We used a transcriptomic approach to study gene expression in whole blood comparing COVID-19 subjects with differing disease severities and investigating transcriptional signatures by comparing SARS-CoV-2 VOC with wildtype strains. Our data show that VOC such as the delta variant downregulate host anti-viral responses through interferon stimulated genes, innate immunity through chemokine activation, T cell development and erythropoiesis by downmodulating erythrocyte and heme function. We VOCs enhanced host inflammatory pathways such as VEGFalpha and ribosomal pathways associated with increased viral mRNA translation. This is the first report describing VOC- induced host transcriptional profiles in a population that experienced a low burden of COVID-19 disease associated morbidity.
We first focused on the comparison of COVID-19 patients with Symptomatic or Asymptomatic disease. The age of Symptomatic COVID-19 cases was older than those who had Asymptomatic COVID-19. Symptomatic cases were those admitted to the hospital for treatment and therefore this trend fits with data showing older age to be associated with increased COVID-19 severity. Transcriptional profiles of those with Symptomatic as compared with Asymptomatic COVID-19 revealed an increase in inflammatory responses as well as cellular signaling associated with increased pathogenesis in COVID-19. The upregulation of neutrophil markers and apoptotic markers (BCL2A1 and ANXA3 likely reflects greater pathogenesis, tissue damage and cell death in symptomatic COVID-19. CXCL8 is reflective of exacerbation of inflammation and has been found to correlate with severity of acute SARS-CoV-2 infection [25]. Interleukins were activated; IL-1 is a pro-inflammatory cytokine involved in the initiation of immune responses, while IL-10 limits excessive inflammation. The TFPI gene (involved in coagulation and the complement pathway) was upregulated. TPFIs has been shown to be raised in delta infections in the macaque model [20]. In Symptomatic cases, lymphocyte differentiation was suppressed and adaptive immunity downmodulated, highlighted by reduced activation of CD28 co-stimulation and ZAP-70 pathways aligning with earlier reports of immune suppression in severe COVID-19 [26, 27]. TCF7, a transcription factor, crucial for T cell development and differentiation was downregulated in symptomatic COVID-19[23]. Previous ARDS data showed downregulation of adaptive immunity and T cell exhaustion, with T-cell apoptosis associated with T-cell depletion [14].
Analysis of interferon-driven responses, showed upregulation of IL1, IL4, IL18, AREG, SOCS3, VEGFA and downregulation of IL12, IL2, IFNAR2, TGF, MAVS, IRF4, TRIM14, SAMDH1 in Symptomatic patients. Interferon-mediated signaling is critical in protection against SARS-CoV-2 and is negatively related to COVID-19 severity [27, 28]. ISGs are proposed as part of predictive biomarker for assessment of disease outcomes [18]. The upregulation of Interferon-genes in the Asymptomatic COVID-19 patients matches earlier reports of from infection with SARS-CoV-2 wildtype variants [17].
Comparison of the VOC-induced transcriptional profiles with wildtype strains revealed differing host genetic perturbations between variant types. Overall, the majority of DEGs were upregulated. Those induced by alpha were associated with cellular signaling and inflammatory factors SYVN1 (regulated the signal transducer and activator of transcription 3 (STAT3)/vascular endothelial growth factor (VEGF) axis) and CH25H (inflammatory factor present in macrophage populations) [24]. Down-DEGs included those involved in NK cell activation and CD*T cell activation (CST7), IL1RAPL2, as well as cellular signaling molecules associated with innate immunity such as, endocytosis (GBP1/2/5), TRIM22 involved in antiviral killing [29] and, FCR3E involved in humoral responses. The reduction of innate immune and defense response related processes may explain the increased virulence of alpha strains, previously reported to suppress innate responses to escape initial stages of host immunity [30].
Between delta and wildtype strains, the greatest enhancement was seen in EGR1, a host transcription factor that inhibits SARS-CoV-2 replication through E3 ubiquitin pathway. EGR1 also inhibits the replication of viruses including CMV, SARS-CoV-2 and EBV [31], and facilitates the transcription of immune genes including TNF, IL-2, IL-6, IL-8. Delta variants upregulated IFIT1 and IFI44L expression. This fits with earlier report that delta variants induce lower interferon expression than wildtype SARS-CoV-2 in vitro [32]. IFIT downregulation would likely negatively affect inflammatory and interferon responses as IFI44L as part of innate immune responses [33]. CCL2 along with cytoplasmic ribosomal proteins and RNA processing pathways and VEGFA-VEFFR2 pathway genes were upregulated. Ribosomal proteins play a critical role viral propagation [34] andVEGF signalling indicates inflammatory pathway activation and viral replication. These data are concordant with increased inflammation and disease severity observed during the delta virus waves, globally [35, 36] and at our center in Karachi [37]. Delta infections downregulated genes related to erythrocyte synthesis and development (HBB, DMTN, SCL4A1, HBD9, GYPC). Of note, ALAS2, the most downregulated gene, is the enzyme responsible for the rate-limiting step in heme biosynthesis, catalyzing the synthesis of aminolaevulinic acid (ALA), the precursor of heme, from glycine and succinyl-CoA. Downregulation of ALAS2 could decrease in heme biosynthesis by feedback inhibition by high levels of heme or regulatory signals that suppress ALAS2 expression. Our results describing the impact of delta variants on RBC development align with earlier reports indicating that delta variant infections resulted in reduced RBC width, correlating with COVID-19 severity [38].
