A new breakthrough study by Canadian researchers from the University of Manitoba, University of Toronto, Canada University Health Network and University of Alberta, Edmonton has found that treating SARS-CoV-2 infected individuals with interferon Lambda helps in rapid clearance of the virus even in those with age-related delays in the induction of T cell immunity.
The study team previously demonstrated that injection of pegylated interferon-lambda accelerated viral clearance in COVID-19 patients (NCT04354259). https://www.sciencedirect.com/science/article/pii/S221326002030566X
Treatment with a single subcutaneous injection of peginterferon lambda accelerated viral load decline and, after controlling for baseline viral load, reduced time to viral clearance in outpatients with COVID-19. The treatment effect was most apparent in those with high baseline viral loads. Peginterferon lambda was well tolerated, with similar symptoms to those treated with placebo.
Results for SARS-CoV-2 diagnostic testing are routinely reported dichotomously as positive or negative, without viral load quantification. Cycle threshold (Ct) values are sometimes reported, but are only semi-quantitative, and vary by assay and even by run, limiting comparisons between samples.
Our use of plasmid-derived cDNA standards with every PCR run allowed for quantification of SARS-CoV-2 RNA in each specimen, permitting direct comparison between samples. Quantification is useful clinically because higher viral levels have been correlated with greater severity of COVID-1918, 19 and increased infectivity.20 When individuals have recovered from infection, they might have persistently very low levels of RNA detected at very high Ct values (>33), which are not infectious.21
We found a clear antiviral effect of peginterferon lambda. Type III interferons lead to a slower but more sustained induction of interferon-stimulated genes, with peak responses seen after approximately 72 h, aligning with the onset of the antiviral response.22 Studies of monoclonal antibodies for outpatients with COVID-19 have also shown antiviral effects. In the early reports of the Regeneron monoclonal antibody cocktail, the difference in the decline in viral load with treatment compared with placebo by day 7 was 0·51 log copies per mL for the high dose and 0·23 log for the low dose group.23
The differences were greater in those who were seronegative for SARS-CoV-2 antibodies at baseline, with a difference of 0·60 log copies per mL with the high dose and 0·51 log copies per mL with the low dose, compared with the placebo group at day 7.23 Similar to our findings, greater antiviral effects were seen in those with high baseline viral load. Chen and colleagues reported that an intravenous infusion of neutralising antibody LY-CoV555 led to a reduction in viral load that was 0·53 log copies per mL greater with treatment than with placebo at day 11.3 Notably, in both of these studies, clinical benefits were seen with treatment in terms of reduced medical visits or hospitalisation,3, 23 highlighting the importance of even modest acceleration in viral load decline.
We found that the odds of clearance were greater in all study participants with peginterferon lambda than with placebo after controlling for baseline viral load. The effect of peginterferon lambda was most evident when the baseline viral load was above 106 copies per mL. Although the specific threshold for transmissible virus is unknown, using a standard infectivity assay, Bullard and colleagues reported that at Ct values above 24, corresponding to approximately 106–107 copies per mL, infectious virus could not be detected.20
We observed that in individuals with low viral load, irrespective of their assigned group, spontaneous clearance occurred rapidly and almost universally by day 7. This finding does not indicate a lack of effectiveness of peginterferon lambda at low viral loads, but rather that when low levels of virus were detected, treatment was not required because clearance was imminent.
Rapid clearance in those with low baseline viral loads explains the apparent absence of an antiviral effect seen by Jagannathan and colleagues24 in another trial of peginterferon lambda in outpatients, as the median Ct value at baseline was 30 with at least 75% of viral loads below 5·5 log copies per mL in their study compared with median baseline Ct value of 23·7 and 35 (58%) of 60 patients with viral loads above 6·0 log copies per mL in our trial.
