VIRUS PANDEMICI EMERGENTI: VALUTAZIONE IMMUNOLOGICA DEL VIRUS INFLUENZALE A/VIETNAM/1194/2004 (H5N1) E DEL NUOVO SARS-COV-2
Creators: name: MAZZINI, LIVIA
Publisher: name: Universit� degli Studi di Siena
Year: 2021

description: The historical moment we are experiencing has raised global awareness about the importance of identifying the main characteristics of emerging pandemic viruses in order to limit the damage caused by infection-associated diseases and safeguard global health. This work included two main studies focused on pandemic A/Vietnam/1194/2004 (H5N1) and the novel SARS-CoV-2, respectively. 
H5N1 is a type of influenza virus that causes a highly infectious, severe respiratory disease in birds called avian influenza (or "bird flu"). Influenza A viruses circulating among poultry have the potential to recombine with human influenza A viruses and become transmissible among humans. The aim of the first study was to determine the efficacy of pandemic influenza vaccine against influenza strain A/Vietnam/1194/2004 (H5N1).
Immunogenicity for the homologous strain A/Vietnam/1194/2004 (H5N1) was investigated using Haemagglutination Inhibition assay (HAI), Micro-Neutralization (MN) and Single Radial Hemolysis (SRH). In order to evaluate cross-protective immunity, serum samples have been assessed to determine the antibody response versus heterologous flu strains (A/Turkey/Turkey/1/2005 (H5N1) and A/Anhui/1/2005 (H5N1)), applying the same tests (HAI, MN, SRH) plus Enzyme Linked Lectin Assay (ELLA) for the antibody response against the Neuraminidase (NA) protein. A first analysis has been carried out considering the seroconversions for two distinguished cohorts: vaccine dose 15�g and 30�g, and age dependent groups 18-40 and 41-60 years old. In the second analysis, all the study methods previously shown were assessed, by evaluating the seroprotection. The results obtained showed the dominance of homologous response anti-A/Vietnam/1194/2004 compared to the heterologous A/H5 strains. In the dose-related evaluation, the same MN seroconversion rate is provided for A/Vietnam/1194/2004, but the 30�g dosage seemed to provide advantage for heterologous strain. ELLA assay revealed that, the administration of A/H5N1/Vietnam vaccine evoke a high response also for the N1 A/California. Regarding the age-effect, the group of younger people achieved a greater effect from each of the two doses received, compared to the group of older subjects.
The newly identified coronavirus, named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in Wuhan, Hubei province, China, in December 2019 and quickly spread throughout the world and was declared a pandemic on March 11th 2020. The diagnosis of Coronavirus associated disease (COVID-19) is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Serological assays aimed at detecting different classes of antibodies could be the best surveillance strategy and can be useful in evaluating of immunogenicity of novel future vaccines for the prevention of COVID-19 disease.
The aim of this second study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. Before starting, a set-up and validation of in-house ELISA was developed; several purified recombinant S-proteins (S1 and RBD domain) were tested for their ability to detect specific human antibodies and ELISA test was standardized to be as sensitive and specific as possible.
For this second study 181 human serum samples were tested by means of in-house ELISA S1 and RBD-specific IgG, IgM and IgA and by means of the Euroimmun S1 Commercial ELISA kit, along with the functional MN assay. Our results showed, the highest agreement between the in-house ELISA IgG and IgA RBD and MN and confirm that the antibodies targeting the RBD domain have the highest probability of being neutralizing. We also noticed that the IgA response was closely linked with a positive MN response and in those subjects in whom we registered a high neutralization titer, we always observed a positive IgA signal. We also evaluated the ELISA IgG subtyping response and our results, showed a strong reactivity for IgG1 and IgG3 against RBD in almost all samples. This second study had two major goals: to standardize ELISAs able to detect different classes of immunoglobulins, and to broaden the data-set of information on comparisons between the results of different serological tests, which could be precious for future evaluations of serological diagnoses and vaccine assessments. The present study constitutes preliminary research into the development of an ELISA that can semi-quantify anti-SARS-CoV-2 human antibodies in a specific and repeatable way.