nsp, nonstructural protein; ORF, open reading frame; RNF41, ring finger protein 41; S, spike; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; TANK, TRAF family member-associated NF-κB activator; TBK1, TANK-binding kinase 1; Tom70, translocase of outer mitochondrial membrane 70; UTR, untranslated region.

SARS-CoV interfering with IFN induction and signaling

On this cartoon are schematically represented the signaling pathways triggered by SARS-CoV RNA recognition by the cytoplasmic RNA sensors RIG-I and MDA5, which leads to IFN induction (A) and subsequent IFN signaling in surrounding cells, resulting in the

expression of ISGs (B).  SARS-CoV proteins that have been reported to interfere with these pathways are indicated.

IFN, interferon; IFNAR, interferon alpha and beta receptor; IκB, inhibitor of nuclear factor κB; IKKε, IκB kinase-ε; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; JAK, Janus kinase; M, membrane; MAVS, mitochondrial antiviral signaling protein; MDA5, melanoma differentiation-associated gene 5; N, nucleocapsid; Nsp, nonstructural protein; ORF, open reading frame;

P, phosphate; PLP, papain-like protease; RIG-I, retinoic acid-inducible gene 1; SARS-CoV, severe acute respiratory syndrome coronavirus; STAT, signal transducer and activator of transcription; TANK, TRAF family member associated NF-κB activator; TBK1, TANK-binding kinase 1; TRAF3, tumor necrosis factor receptor-associated factor 3; TYK2, tyrosine kinase 2.

1. 5. Differential Properties of Type I- and III Interferons

Ep 316-7: Jafarzadeh  Viral Immunology 2021  

Despite the many common molecular and functional properties of type I and III just described, there are many differences, as shown in the Table below with the most important outlined in red:

Type III is produced more stably at the mucosal site as a first line of defense, thus limiting the spread of the virus with less pro-inflammatory systemic side effects    

PART 2 Clinical studies with IFN type I and III in COVID

2.1. Controversial findings with type 1 IFN

Ep 316-3: Fatemeh Sodeifian (Iran) Med Virol Aug 2021 provides a large table with phase2/3 studies, with as overall conclusion that timing is crucial:

• IFNs' administration before the viral peak and inflammatory phase of disease could be highly protective
• However, IFNs' treatment during the inflammatory and severe stages of the disease causes immunopathology and long‐lasting harm for patients

Ep 316-7:  Ling-Ying Lu (Taiwan) Cytokine and Growth Factor Rev 2022 makes a positive, but not uniform balance on the use of interferon alpha in a large number and variety of studies: No evidence of safety issues

Early intervention, either within five days from the onset of symptoms or at hospital admission, confers better clinical outcomes, whereas late intervention may result in prolonged hospitalization.

Ep 316-8: Juan Pablo Sosa (USA Infect Chemother 2021: a systematic review on the use of interferon-beta in  8 selected studies (from 66). The conclusion is cautiously positiver

IFN-β has been shown to decrease hospital stay's overall length and decrease the severity of respiratory symptoms when added to the standard of care. Also, in some studies, it has been demonstrated to reduce the length of ICU stay, enhance survival rate, and decrease the need for invasive mechanical ventilation. There were minor side effects reported (neuropsychiatric symptoms and hypersensitivity reaction).

However, randomized clinical trials with a large sample size are needed to assess IFN-β's benefit precisely

Ep 316-9: Abuzar Asif (US) J. Comm Hosp Internal Med Perspectives 2021 makes a Cochrane-type analysis on 7 carefully selected studies and concludes that use of subcutaneous interferon-beta is futile in COVID-19.

The IFN-beta group did not improve the 28-day mortality (RR = 1.276; 95% CI: 1.106–1.472, p = 0.001) or the discharge rate (RR = 0.906; 95% CI = 0.85–0.95, p = < 0.001).

The mean hospital stay was similar 11.95± 2.5 days in the interferon-beta group and 11.43 ± 3.74 days in the traditional treatment group.

Likewise, interferon-beta did not add any advantage to post-intervention intubation rate (RR = 0.92; 95% CI = 0.7841–1.0816, p = 0.3154).

Preliminary conclusion: Early treatment is warranted, but the use of type I interferon has rather variable clinical results. 

2.2. What about type III IFN?

Clinical studies with pegylated interferon lambda  (always 180 microgran subcutaneous)

2.2.1. Two contrasting double blind placebo controlled phase 2 trials in outpatients:

• No clear clinical effect
• No more side effect than placebo
• Is there an effect on viral load?

Ep 316-10: Jonathan Feld Lancet Resp Dis May 2021:

Patients within 7 days of symptom onset or first positive test if asymptomatic.  