Five (10%) of 51 participants, all in the placebo group, had already developed SARS-CoV-2-specific antibodies by the day of randomisation. Similarly, in the REGN-COV2 monoclonal antibody cocktail trial,23 45% of the study population was positive for SARS-CoV-2 IgG antibodies by day 0, which was associated with low viral loads and no benefit from therapy.
Understanding why some but not all infected individuals develop rapid IgG antibodies that are associated with milder disease course clearly warrants further investigation. Antivirals will probably be most effective early in infection and maximally beneficial to those with high viral loads. Ideally, antivirals would be given shortly after disease onset because rapid reduction of viral load is likely to lower the risk of clinical deterioration and might reduce transmission, translating into important public health benefits.
Several studies have investigated the interaction between SARS-CoV-2 and the endogenous interferon response. Like many viral pathogens, SARS-CoV-2 appears to impair induction of interferon, with low levels of type I and type III interferon production seen with ex-vivo infection of lung slices by severe acute respiratory syndrome coronavirus and to a greater extent with SARS-CoV-2.25 This finding is consistent with detailed immune profiling in patients showing an impaired type I interferon response in patients with severe, compared with mild, COVID-19.26 Patients with inborn errors of interferon production and those with antibodies to interferon alfa also have a much higher risk of severe disease with SARS-CoV-2 infection.27, 28
On the basis of the rationale that low interferon production is associated with severe disease, interferon treatment has been proposed. Early studies from China investigated subcutaneous interferon beta treatment. Although studies found a suggestion of accelerated viral clearance with interferon beta treatment, the studies were uncontrolled, non-randomised, and included co-interventions with other drugs (eg, hydroxychloroquine, lopinavir and ritonavir, umifenovir), making drawing strong conclusions difficult.16, 29, 30
A randomised open-label study of lopinavir and ritonavir with or without interferon beta in patients admitted to hospital showed that interferon treatment decreased the duration of viral shedding and shortened the duration of symptoms.31
However, early reports from the WHO SOLIDARITY trial32 found no clinical benefit to interferon beta treatment in hospitalised patients. Whether the lack of benefit in individuals with more severe manifestations of COVID-19 relates to introduction of therapy late in the course of illness or possibly to the proinflammatory side-effects of type I interferon is unknown. In mice infected with severe influenza, treatment with interferon alfa increased mortality compared with control mice, by promoting cytokine release syndrome, whereas treatment with interferon lambda was associated with improved survival.8
To diminish the risk of systemic inflammation from type I interferon use, Monk and colleagues33 investigated inhaled nebulised interferon beta-1a in patients admitted to hospital with moderate COVID-19. The authors showed a substantial reduction in clinical worsening by day 15 with good tolerability.33 Antiviral effects were not reported.
Because of the limited distribution of the interferon lambda receptor, we felt that interferon lambda might provide a safer approach to interferon treatment in patients with COVID-19 with similar antiviral effects but a reduced risk of potentially harmful proinflammatory responses.7
Despite the clear antiviral effect of peginterferon lambda, we did not see a marked difference in clinical outcomes. To translate acceleration of viral clearance to clear clinical benefit, the study would likely need to be enriched for those at higher risk of severe disease, such as individuals older than 65 years and those with comorbidities.34
Notably, the antiviral effect of peginterferon lambda compared with that of placebo was similar in those with comorbidities and the entire study population. Interestingly, among those still RNA positive at day 7, the viral concentrations were lower in the peginterferon lambda group than in the placebo group, which might have clinical relevance given the finding in the monoclonal antibody outpatient trial that patients with a higher viral load at day 7 were more likely to require hospitalisation.3 In addition to the risk of disease progression, lowering viral loads might reduce the risk of transmission.
In those with high baseline viral load, most participants treated with placebo had detectable virus at day 7, with most of these continuing to exceed 105 copies per mL, raising concern of persistent shedding of competent virus. By contrast, few participants who received peginterferon lambda had detectable virus at day 7, all with viral loads below 106 copies per mL.