Omicron versus wildtype infections displayed the greatest number of DEGs, including activation of viral mRNA translation, cytoplasmic ribosomal pathways, and RNA processing pathways while inflammatory and immune response pathways were suppressed. EGR1 was the most upregulated DEG whilst ALAS2 was most downregulated. Notably, chemokine receptors, CXCR1 and CXCR2, as well as MGAM, markers of innate immunity, were also found downregulated. The upregulation of viral RNA translation and ribosomal pathways fits with the increased host protein synthesis observed during infections with higher viral loads. Previously it was shown that those infected with omicron had lower CT values indicating higher viral loads compared to the alpha variants, but omicron infections were associated with less severe in-patient mortality [12]. Mutations across various sites in Spike glycoprotein genes such as D614G, have facilitated increased viral entry and replication[39], leading to immune escape [30].
Comparing the ISG expression patterns between Symptomatic and Asymptomatic cases, as well as in the context of alpha, delta, and omicron variants, provides insights into the host immune response to SARS-CoV-2. Delta variants caused the greatest upregulation of ISGs (IFI44, IFI44L) [40]. ISGs control pathways induced during pathogen life cycles and are induced by IRF independent of JAK/STAT pathways [41]. The high fold change observed in IRAK3 in individuals with Symptomatic COVID-19 compared to Asymptomatic cases suggests a potential association between IRAK3 expression and disease severity. Expression of IRAK3 may reflect an attempt by the host immune system to regulate inflammation and outcome on disease severity.
Vaccinations also impact transcriptional profiling of COVID-19 patients [42] [43].To focus on the mechanisms that are protective against controlling infection with VOC to prevent confounders due to vaccination, we focused only on unvaccinated Asymptomatic cases. Transcriptional profiles of those infected with VOC as compared with wildtype strains revealed upregulation of ribosomal genes, whilst downregulated genes involved in heme synthesis (ALAS2, HBB) as well as MAVS, an ISG. A strong association is shown between anemia and increased mortality in COVID-19 patients [44]. Therefore, altered erythropoietic pathways as seen through transcriptional profiles of COVID-19 patients in our study may be a biomarker of severe disease in the patients. The activation of ribosomal pathways in COVID-19 patients infected with VOCs, indicating that the ribosomes are engaged in translating viral RNA into viral proteins thus increasing viral replication [45]. This suggests the delta and omicron variants replicate more efficiently than the wild type, potentially contributing to their increased transmissibility or virulence [46].
Our data is unique that it sheds light on VOC infections in Asymptomatic individuals in a population which experienced relatively low COVID-19 morbidity through the pandemic. The reduced trend of ISGs after VOC infection fits data shown in the rhesus macaque model [20]. However, reports of infection with SARS-CoV-2 variants have varied in other models. A comparison of transcriptional responses to SARS-CoV-2 variants as compared with wildtype strains in rhesus macaques noted that VOC induced greater upregulation of RNAs involved in gene expression and metabolic pathways, but downregulated RNAs involved in cardiac contraction [19]. However, this study noted differential transcriptional changes in the study of tissues but not in blood gene expression profiles. Another study of SARS-CoV-2 variants conducted in cynomolgus macaques infected with delta and omicron variants showed differences only in TFPI2 gene which was upregulated in the alveolar ROIs of the delta-infected group compared to those of the omicron-infected group [20]. Of note, we found TFPI to be upregulated only in the comparison between Symptomatic as compared with Asymptomatic infections, but not in a VOC-specific manner.
Our data supports the view that that alpha and delta have progressively evolved over the ancestral variants by silencing the innate immune response [47]. Reduced neutralizing activity against variants has been demonstrated in the context of alpha, delta and omicron infections with mutations such as, N501Y and E484K which were first shown to be associated with antibody escape in the host [48]. Increased inflammation in individuals infected with alpha and delta as compared with wildtype variants fits with COVID-19 trends observed in the population from the UK, with an increase in deaths due to alpha variants [49]. Studies from Brazil and Singapore also reported increased severity of disease in COVID-19 patients with the gamma variant and delta variant respectively [50]. In Europe, the CFR from infections with omicron was significantly lower as compared with delta [36]. In Pakistan, studies showed that delta variants were associated with increased risk of severe disease [37], and also that omicron was associated with reduced in-hospital mortality [12].
A limitation of our study was that the sample size for VOC was relatively small. However, this was limited by the number of individuals on whom we had SARS-CoV-2 variant data based on NGS or RT-PCR, and on the number of individuals who consented to the study. Unfortunately, we were unable to include omicron infected individuals who were unvaccinated and Asymptomatic to compare in this cohort. By the time omicron infections were introduced in Pakistan, a large majority of the population had received COVID-19 vaccinations [51]. Also, we do not have longitudinal transcriptional profiles of the COVID-19 cases, to see what changes may occur as their infections progress or resolve. Whilst we have results for only a single sample from each COVID-19 case, we do know that of the 32 individuals with asymptomatic infections, only two progressed to have severe disease whilst the others recovered well. Although our data set was small, it is robust in the classification of COVID-19 cases and therefore provides insights into infections wildtype and VOCs.
Overall, our data highlight the role of interferon-pathways in protection against viral diseases and ISGs as markers predictive of immune clearance. Our work describing the effect of VOCs provides key insights by identifying genes involved in pathways that related to erythrocyte development and suggest these as potential targets which can be explored further to reduce the negative impact observed in patients infected with SARS-CoV-2 strains.