The sleep disorder narcolepsy is linked to the HLA-DQB1*0602 haplotype and dysregulation of the hypocretin ligand-hypocretin receptor pathway. Narcolepsy was associated with Pandemrix vaccination (an adjuvanted, influenza pandemic vaccine) and also with infection by influenza virus during the 2009 A(H1N1) influenza pandemic. In contrast, very few cases were reported after Focetria vaccination (a differently manufactured adjuvanted influenza pandemic vaccine). We hypothesized that differences between these vaccines (which are derived from inactivated influenza viral proteins) explain the association of narcolepsy with Pandemrix-vaccinated subjects. A mimic peptide was identified from a surface-exposed region of influenza nucleoprotein A that shared protein residues in common with a fragment of the first extracellular domain of hypocretin receptor 2. A significant proportion of sera from HLA-DQB1*0602 haplotype-positive narcoleptic Finnish patients with a history of Pandemrix vaccination (vaccine-associated narcolepsy) contained antibodies to hypocretin receptor 2 compared to sera from nonnarcoleptic individuals with either 2009 A(H1N1) pandemic influenza infection or history of Focetria vaccination. Antibodies from vaccine-associated narcolepsy sera cross-reacted with both influenza nucleoprotein and hypocretin receptor 2, which was demonstrated by competitive binding using 21-mer peptide (containing the identified nucleoprotein mimic) and 55-mer recombinant peptide (first extracellular domain of hypocretin receptor 2) on cell lines expressing human hypocretin receptor 2. Mass spectrometry indicated that relative to Pandemrix, Focetria contained 72.7% less influenza nucleoprotein. In accord, no durable antibody responses to nucleoprotein were detected in sera from Focetria-vaccinated nonnarcoleptic subjects. Thus, differences in vaccine nucleoprotein content and respective immune response may explain the narcolepsy association with Pandemrix.

The micro-neutralization assay is a fundamental test in virology, immunology, vaccine assessment, and epidemiology studies. Since the SARS-CoV-2 outbreak at the end of December 2019 in China, it has become extremely important to have wellestablished and validated diagnostic and serological assays for this new emerging virus. Here, we present a micro-neutralization assay with the use of SARS-CoV-2 wild type virus with two different methods of read-out. We evaluated the performance of this assay using human serum samples taken from an Italian seroepidemiological study being performed at the University of Siena, along with the human monoclonal antibody CR3022 and some iper-immune animal serum samples against Influenza and Adenovirus strains. The same panel of human samples have been previously tested in enzyme-linked immunosorbent assay (ELISA) as a pre-screening. Positive, borderline, and negative ELISA samples were evaluated in neutralization assay using two different methods of read-out: subjective (by means of an inverted optical microscope) and objective (by means of a spectrophotometer).Our findings suggest that at least 50% of positive ELISA samples are positive in neutralization as well, and that method is able to quantify different antibody concentrations in a specific manner. Taken together, our results confirm that the colorimetric cytopathic effect-based microneutralization assay could be used as a valid clinical test method for epidemiological and vaccine studies. K E Y W O R D S epidemiology, humoral immunity, neutralization, pandemic, SARS coronavirus

Serological techniques commonly used to quantify influenza-specific antibodies include the Haemagglutination Inhibition (HI), Single Radial Haemolysis (SRH) and Virus Neutralization (VN) assays. HI and SRH are established and reproducible techniques, whereas VN is more demanding. Every new influenza vaccine needs to fulfil the strict criteria issued by the European Medicines Agency (EMA) in order to be licensed. These criteria currently apply exclusively to SRH and HI assays and refer to two different target groups—healthy adults and the elderly, but other vaccine recipient age groups have not been considered (i.e., children). The purpose of this timely review is to highlight the current scenario on correlates of protection concerning influenza vaccines and underline the need to revise the criteria and assays currently in use. In addition to SRH and HI assays, the technical advantages provided by other techniques such as the VN assay, pseudotype-based neutralization assay, neuraminidase and cell-mediated immunity assays need to be considered and regulated via EMA criteria, considering the many significant advantages that they could offer for the development of effective vaccines.