To identify those most likely to benefit from this therapy, either quantitative testing could be introduced or a qualitative assay, ideally a point-of-care test, could be titrated to achieve an analytical sensitivity of approximately 105–106 copies per mL, allowing for immediate risk stratification and identification of the need for treatment. Indeed, this quantitative detection of SARS-CoV-2 could probably already be achieved using available rapid antigen tests, with detection sensitivities in the range of 10–50 000 copies per mL, safely below the infectious threshold but avoiding those with very low viral loads who are unlikely to require any intervention.35
More Black participants were in the placebo group than in the treatment group, a population typically shown to have reduced responsiveness to type I interferon for treatment of viral hepatitis.36, 37 However, similar proportions of patients in each group had the treatment-responsive interferon lambda genotype (TT), which is strongly associated with response to interferon alfa for hepatitis C infection and thought to explain most of the differential response to interferon by race.14 No effect of the interferon lambda genotype was observed on baseline viral load or response to treatment in the interferon lambda group. A high proportion of eligible individuals declined to participate in the study, probably because of the listed adverse event profile, which reflected weekly injections for a year of treatment for hepatitis B and C infections.11, 12
Peginterferon lambda was well tolerated with no identified safety concerns. Side-effects of peginterferon lambda overlap with COVID-19 symptoms, making distinguishing whether adverse events were related to treatment or the infection difficult. As has been reported previously,3 symptoms were more prominent in those with higher viral loads. With detailed serial symptom assessment, we found that symptoms improved in both groups over time.
Notably, among those who were asymptomatic at baseline, we found no difference in the number of adverse events between the treatment and placebo groups. Mild and transient aminotransferase increases were seen more frequently in the peginterferon lambda group than in the placebo group, which has been reported previously.11 D-dimer concentrations reduced with peginterferon lambda treatment, which might be relevant given the association of high concentrations with more severe disease and increased all-cause mortality.34, 38, 39 The side-effect profile and absence of haematological toxicity is consistent with the improved tolerability of type III interferons compared with that of type I interferons.11
Study limitations include the small sample size, although clearance rates in those with high viral loads were consistent with the power calculations. Based on viral load and antibody data at the baseline visit, several participants were probably clearing the infection, an observation reported in other studies of outpatients with COVID-19.3, 23 Ideally with the introduction of point-of-care testing, treatment could be initiated promptly at the time of diagnosis, which was not possible in this study because of delays in reporting times of positive results and required time for recruitment and consent.
The benefit of treatment was more pronounced in the group with high baseline viral load than in those with low baseline viral load. Quantitative assays or calibrated qualitative tests for COVID-19 could identify those most likely to benefit from therapy. As a phase 2 trial, the study was not powered to show differences in transmission, which are hard to document, or hospitalisation and mortality, which would require a larger study enriched for those at high risk of complications. However, as a first step to confirm efficacy, viral clearance is a key relevant endpoint. Having shown safety and efficacy in an ambulatory cohort, we and others are now investigating the clinical benefit of peginterferon lambda in patients admitted to hospital with COVID-19.
In conclusion, peginterferon lambda is among the first antiviral therapies to show benefit among outpatients with COVID-19. Peginterferon lambda accelerated viral clearance, particularly in those with high baseline viral load. This treatment might have potential to avert clinical deterioration, shorten the duration of infectiousness, and reduce isolation time, with substantial public health and societal effects.
The study findings were published in the peer reviewed journal: Nature Communications.
Our Phase II clinical trial data demonstrated that a single subcutaneous injection of PEG-IFN-λ (180 µg) showed efficacy as an early antiviral treatment for COVID-19. Here, we show that specific immune cells in the peripheral blood were responsive to PEG-IFN-λ, but this responsiveness did not modulate peripheral adaptive immunity to SARS-CoV-2, either positively or negatively.
