Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context

Influenza anti-stalk antibodies: Development of a new method for the evaluation of the immune responses to universal vaccine

Growing interest in universal influenza vaccines and novel administration routes has led to the development of alternative serological assays that are able to detect antibodies against conserved epitopes. We present a competitive ELISA method that is able to accurately determine the ratio of serum immunoglobulin G directed against the different domains of the hemagglutinin, the head and the stalk. Human serum samples were treated with two variants of the hemagglutinin protein from the A/California/7/2009 influenza virus. The signals detected were assigned to different groups of antibodies and presented as a ratio between head and stalk domains. A subset of selected sera was also tested by hemagglutination inhibition, single radial hemolysis, microneutralization, and enzyme-linked lectin assays. Pre-vaccination samples from adults showed a quite high presence of anti-stalk antibodies, and the results were substantially in line with those of the classical serological assays. By contrast, pre-vaccination samples from children did not present anti-stalk antibodies, and the majority of the anti-hemagglutinin antibodies that were detected after vaccination were directed against the head domain. The presented approach, when supported by further assays, can be used to assess the presence of specific anti-stalk antibodies and the potential boost of broadly protective antibodies, especially in the case of novel universal influenza vaccine approaches

Comparative analyses of SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from human serum samples

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

Cross-talk between endogenous H2S and NO accounts for vascular protective activity of the metal-nonoate Zn(PipNONO)Cl

Nitric oxide (NO) and hydrogen sulfide (H2S) are now recognized as gaseous transmitters with many cardiovascular protective properties. The present study concerns the possibility that NO donors can also function through endogenous activation of NO and H2S pathways. Based on the previous characterization of a novel metal-nonoate, Ni(PipNONO)Cl, our aim was: 1) to study the effects of a zinc based compound, Zn(PipNONO)Cl, on vascular endothelial and smooth muscle cells, and 2) to assess the role and interplay between endogenous NO and H2S promoted by the nonoate. Zn(PipNONO)Cl completely reproduced the vasodilation elicited by Ni(PipNONO)Cl. In the presence of endothelium, preincubation with Zn(PipNONO)Cl sensitized the intima to acetylcholine-induced vasodilation. When tested on cultured endothelial cells, Zn(PipNONO)Cl promptedPI-3K/Akt- and MAPK/ERK1/2-mediated survival. Nitrite levels indicated fast NO release (due to the molecule) and delayed (1-6 h) NO production linked to PI-3K/Akt-dependent eNOS activation. In the same time frame (1-6 h), significant CSE-dependent H2S levels were detected in response to Zn(PipNONO)Cl. The mechanisms responsible for H2S increase seemed to depend on the NONO moiety/sGC/cGMP pathway and zinc-associated ROS production. Our results indicate that endogenous H2S and NO were produced after fast NO release from Zn(PipNONO)Cl, contributing to the vascular endothelium protective effect. The effect was partially reproduced on smooth muscle cells, where Zn(PipNONO)Cl inhibited cell proliferation and migration. In conclusion, vasorelaxant effects, with complementary activities on endothelium and smooth muscle cells, are elicited by the novel metal-nonoate Zn(PipNONO)Cl

SARS-CoV-2 escape from a highly neutralizing COVID-19 convalescent plasma

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed