Immune profiling of SARS-CoV-2 epitopes in asymptomatic and symptomatic pediatric and adult patients.

BACKGROUND: The infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unpredictable manifestations of coronavirus disease (COVID-19) and variable clinical course with some patients being asymptomatic whereas others experiencing severe respiratory distress, or even death. We aimed to evaluate the immunoglobulin G (IgG) response towards linear peptides on a peptide array containing sequences from SARS-CoV-2, Middle East respiratory syndrome-related coronavirus (MERS) and common-cold coronaviruses 229E, OC43, NL63 and HKU1 antigens, in order to identify immunological indicators of disease outcome in SARS-CoV-2 infected patients. METHODS: We included in the study 79 subjects, comprising 19 pediatric and 30 adult SARS-CoV-2 infected patients with increasing disease severity, from mild to critical illness, and 30 uninfected subjects who were vaccinated with one dose of SARS-CoV-2 spike mRNA BNT162b2 vaccine. Serum samples were analyzed by a peptide microarray containing 5828 overlapping 15-mer synthetic peptides corresponding to the full SARS-CoV-2 proteome and selected linear epitopes of spike (S), envelope (E) and membrane (M) glycoproteins as well as nucleoprotein (N) of MERS, SARS and coronaviruses 229E, OC43, NL63 and HKU1 (isolates 1, 2 and 5). RESULTS: All patients exhibited high IgG reactivity against the central region and C-terminus peptides of both SARS-CoV-2 N and S proteins. Setting the threshold value for serum reactivity above 25,000 units, 100% and 81% of patients with severe disease, 36% and 29% of subjects with mild symptoms, and 8% and 17% of children younger than 8-years reacted against N and S proteins, respectively. Overall, the total number of peptides in the SARS-CoV-2 proteome targeted by serum samples was much higher in children compared to adults. Notably, we revealed a differential antibody response to SARS-CoV-2 peptides of M protein between adults, mainly reacting against the C-terminus epitopes, and children, who were highly responsive to the N-terminus of M protein. In addition, IgG signals against NS7B, NS8 and ORF10 peptides were found elevated mainly among adults with mild (63%) symptoms. Antibodies towards S and N proteins of other coronaviruses (MERS, 229E, OC43, NL63 and HKU1) were detected in all groups without a significant correlation with SARS-CoV-2 antibody levels. CONCLUSIONS: Overall, our results showed that antibodies elicited by specific linear epitopes of SARS-CoV-2 proteome are age dependent and related to COVID-19 clinical severity. Cross-reaction of antibodies to epitopes of other human coronaviruses was evident in all patients with distinct profiles between children and adult patients. Several SARS-CoV-2 peptides identified in this study are of particular interest for the development of vaccines and diagnostic tests to predict the clinical outcome of SARS-CoV-2 infection.

Bimodal antibody-titer decline following BNT162b2 mRNA anti-SARS-CoV-2 vaccination in healthcare workers of the INT - IRCCS "Fondazione Pascale" Cancer Center (Naples, Italy).

BACKGROUND: Both SARS-CoV-2 mRNA-based vaccines [BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna)] have shown high efficacy, with very modest side effects in limiting transmission of SARS-CoV-2 and in preventing the severe COVID-19 disease, characterized by a worrying high occupation of intensive care units (ICU), high frequency of intubation and ultimately high mortality rate. At the INT, in Naples, only the BNT162b2/Pfizer vaccine has been administered to cancer patients and healthcare professionals aged 16 and over. In the present study, the antibody response levels and their decline were monitored in an interval of 6-9 months after vaccine administration in the two different cohorts of workers of the INT - IRCCS "Fondazione Pascale" Cancer Center (Naples, Italy): the group of individuals previously infected with SARS-CoV-2 and vaccinated with a single dose; and that of individuals negative for previous exposure to SARS-CoV-2 vaccinated with two doses 21 days apart. METHODS: Specific anti-RBD (receptor-binding domain) titers against trimeric spike glycoprotein (S) of SARS-CoV-2 by Roche Elecsys Anti-SARS-CoV-2 S ECLIA immunoassay were determined in serum samples of 27 healthcare workers with a previously documented history of SARS-CoV-2 infection and 123 healthcare workers without, during antibody titers' monitoring. Moreover, geometric mean titers (GMT) and relative fold changes (FC) were calculated. RESULTS: Bimodal titer decline was observed in both previously infected and uninfected SARS-CoV-2 subjects. A first rapid decline was followed by a progressive slow decline in the 6/9 month-period before the further vaccine boost. The trend was explained by 2 different mathematical models, exponential and power function, the latter revealing as predictive of antibody titer decline either in infected or in not previously infected ones. The value of the prolonged lower vaccine titer was about 1 log below in the 6/9-month interval after the single dose for previously infected individuals with SARS-CoV-2 and the two doses for those not previously infected. The titer change, after the boost dose administration, on the other hand, was ≥ 1.5 FC higher than the titers at the 6/9-month time-points in both cohorts. A similar quantitative immune titer was observed in both cohorts 8 days after the last boost dose. The subsequent immunoresponse trend remains to be verified. DISCUSSION: The results show that a very rapid first decline, from the highest antibody peak, was followed by a very slow decline which ensured immune protection lasting more than 6 months. The apparent absence of adverse effects of the rapid decline on the vaccine's immune protective role has been related to a large majority of low avidity antibodies induced by current vaccines. High avidity antibodies with prolonged anti-transmission efficacy show a longer half-life and are lost over a longer interval period. The cellular immunity, capable of preventing severe clinical diseases, lasts much longer. The unbalanced dual activity (cellular vs humoral) while effective in limiting ICU pressure and overall mortality, does not protect against transmission of SARS-CoV-2, resulting in high circulation of the virus among unvaccinated subjects, including the younger population, and the continuous production of variants characterized by changes in transmissibility and pathogenicity. The high mutation rate, peculiar to the RNA virus, can however lead to a dual opposite results: selection of defective and less efficient viruses up to extinction; risk of more efficiently transmitted variants as the current omicron pandemic. CONCLUSIONS: In conclusion the current bimodal antibody-titer decline, following BNT162b2 mRNA anti-SARS-CoV-2 vaccination, needs a further extended analysis to verify the protective borderline levels of immunity and the optimal administration schedule of vaccine boosters. Our current results can contribute to such goal, besides a direct comparison of other FDA-approved and candidate vaccines.

