Combined Prophylactic and Therapeutic Use Maximizes Hydroxychloroquine Anti-SARS-CoV-2 Effects in vitro.

While the SARS-CoV-2 pandemic is heavily hitting the world, it is of extreme importance that significant in vitro observations guide the quick set up of clinical trials. In this study, we evidence that the anti-SARS-CoV2 activity of a clinically achievable hydroxychloroquine concentration is maximized only when administered before and after the infection of Vero E6 and Caco-2 cells. This suggests that only a combined prophylactic and therapeutic use of hydroxychloroquine may be effective in limiting viral replication in patients.

Detection of low-level HCV variants in DAA treated patients: comparison amongst three different NGS data analysis protocols.

BACKGROUND: Notwithstanding the efforts of direct-acting antivirals (DAAs) for the treatment of chronically infected hepatitis C virus (HCV) patients, concerns exist regarding the emergence of resistance-associated substitutions (RAS) related to therapy failure. Sanger sequencing is still the reference technique used for the detection of RAS and it detects viral variants present up to 15%, meaning that minority variants are undetectable, using this technique. To date, many studies are focused on the analysis of the impact of HCV low variants using next-generation sequencing (NGS) techniques, but the importance of these minority variants is still debated, and importantly, a common data analysis method is still not defined. METHODS: Serum samples from four patients failing DAAs therapy were collected at baseline and failure, and amplification of NS3, NS5A and NS5B genes was performed on each sample. The genes amplified were sequenced using Sanger and NGS Illumina sequencing and the data generated were analyzed with different approaches. Three different NGS data analysis methods, two homemade in silico pipeline and one commercially available certified user-friendly software, were used to detect low-level variants. RESULTS: The NGS approach allowed to infer also very-low level virus variants. Moreover, data processing allowed to generate high accuracy data which results in reduction in the error rates for each single sequence polymorphism. The results improved the detection of low-level viral variants in the HCV quasispecies of the analyzed patients, and in one patient a low-level RAS related to treatment failure was identified. Importantly, the results obtained from only two out of the three data analysis strategies were in complete agreement in terms of both detection and frequency of RAS. CONCLUSIONS: These results highlight the need to find a robust NGS data analysis method to standardize NGS results for a better comprehension of the clinical role of low-level HCV variants. Based on the extreme importance of data analysis approaches for wet-data interpretation, a detailed description of the used pipelines and further standardization of the in silico analysis could allow increasing diagnostic laboratory networking to unleash true potentials of NGS.

Interferon-ß-1a Inhibition of Severe Acute Respiratory Syndrome-Coronavirus 2 In Vitro When Administered After Virus Infection.

The ongoing coronavirus disease 2019 pandemic has forced the clinical and scientific community to try drug repurposing of existing antiviral agents as a quick option against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Under this scenario, interferon (IFN) ß-1a, whose antiviral potential is already known, and which is a drug currently used in the clinical management of multiple sclerosis, may represent as a potential candidate. In this report, we demonstrate that IFN-ß-1a was highly effective in inhibiting in vitro SARS-CoV-2 replication at clinically achievable concentration when administered after virus infection.

Divergent Trends of Anti-JCPyV Serum Reactivity and Neutralizing Activity in Multiple Sclerosis (MS) Patients during Treatment with Natalizumab.

The association between natalizumab and progressive multifocal leukoencephalopathy (PML) is established, but a reliable clinical risk stratification flow-chart is lacking. New risk factors are needed, such as the possible role of the anti-JC polyomavirus (JCPyV) neutralizing antibody. In this pilot study, we analyzed this parameter during natalizumab treatment. Sequential sera of 38 multiple sclerosis patients during their first year of natalizumab treatment were collected, and grouped according to the number of infusions. For 11 patients, samples were also available after 24 infusions (T24), when progressive multifocal leukoencephalopathy (PML) risk is higher. The reactivity against VP1, the main JCPyV surface protein, and the anti-JCPyV neutralizing activity were evaluated. During the first year, a lack of correlation between anti-JCPyV antibody response and its neutralizing activity was observed: a significant decrease in anti-JCPyV antibody response was observed (p = 0.0039), not paralleled by a similar trend in the total anti-JCPyV neutralizing activity (p = 0.2239). This lack of correlation was even more evident at T24 when, notwithstanding a significant increase in the anti-JCPyV response (p = 0.0097), a further decrease of the neutralizing activity was observed (p = 0.0062). This is the first study evidencing, prospectively, the lack of correlation between the anti-JCPyV antibody response and its neutralizing activity during natalizumab treatment.

Cloning of the first human anti-JCPyV/VP1 neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy.

JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment.

