The use of SARS-CoV-2 pseudoviruses to investigate the neutralizing immunity and the pathogenesis of COVID-19

The Coronavirus Disease – 19 (COVID-19) occurs following SARS-CoV-2 infection with a wide spectrum of symptoms, which degenerate into severe acute respiratory syndrome (SARS) in susceptible patients or those with comorbidities. Although innovative vaccine technologies have been developed, the mutability of the virus, coupled with the natural decrease of neutralising antibodies (nAbs) that protect the organism by blocking the viral glycoprotein-human receptor interaction, has led to the administration of a booster dose of vaccine to increase the levels of protection against the newer variants (Omicron variants). nAbs targeting a virus are measured by enzymatic or cell-based serological tests. Among these, neutralisation assays that use pseudoviruses (PVNA) contributed to the investigation of anti-SARS-CoV-2 humoral immunity. PVs borrow the core from a known virus, engineered to be safe, whose envelope is replaced with that of a virus of interest. In this way, the infection mechanism is replicated in a safe, measurable and standardised manner. The PVNA on serum samples from 17 subjects receiving the third dose revealed that A) the vaccine stimulates the production of high specific antibody titres against the Spike protein, which wane after 4 months, B) these antibodies protect against the Omicron variants, C) older people produce fewer antibodies than younger people, D) hybrid immunity (having contracted the virus before or after the administration of the vaccine) provides better protection. These results are in line with what the literature reported. Although the technology of PVs contributed to the research on neutralization of SARS-CoV-2, it still suffers from the problems of using a cell line. An alternative strategy is to couple an enzymatic serological assay with the quantification of other biomarkers, such as circulating microRNAs, which are involved in the regulation of multiple processes, including the production of nAbs. The overexpression of miR-155-5p and downregulation of miR-148a-3p in serum are studied in the context of infections or following vaccination. Both are valuable candidates as potential biomarkers within a panel for the rapid assessment of the neutralising responses. Unfortunately, miR-155-5p was present in a few samples but its trend was similar to that of the literature. The analysis of miR-148a-3p also reflected what the literature reported. Despite this, no difference in expression over time was observed, nor did a direct correlation with specific antibody titre or protection levels emerge. PVs are also used to study cellular mechanisms, if they depend on the viral protein-host receptor interaction. Most severe COVID-19 patients show high levels of the SARS-CoV-2 receptor, ACE2, in soluble form in the circulation. ACE2 is normally released in circulation by proteases, such as ADAM17. However, excessive levels of ACE2 occur in cardiovascular diseases. A study from 2021found that the SARS-CoV-2 spike-induced reduction in intracellular miR-28-3p expression corresponded to altered ADAM17 expression levels. This mechanism could explain one of the main causes of the pathogenesis of COVID-19. The experiments I performed showed that A) the original variant (Wuhan) and the BA.5 induce the reduction of miR-28-3p; B) the Wuhan variant does not induce alteration of ACE2 or ADAM17, C) the Omicron BA.5 variant appears to reduce both the number of ACE2-positive and ADAM17-positive cells at 12 h incubation, although the alteration is minimal and not significant, D) there is no correlation between miR-28-3p expression levels and the number of ACE2- and ADAM17-positive cells. In conclusion, this thesis presents the advantageous aspects and limitations of using SARS-CoV-2 PVs technology to study neutralising immunity, in comparison to other serological tests, proposing an alternative approach, and understanding the pathogenesis of COVID-19.