HLA Does Not Impact on Short-Medium-Term Antibody Response to Preventive Anti-SARS-Cov-2 Vaccine.

The response to anti-SARS-Cov-2 preventive vaccine shows high interpersonal variability at short and medium term. One of the explanations might be the individual HLA allelic variants. Indeed, B cell response is stimulated and sustained by CD4+ T helper cells activated by antigens presented by HLA-class II alleles on antigen-presenting cells (APCs). The impact of the number of antigens binding to HLA class-II alleles on the antibody response to the COVID vaccine has been assessed in a cohort of 56 healthcare workers who received the full schedule of the Pfizer-BioNTech BNT162b2 vaccine. Such vaccine is based on the entire spike protein of the SARS-CoV-2. Ab titers have been evaluated 2 weeks after the first dose as well as 2 weeks and 4 months after the boosting dose. HLA-DRB1 and DBQ1 for each of the vaccinees have been assessed, and strong binders have been predicted. The analysis showed no significant correlation between the short-medium-term Ab titers and the number of strong binders (SB) for each individual. These results indicate that levels of Ab response to the spike glycoprotein is not dependent on HLA class II allele, suggesting an equivalent efficacy at global level of the currently used vaccines. Furthermore, the pattern of persistence in Ab titer does not correlate with specific alleles or with the number of SBs.

The clinical and translational research activities at the INT - IRCCS "Fondazione Pascale" cancer center (Naples, Italy) during the COVID-19 pandemic.

COVID-19 pandemic following the outbreak in China and Western Europe, where it finally lost the momentum, is now devastating North and South America. It has not been identified the reason and the molecular mechanisms of the two different patterns of the pulmonary host responses to the virus from a minimal disease in young subjects to a severe distress syndrome (ARDS) in older subjects, particularly those with previous chronic diseases (including diabetes) and cancer. The Management of the Istituto Nazionale Tumori - IRCCS "Fondazione Pascale" in Naples (INT-Pascale), along with all Health professionals decided not to interrupt the treatment of those hospitalized and to continue, even if after a careful triage in order not to allow SARS-CoV-2 positive subjects to access, to take care of cancer patients with serious conditions. Although very few (n = 3) patients developed a symptomatic COVID-19 and required the transfer to a COVID-19 area of the Institute, no patients died during the hospitalization and completed their oncology treatment. Besides monitoring of the patients, all employees of the Institute (physicians, nurses, researchers, lawyers, accountants, gatekeepers, guardians, janitors) have been tested for a possible exposure. Personnel identified as positive, has been promptly subjected to home quarantine and subdued to health surveillance. One severe case of respiratory distress has been reported in a positive employees and one death of a family member. Further steps to home monitoring of COVID-19 clinical course have been taken with the development of remote Wi-Fi connected digital devices for the detection of early signs of respiratory distress, including heart rate and oxygen saturation.In conclusion cancer care has been performed and continued safely also during COVID-19 pandemic and further remote home strategies are in progress to ensure the appropriate monitoring of cancer patients.

SARS-CoV-2 RNA polymerase as target for antiviral therapy.

A new human coronavirus named SARS-CoV-2 was identified in several cases of acute respiratory syndrome in Wuhan, China in December 2019. On March 11 2020, WHO declared the SARS-CoV-2 infection to be a pandemic, based on the involvement of 169 nations. Specific drugs for SARS-CoV-2 are obviously not available. Currently, drugs originally developed for other viruses or parasites are currently in clinical trials based on empiric data. In the quest of an effective antiviral drug, the most specific target for an RNA virus is the RNA-dependent RNA-polymerase (RdRp) which shows significant differences between positive-sense and negative-sense RNA viruses. An accurate evaluation of RdRps from different viruses may guide the development of new drugs or the repositioning of already approved antiviral drugs as treatment of SARS-CoV-2. This can accelerate the containment of the SARS-CoV-2 pandemic and, hopefully, of future pandemics due to other emerging zoonotic RNA viruses.