However, early sampling revealed that older patients displayed a delayed T cell response towards SARS-CoV-2, showing a less diverse and less functional early response. Overall, our findings show that accelerated clearance of SARS-CoV-2 by PEG-IFN-λ was mediated by induction of the antiviral ISG response without major effects on B and T cell immunity, an advantage in older patients where the T cell immune response was delayed.
Our results revealed that subsets of immune cells within PBMCs from COVID-19 patients expressed IFNLR1 and were able to respond to PEG-IFN-λ treatment by upregulating ISGs. To our knowledge, this is the first report demonstrating specific human immune cell subsets are sensitive to PEG-IFN-λ treatment in vivo. PEG-IFN-λ was not given to healthy individuals.
Therefore, we were not able to determine if the ISG response was dampened by SARS-CoV-2 proteins implicated from in vitro studies but the clinical data show clear benefit of IFN-λ therapy8,36,37. Our in vivo results agree with earlier healthy donor in vitro studies demonstrating IFN-λ responsiveness, with purified pDCs and B cells as the top responders and monocytes and natural killer cells as non-responders20,23.
Subsets of CD8 + T cells, despite expressing IFNLR1, did not respond to PEG-IFN-λ in vivo. This is in contrast to our previous results where in vitro IFN-λ3 treatment of healthy donor CD8 + T cells led to an upregulation of antiviral ISGs measured by reverse transcription quantitative real-time PCR (RT-qPCR)20. Deeper analysis of the IFNLR1 + CD8 T cells revealed relatively low expression levels for both receptor chains, which may induce a response that falls below the sensitivity of scRNAseq.
This would be in line with our in vitro studies, where IFN-λ induced ~10-100 fold greater ISG fold changes in primary bronchial lung epithelial cells compared to purified B cells20, confirming the potent nature of IFN-λs at the site of SARS-CoV-2 infection. We also noted high baseline ISG expression in all patients, indicating the presence of endogenous IFNs.
The endogenous IFNs were likely type I IFN, which would explain the variability in ISG module scores seen in monocytes from placebo control patients enrolled at different days after symptom onset. This was anticipated due to the acute virus infection. Despite IFN-λ being exempt from desensitization typical of IFN-α repeat responses38, the high expression of ISGs likely limited the magnitude of ISG induction upon PEG-IFN-λ treatment in immune cells.
To gain further insight into the immunomodulatory properties of IFN-λ therapy, we also considered investigating differences between responders and non-responders in the IFN-λ treatment arm. However, only one IFN-λ treated patient was classified as a non-responder in the clinical study, limiting our ability to draw any conclusions related to non-response. Overall, we demonstrate that immune cells respond to IFN-λ in vivo despite an ongoing SARS-CoV-2 infection. However, viral clearance was likely driven by direct ISG activation, as demonstrated in our recently published paper measuring OAS1 activity, rather than promotion of adaptive immune responses39.
Similar to previous reports, we detected virus-specific T cell responses in acute and convalescent COVID-19 patients targeting SARS-CoV-2 structural proteins, including spike, nucleocapsid, and membrane40,41,42,43,44,45. Spike- and nucleocapsid-specific T cells dominated the T cell response, peaking at D7 and displaying the broadest functionality. Membrane-specific T cells displayed different kinetics for IFN-γ, with detectable responses at D0 that did not change significantly over time.
PEG-IFN-λ treatment had no impact on the kinetics, magnitude, functionality, or maintenance of a functional memory T cell pool compared to placebo. However, age, a key variable associated with severe COVID-19 disease outcomes, impacted the generation of SARS-CoV-2-specific T cell immunity28,46,47,48. Both IFN-γ and IL-2 responses were significantly delayed in patients over 45 years old. Notably all five patients in the trial who required hospitalization were over this age threshold. Our data are consistent with a recent report showing impaired naïve CD8 + T cell priming in older patients49,50.