Peptide-based vaccinology: experimental and computational approaches to target hypervariable viruses through the fine characterization of protective epitopes recognized by monoclonal antibodies and the identification of T-cell-activating peptides.

Defining immunogenic domains of viral proteins capable of eliciting a protective immune response is crucial in the development of novel epitope-based prophylactic strategies. This is particularly important for the selective targeting of conserved regions shared among hypervariable viruses. Studying postinfection and postimmunization sera, as well as cloning and characterization of monoclonal antibodies (mAbs), still represents the best approach to identify protective epitopes. In particular, a protective mAb directed against conserved regions can play a key role in immunogen design and in human therapy as well. Experimental approaches aiming to characterize protective mAb epitopes or to identify T-cell-activating peptides are often burdened by technical limitations and can require long time to be correctly addressed. Thus, in the last decade many epitope predictive algorithms have been developed. These algorithms are continually evolving, and their use to address the empirical research is widely increasing. Here, we review several strategies based on experimental techniques alone or addressed by in silico analysis that are frequently used to predict immunogens to be included in novel epitope-based vaccine approaches. We will list the main strategies aiming to design a new vaccine preparation conferring the protection of a neutralizing mAb combined with an effective cell-mediated response.

JC polyomavirus (JCV) and monoclonal antibodies: friends or potential foes?

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS), observed in immunodeficient patients and caused by JC virus ((JCV), also called JC polyomavirus (JCPyV)). After the HIV pandemic and the introduction of immunomodulatory therapy, the PML incidence significantly increased. The correlation between the use of natalizumab, a drug used in multiple sclerosis (MS), and the PML development of particular relevance. The high incidence of PML in natalizumab-treated patients has highlighted the importance of two factors: the need of PML risk stratification among natalizumab-treated patients and the need of effective therapeutic options. In this review, we discuss these two needs under the light of the major viral models of PML etiopathogenesis.

New therapeutic options for HCV infection in the monoclonal antibody era.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease and the most common indication for liver transplantation. Current therapies are ineffective in a relevant percentage of patients raising the urgent medical need to develop adequate therapies for this infection. Broadly neutralizing human monoclonal antibodies (mAbs) directed against the HCV E2 glycoprotein (HCV/E2), the major target of the neutralizing humoral immune response, are considered as a possible novel therapeutic strategy for this infection. In the last few years, several anti-HCV/E2 human mAbs have been described in literature to be possibly used for therapeutic or prophylactic purposes. In this review, we illustrate the best candidates for an anti-HCV mAb-based therapy, considering their cross-neutralization profiles and their ability to overcome possible viral escape mechanisms.

Neutralization activity and kinetics of two broad-range human monoclonal IgG1 derived from recombinant Fab fragments and directed against Hepatitis C virus E2 glycoprotein.

Hepatitis C virus (HCV) is the major cause of chronic liver disease worldwide. There is evidence that neutralizing anti-HCV antibodies may find potential applications in novel prophylactic and therapeutic strategies. This paper describes the very high neutralization activity and unique biological features of two broadly cross-reactive and cross-neutralizing anti-HCV human monoclonal IgG1 derived from human monoclonal recombinant Fab fragments.

A phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments.

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III and light chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide, whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was used to generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragment antibody directed against the nucleoprotein (NP) of Influenza A virus.

Monoclonal antibodies isolated from human B cells neutralize a broad range of H1 subtype influenza A viruses including swine-origin Influenza virus (S-OIV).

The new H1N1 swine-origin influenza virus (S-OIV) strain is a global health problem. The elucidation of the virus-host relationship is crucial for the control of the new infection. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned before the emergence of S-OIV from a patient who had a broad-range H1N1 serum neutralizing activity. The two HMabs neutralized all tested H1N1 strains, including S-OIV and a swine strain with IC(50) ranging from 2 to 7 microg/ml. Data demonstrate that infection with previously circulating H1N1 strains can elicit antibodies neutralizing S-OIV. Finally, the human genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered being potentially useful in the prophylaxis and the treatment of this human infection.

Molecular cloning of the first human monoclonal antibodies neutralizing with high potency swine-origin influenza A pandemic virus (S-OIV).

The pandemic caused by the new H1N1 swine-origin influenza virus (S-OIV) strain is a worldwide health emergency and alternative therapeutic and prophylactic options are greatly needed. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned from a patient who had a broad-range anti-H1N1 serum neutralizing activity. The two HMabs neutralized S-OIV with an IC50 of 2.8 and 4 microg/mL. The genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered with the potentially of representing a novel drug to be used in the prophylaxis and the treatment of this human infection. This is the first report of molecular cloning of human monoclonal antibodies against the new pandemic swine-origin influenza virus.