Peginterferon lambda accelerated viral decline in outpatients with COVID-19, increasing the proportion of patients with viral clearance by day 7, particularly in those with high baseline viral load (> 10exp 6 copies):

Undetectable viral load by Day 7:  15/19 (79 %à in active group versus 6/16 (38 % ) in placebo group (p = 0.012)

As can be seen, overall effect and effect in patients with low viral load not so convincing.

Ep 316-11: Prasanna Jagannathan Nat Comm March 2020

Patients within 3 days of symptoms onset:

No effect on viral clearance irrespective of:

- presence of antibodies (seronegative probably earlier in the infection)

- baseline viral load

2.2.2 Phase 3 trial

Ep 316-12: G. Reis NEJM Feb 2023

Trial in almost 2000 severe patients, most partly vaccinated, within 7 days of symptoms onset 

→ Primary outcome: hospitalization, transfer ton tertiary hospital, or emergency dep.

= rather clearcut decrease of about 50 %: 2.7 % in IFN versus 5.6 % in placebo:

• Irrespective of risk group
• Apparently more clear-cut with omicron than with alpha, gamma or delta

→ Effect on viral clearance? 

Overall IFN greater log10 reduction: 8.29 vs 5.16 in placebo

Effect on clearance more pronounced in patients on IFN

2.3. Guidelines on type 1 and type 3 interferon

Ep 316-13: Recommendations NIH/ FDA 16 Dec 2021: https://www.covid19treatmentguidelines.nih.gov/therapies/antivirals-including-antibody-products/interferons/

• The COVID-19 Treatment Guidelines Panel (the Panel) recommends against the use of systemic interferon beta for the treatment of hospitalized patients with COVID-19 .
• The Panel recommends against the use of interferon alfa or lambda for the treatment of hospitalized patients with COVID-19, except in a clinical trial 
• The Panel recommends against the use of interferons for the treatment of nonhospitalized patients with mild or moderate COVID-19, except in a clinical trial 

Ep 316-14: Last update of WHO guidelines 13 Jan 2023: does not even mention interferon https://www.who.int/publications/i/item/WHO-2019-nCoV-therapeutics-2023.1

Ep 316-15: EMA does not consider interferon treatment.  See https://www.ema.europa.eu/en/human-regulatory/overview/public-health-threats/coronavirus-disease-covid-19/treatments-vaccines/covid-19-treatments

HOWEVER:

Ep 316-16: NIH 14 July 2022:  interferon treatment may reduce the severity of COVID-19 in people with genetic polymorphism in the OAS (oligo-adenylate-cyclase gene)!  Based on a study in Nature Genetics (see Ep 316-14).  https://www.nih.gov/news-events/news-releases/interferon-treatment-may-reduce-severity-covid-19-people-certain-genetic-factors

Par 3: Genetic background literature

Ep 316-17: Leonardo Ferreira  Infection, Genetics and Evolution 2022:  

Mechanisms underlying COVID-19 based on the genetic variants identified by genome-wide association studies

The genes associated with COVID-19 phenotypes are highlighted in bold.

In the top panel, biological processes predominantly occurring in the upper respiratory tract, such as

1. Mucociliary clearance: involved with ciliogenesis and mucus production (LZTFL1, CEP97, FOXP4, TULP2) ,
2. Viral-entry and viral replication processes (SLC6A20, FYCO1, ACE2).
3. Mucosal immunity: 6p21 (rs143334143 and rs3131294) seems to influence antigen presentation and IgA production..

The genetic variants related to those genes are associated with susceptibility to infection (IS phenotype, Table 3). Impairment in these processes caused by the combined effect of risk alleles may favor the passage of higher viral loads to the lower airways until the alveolar space, contributing to the disease worsening.

Lower panels Immune modulatory processes, including

4. Antiviral immune response: parts of IFN type 1 and type 3 receptors and interferon response genes OAS (2’5’ oligo-adenylate synthetase) and SHFL (Shiftless Antiviral Inhibitor of Ribosomal Frameshifting) , , inflammasome/piroptosis, leukocyte trafficking and migration (E), drive the mechanism underlying severe disease. The genetic variants controlling those processes are associated with disease severity (DS phenotype, Table 3). Variants rs643434 and rs687289 are in linkage disequilibrium with the variants in ABO locus (9q34.2), and associated with the concentration of blood markers of coagulation (F). The other variants are associated with the amount of circulating leukocytes and platelets. Altogether, this quantitative effect on blood cells and proteins potentially links these genetic loci to immunothrombosis (F), an event frequently found in COVID-19 patients.