Other studies have also assessed age-related differences in T cell responses, observing decreased cytotoxic CD8 + responses, lower IFN-γ/higher IL-2 secreting CD4 + T cells, and uncoordinated adaptive immune responses in older patients51,52,53. In assessing the interaction between treatment and age groups, we found viral load decline was similar in younger and older groups when stratified by treatment, suggesting that PEG-IFN-λ treatment can have an antiviral effect despite the differences in T cell responses.
In one fatal COVID-19 case, the patient did not exhibit detectable SARS-CoV-2-specific CD4 + or CD8 + responses after 2 weeks post-symptom onset, highlighting the importance of mounting T cell responses during early infection53. However, some of these studies either had a lower number of participants or recruited participants during a wide range of time after symptom onset.
A strength of our study was baseline samples were collected within 7 days of symptom onset in patients with a laboratory-confirmed diagnosis, providing better insight into the immune response early in the infection. Altogether, our findings show delayed T cell responses early after infection in older individuals, potentially exposing them to greater severity of outcomes, which may be compensated by early therapeutic intervention with PEG-IFN-λ.
Given our previous study showed in vitro exposure to IFN-λ negatively impacted influenza vaccine antibody responses22, we anticipated a negative impact on antibody production. However, despite detecting B cells were responsive to PEG-IFN-λ in peripheral blood, no difference in the levels of RBD-specific IgM, IgA, or IgG were measured in patient plasma between placebo and PEG-IFN-λ groups.
This indicates that one dose of PEG-IFN-λ was not sufficient to alter systemic antibody levels.
RBD-specific IgG antibodies were still elevated above baseline in most patients at D90 + , indicating long-term circulating levels in plasma. Unlike T cell responses, there was no significant impact of age on antibody levels when we compared those above or below the median of age 45.
Age has been negatively correlated with SARS-CoV-2 antibody levels, although greatest differences have been documented in those over 6054,55 and we only had 2 participants enrolled over this age. Whether multiple injections of PEG-IFN-λ could impact B cell function or responses by age, or whether memory B cell persistence and function at mucosal sites were altered requires further investigation.
We also assessed sex and IFNL4 genotype during our analysis since both have been associated with COVID-19 disease outcomes29,30,31,32,33. Multiple groups have found a greater proportion of male patients suffer from more severe COVID-19 outcomes29,30,31.
While we did not find sex differences in T cell responses in our patient cohort, we were able to see some significant differences in antibody levels (i.e., Total IgM and RBD-specific IgA) but the relevance of these differences remains unclear. In chronic HCV infections, IFNL4 genotype has been found to negatively affect the efficacy of PEG-IFN-α treatment and the probability of spontaneous viral clearance in those with the ΔG rs368234815 genotype34,35,56. Similar to our findings, a recent study found no associations with SARS-CoV-2-specific CD8 + T cell responses or antibody levels and IFNL4 genotype57.
However, IFNL4 variants have been found to be associated with the severity of and predisposition to acquiring COVID-1932,33, suggesting that IFNL4 genotype may affect innate immune responses, rather than adaptive responses. Of the five patients in our cohort who required hospital care, four had the risk-associated genotype at rs368234815. Additional analyses of the effects of sex and IFNL4 genotype on SARS-CoV-2-specific T cell and antibody responses would be useful, as our findings are limited by small sample size.
To summarize, our analyses demonstrate that a single dose of PEG-IFN-λ accelerates SARS-CoV-2 clearance without affecting virus-specific T cell responses or antibody production in mild-to-moderate acute SARS-CoV-2 infection.
Compared to current antiviral treatments for COVID-19, PEG-IFN-λ treatment is broad-acting, effective with a single dose, and is less likely to be affected by new variants or resistance mutations. This supports future use of PEG-IFN-λ as an early treatment option because it provides beneficial antiviral effects without negative consequences on adaptive immunity.
This aspect may be particularly relevant for older COVID-19 patients who may have naturally delayed T cell responses to SARS-CoV-2 early in an infection.