Vaccination is the most effective method of controlling seasonal influenza infections and preventing possible pandemic events. Although influenza vaccines have been licensed and used for decades, the potential correlates of protection induced by these vaccines are still a matter of discussion. Currently, inactivated vaccines are the most common and the haemagglutination inhibition antibody titer is regarded as an immunological correlate of protection and the best available parameter for predicting protection from influenza infection. However, the assay shows some limitations, such as its low sensitivity to B and avian strains and inter-laboratory variability. Additional assays and next-generation vaccines have been evaluated to overcome the limitations of the traditional serological techniques and to elicit broad immune responses, underlining the need to revise the current correlates of protection. The aim of this review is to provide an overview of the current scenario regarding the immunological evaluation and correlates of protection of influenza vaccines.

A newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 49.7 million cases of disease and 1,2 million deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays detecting different classes of antibodies constitute an excellent surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population. In addition, it can contribute to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease. The aim of this 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. It is important to identify the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict human population immunity, possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects. In addition, in a small sub-group of samples, a subtyping IgG ELISA has been performed. Our findings showed a notable statistical correlation between the neutralization titers and the IgG, IgM and IgA ELISA responses against the receptor-binding domain of the spike protein. Thus confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories that do not have biosecurity level-3 facilities.

BackgroundThe immunological response to influenza vaccine and/or natural infection is evaluated by serological techniques, the most common being hemagglutination inhibition (HI), single radial hemolysis (SRH), and virus neutralization assays, which is commonly used in a micro‐neutralization (MN) format. ELISA is not officially required; however, this assay is able to measure different class‐specific antibodies. The four assays identify different sets or subsets of antibodies.ObjectivesThe aim of this study was to establish the correlation among four serological assays using four seasonal influenza strains.MethodsThe HI, SRH, MN assays, and ELISA were performed on four seasonal influenza strains.ResultsA strong positive correlation was found between HI and MN and between SRH and MN assays for influenza A strains. The B strains also showed good correlations among the three assays. A positive correlation was also found between ELISA and the “classical” assays for all strains. Concerning the correlates of protection, as defined by HI ≥ 40 and SRH ≥ 25 mm2, good agreement was observed for the influenza A strains. By contrast, the agreement for the B strains was very low.ConclusionsThere is a positive strong correlation among the four serological assays for both A and B strains, especially for the HI and MN assays. There is good agreement on correlates of protection between HI and SRH assays for the A strains, but very low agreement for the B strains, suggesting higher sensitivity of SRH than HI assay in detecting antibodies against the influenza B viruses.

In the 20th century, three influenza pandemics killed approximately 100 million people. The traditional method of influenza vaccine manufacturing is based on using chicken eggs. However, the necessity of the availability of millions of fertile eggs in the event of a pandemic has led research to focus on the development of cell culture-derived vaccines, which offer shorter lead-in times and greater flexibility of production. So far, the cell substrates being evaluated and in use include Vero, Madin-Darby canine kidney, PER.C6 and insect cells. However, Vero cells are the most widely accepted among others. This review introduces briefly the concepts of advanced cell culture-derived influenza vaccine production and highlights the advantages of these vaccines in terms of efficiency, speed and immunogenicity based on the clinical data obtained from different studies.

Vaccination remains the principal way to control seasonal infections and is the most effective method of reducing influenza-associated morbidity and mortality. Since the 1940s, the main method of producing influenza vaccines has been an egg-based production process. However, in the event of a pandemic, this method has a significant limitation, as the time lag from strain isolation to final dose formulation and validation is six months. Indeed, production in eggs is a relatively slow process and production yields are both unpredictable and highly variable from strain to strain. In particular, if the next influenza pandemic were to arise from an avian influenza virus, and thus reduce the egg-laying hen population, there would be a shortage of embryonated eggs available for vaccine manufacturing. Although the production of egg-derived vaccines will continue, new technological developments have generated a cell-culture-based influenza vaccine and other more recent platforms, such as synthetic influenza vaccines.