Multiple role for ABO:

• ABO encodes the glycosyltransferase responsible for the ABO blood group system. There is evidence that Anti-A and anti-B antibodies in individuals of blood group O might target glycoproteins in the virus surface, impairing the viral-entry process.
• In addition, variants at the ABO locus influence the concentration of blood markers of coagulation and thus may be involved with the risk of thromboembolitic events in COVID-19 patients

OAS (2,5 Oligo-Adenylate Syntherase) is an antiviral enzyme

• OAS is induced by type 1 and type 3 IFN (as one of the ISRE (Interferon Stimulated Response Elements)
• OAS is activated by double stranded RNA (intermediary product during viral replication)
• It generates 2’5’ adenylate from ATP, which activates ribonuclease L, leading to viral RNA degradation

The OAS/RNase L pathway in an innate immune response against viruses. dsRNA, double-stranded RNA; OAS, 2'-5'-oligoadenylate synthetase; 2-5A, 5'-phosphorylated, 2'-5'-linked oligoadenylates; RNase L, 2'-5'-oligoadenylate-dependent ribonuclease L.

Ep 316-18: A Rouf Banday Nature Genetics Aug 2022: Genetic regulation of OAS1 nonsense-mediated

decay underlies association with COVID-19 hospitalization

• Decreased OAS1 expression due to a common variant haplotype contributes to COVID-19 severity: risk of hospitalization
• The effects of genetic variants on OAS1 expression can be compensated in vitro by IFN-λ treatment to overcome impaired viral clearance and (potentially) prevent progression to severe COVID-19 requiring hospitalization.

A very complex model the authors propose (only understandable with a lot of knowledge on genetics that I lack…)

Ep 316-19:  JL Cassanova  J Clin Invest Jan 2023: The broader perspective on convergent genetic defects

• In T cell tolerance, leading to auto-antibodies to type 1 IFN
• In Type 1 Interferon induction and effector pathways

Both pathways result in high susceptibility to severe COVID and other virus infections or live virus vaccination

AR autosomal-recessive; AD = autosomal dominant

YFV-17D: Yellow fever; MMR(V): measles, mumps, rubella, varicella: severe infections after these life-attenuated vaccines

HSE: Herpes Simplex Encephalitis

Ep 316-20: Qian Zhang J Exp Med 2022

Some of these genetic deficiencies are associated with severe COVID in children, because they represent recessive and biochemically complete inborn errors of type I IFN immunity: X-linked recessive TLR7 deficiency and autosomal recessive IFNAR1, STAT2 or TYK2 deficiencies

Ep 316-21 Zhang Science 2022

Some functionally important polymorphisms partly in the same genes are probably less completely deleterious and were seen in adults who have a less vivid IFN response than children and therefore might be rather relatively deficient.

Inborn errors of type I IFN production and amplification underlie life-threatening COVID-19 pneumonia

Molecules in red are encoded by genes, deleterious variants underlie critical influenza pneumonia with incomplete penetrance, Those in blue are deleterious variants of genes underlie other viral illnesses.

Molecules represented in bold are encoded by genes with variants that also underlie critical COVID-19 pneumonia

What is the therapeutic implication?

1. Logically, exogenous type 1 (or type 3) IFN could substitute for genetically (or age-dependent?) deficient production of IFN e.g. IRAK, MyD88, MDA5, IRF3, IRF7 etc
2. If the defect is localized in the IFN response (e.g. IFNAR, STAT1/3, JAK, Tyk or downstream in the interferon-stimulated response genes (ISRG), exogenous IFN might still be useful if the defect is not complete   

CONCLUSION

In principle, both type I and III could be therapeutically useful in SARS-CoV-2 infection:

• Genetic errors in type I/III pathways are associated with severe COVID
• SARS-CoV-2 shows multiple IFN escape mechanism.
• The age-related worsening of COVID can partly be explained by decreased IFN activity

Although both types of IFN share a lot of characteristics, type III is more mucosa-oriented and most likely has less systemic side effects (if applied locally). Conceptually, type I/III IFN should be applied locally very early in the infection, best before the symptoms are apparent, to effectively block viral replication, before the virus itself has the chance to block IFN production/action.  

Until now, most clinical trials were done with type I (either alpha or beta) using various administration routes (including inhalation) and mostly within 1 week after symptoms onset.  The variable results failed to convince the regulatory agencies NIH, WHO and EMA (see Ep 316-12, -14, -15).

Theoretically type III IFN could be better than type I, but one would prefer local application, while the first clinical studies use the subcutaneous route. The phase 2 data are not very convincing, but the first phase 3 trial shows clear beneficial effects, not unlike several trials with type I IFN.

The paper in Nature Genetics (Ep 316-18), suggests that the deleterious effects of genetic variants on OAS1 expression can be compensated in vitro by IFN-lambda treatment to overcome impaired SARS-CoV-2 suppression by this OAS variant, by increasing its expression. This paper has inspired NIH to become more optimistic about type III IFN (Ep 316-16).

Therefore, it remains possible that inhalation treatment with type I or III IFN very early after onset of symptoms could be useful in some selected patients with either genetic or age-related defects in the IFN pathway that can be overcome by exogenous IFN, but more convincing clinical data should be presented.

I hope this was useful…

Best wishes,

Guido