The pathological mechanisms involved in the interaction of SARS-CoV-2 are of key significance for infection and viral replication. Whilst a well-regulated immune response is essential in controlling SARS‐CoV‐2 infection, the ability of this virus to disrupt the normal immune responses leads to an uncontrolled immune modulation. In this study, we hypothesized that SARS-CoV-2 IgG level, ACE2 G8790A and ACE I/D polymorphism may impact the SARS‐CoV‐2 infection and its outcomes. We observed a significant association of SARS-CoV-2 IgG levels with smoking history, dwelling and age, with comparatively higher levels in non smokers, urban and those aged above 35 years. An interesting observation was that SARS-CoV-2 IgG levels were comparatively higher in subjects who had conspicuous complications coupled with need for hospitalization suggesting that SARS-CoV-2 IgG levels might be helpful in evaluating the course of disease and predicting the prognosis as also suggested by Wu et al., 2019 [26]. Correlation analysis of SARS-CoV-2 IgG levels with the resolution-time revealed insignificant statistical results. However, resolution-time as a dependent variable showed significant association with age and H1N1 vaccination. Subjects aged more than 35 years showed a mean resolution-time of 13.76 which was significantly higher than younger sub group (P < 0.05). In addition, subjects who had vaccinated themselves against H1N1 presented with a lower resolution-time (p < 0.05) suggesting that previous vaccination against H1N1 may cross protect against SARS-CoV-2 infection. Several studies have evaluated immune responses in H1N1 and SARS-CoV-2 co-infected models and have revealed some important considerations. Zhang et al., revealed that simultaneous or sequential co-infection by SARS-CoV-2 and H1N1 caused more severe disease than infection by either virus in hamsters although prior H1N1 infection lowered SARS-CoV-2 pulmonary viral load [27]. In an experimental model, Bao et al., reported that co-vaccination effectively protected K18-hACE2 mice against both H1N1 and SARS-CoV-2 infection [28]. A case report from an Influenza-like illness surveillance site in Egypt reported rapid resolution of SARS-CoV-2 and H1N1 co-infection in a 21-year-old woman and her family without treatment [29]. Thus, exploring the cross-protective role of neutralizing antibodies combined with co-vaccination strategies may hold promise against different variants of SARS virus as also suggested by persistence of lower resolution-time in H1N1 vaccinated subgroup in our study and may be an effective tool in developing more efficacious vaccines to support and succor the management of covid-19 pandemic.
Role of ACE2 G8790A and ACE ID Polymorphisms in SARS-CoV-2
SARS-Cov-2 infection is mediated by its binding with ACE2 receptor, a membrane exopeptidase, present on the host cells. Apparently, it seems concurring that the extent of ACE2 expression may determine the severity of this infection. Various studies have correlated ACE2 expression and its polymorphisms with covid-19 severity and most importantly ACE2 is overexpressed in men suggesting a higher sensitivity against the adverse effects of covid-19 infection [30, 31]. Various studies have investigated the potential for ACE2 polymorphisms to explain population-based differences in Covid-19 severity [30, 32]. In current study, frequency of G8790A GG genotype was observed to be considerably higher in subjects who required hospitalization suggesting GG genotype to be a potential genetic risk which could be predicative of disease severity. Carriers of G8790A GG genotype had higher anti covid-19 IgG titers as compared to those who carried AA genotype; however the results were statistically insignificant. In addition, no significant difference in the resolution-time of covid-19 infection was observed between the carriers of GG and AA genotypes. G8790A is located at the beginning of intron 2 and may play a possible role in splicing process which may in turn alter the expression level of ACE 2 [20, 33]. AA genotype has been observed to be associated with higher expression of ACE2. A study reported A/A genotype to increase the expression level of ACE2 by almost 50% in comparison to the G/G genotype [22]. This variant has been extensively studied as a potential risk factor for hypertension, type-2 diabetes, and coronary artery disease [22, 23, 34]. Contrary to these reports, a study carried out in Indian sub continent showed a strong association of G8790A ‘A’ allele with lower infection rate and lower CFR thus highlighting its protective role which holds true for the current study as we observed an odds ratio of 0.4 associated with this genotype which was statistically significant (OR = 0.40 (0.141–0.717), P = 0.007) [16]. The GG form of rs2285666 binds to heterogeneous ribonucleoprotein D-like (hnRNPDL) which is an RNA-processing prion-like protein with three alternative splicing (AS) isoforms, which lack none, one, or both of its two disordered domains. It has been suggested that alternative splicing might regulate the assembly properties of RNA-processing proteins by controlling the incorporation of multivalent disordered regions in the isoforms which would modulate their activity in the downstream splicing program [35]. Pouladi et al., demonstrated that binding of hnRNP DL to the GG form of rs2285666 enhances the splicing and produce an ACE2 with more binding affinity to the SARS-CoV-2, which may increase susceptibility to infection and aggravation of related complications [36]. The other variant which was genotyped in this study was ACE I/D, where ID genotype was observed to show protective role in comparison to II genotype. DD genotype and D allele although showed an OR of 0.56 and 0.65 respectively in comparison to II genotype, the results were statistically insignificant. There is evidence that ACE I/D genotypes affect the outcomes of acute respiratory distress syndrome (ARDS) treatment which is one of the aggressive outcomes of Covid-19 infection [37, 38]. Adamzik et al reported ACE DD genotype to be associated with increased mortality in Caucasian population [39]. Matteo Bellone et al associated ACE DD genotype with increased serum ACE levels and proposed that ACE DD variant might be predictive of more aggressive outcomes of covid-19 infection [40]. Various other population based studies have demonstrated that COVID-19 may be associated with ACE I/D polymorphism, with increasing D allele frequency correlating to a reduction in prevalence but increase in mortality from COVID-19 infection [41–43]. Currently, it remains unclear whether ACE polymorphisms are associated with the observed variations in COVID-19 mortality among different ethnicities and to what extent does the frequency of the II and DD genotypes contribute to the covid-19 severity. This study surmises that ACE ID genotype (heterozygous) bears protective role against covid-19 which supports the notion that D allele favors reduced prevalence of covid-19. The apparent inconstancy may be because of different ethnicity, and the fact that this study was carried out in covid-19 recovered patients offsets the possibility to observe reported association of ACEI/D DD variant with increased mortality. Based on the close proximity of ACE2 G8790A and ACE ID; these polymorphisms being in linkage disequilibrium, we further carried out their combined genotypic distribution, however the results were statistically insignificant.
In conclusion the aforementioned results suggest that ACE G8790A GG genotype carries a significant risk for covid-19 severity whereas ACE I/D heterozygous genotype bears protective role. The study provides preliminary evidence of a genetic link between the mentioned variants and COVID-19 suggesting a subtle way of COVID-19 risk stratification and utilization of respective variants as predictive biomarkers. In addition, we report a significant association of anti SARS-CoV-2 IgG levels with covid-19 severity. At an individual level, risk stratification involving ACE I/D and ACE2 G8790A combined with status of SARS-CoV-2 IgG titers might be helpful in evaluating the course of disease, predicting the prognosis and may be an effective tool in improving patient outcomes, management of covid-19 and related complications.