The initial interplay between HIV and mucosal innate immunity.

Human Immunodeficiency Virus (HIV) is still one of the major global health issues, and despite significant efforts that have been put into studying the pathogenesis of HIV infection, several aspects need to be clarified, including how innate immunity acts in different anatomical compartments. Given the nature of HIV as a sexually transmitted disease, one of the aspects that demands particular attention is the mucosal innate immune response. Given this scenario, we focused our attention on the interplay between HIV and mucosal innate response: the different mucosae act as a physical barrier, whose integrity can be compromised by the infection, and the virus-cell interaction induces the innate immune response. In addition, we explored the role of the mucosal microbiota in facilitating or preventing HIV infection and highlighted how its changes could influence the development of several opportunistic infections. Although recent progress, a proper characterization of mucosal innate immune response and microbiota is still missing, and further studies are needed to understand how they can be helpful for the formulation of an effective vaccine.

The interferon landscape along the respiratory tract impacts the severity of COVID-19.

Severe coronavirus disease 2019 (COVID-19) is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In contrast, compared to subjects with other infectious or noninfectious lung pathologies, IFNs are overrepresented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites.

Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection.

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent but Neuropilin-1-dependent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients.

Viral Respiratory Pathogens and Lung Injury.

Several viruses target the human respiratory tract, causing different clinical manifestations spanning from mild upper airway involvement to life-threatening acute respiratory distress syndrome (ARDS). As dramatically evident in the ongoing SARS-CoV-2 pandemic, the clinical picture is not always easily predictable due to the combined effect of direct viral and indirect patient-specific immune-mediated damage. In this review, we discuss the main RNA (orthomyxoviruses, paramyxoviruses, and coronaviruses) and DNA (adenoviruses, herpesviruses, and bocaviruses) viruses with respiratory tropism and their mechanisms of direct and indirect cell damage. We analyze the thin line existing between a protective immune response, capable of limiting viral replication, and an unbalanced, dysregulated immune activation often leading to the most severe complication. Our comprehension of the molecular mechanisms involved is increasing and this should pave the way for the development and clinical use of new tailored immune-based antiviral strategies.

Fast inactivation of SARS-CoV-2 by UV-C and ozone exposure on different materials.

The extremely rapid spread of the SARS-CoV-2 has already resulted in more than 1 million reported deaths of coronavirus disease 2019 (COVID-19). The ability of infectious particles to persist on environmental surfaces is potentially considered a factor for viral spreading. Therefore, limiting viral diffusion in public environments should be achieved with correct disinfection of objects, tissues, and clothes. This study proves how two widespread disinfection systems, short-wavelength ultraviolet light (UV-C) and ozone (O3), are active in vitro on different commonly used materials. The development of devices equipped with UV-C, or ozone generators, may prevent the virus from spreading in public places.

Cell-to-Cell Spread Blocking Activity Is Extremely Limited in the Sera of Herpes Simplex Virus 1 (HSV-1)- and HSV-2-Infected Subjects.

Herpes simplex virus 1 (HSV-1) and HSV-2 can evade serum antibody-mediated neutralization through cell-to-cell transmission mechanisms, which represent one of the central steps in disease reactivation. To address the role of humoral immunity in controlling HSV-1 and HSV-2 replication, we analyzed serum samples from 44 HSV-1 and HSV-2 seropositive subjects by evaluating (i) their efficiency in binding both the purified viral particles and recombinant gD and gB viral glycoproteins, (ii) their neutralizing activity, and (iii) their capacity to inhibit the cell-to-cell virus passage in vitro All of the sera were capable of binding gD, gB, and whole virions, and all sera significantly neutralized cell-free virus. However, neither whole sera nor purified serum IgG fraction was able to inhibit significantly cell-to-cell virus spreading in in vitro post-virus-entry infectious assays. Conversely, when spiked with an already described anti-gD human monoclonal neutralizing antibody capable of inhibiting HSV-1 and -2 cell-to-cell transmission, each serum boosted both its neutralizing and post-virus-entry inhibitory activity, with no interference exerted by serum antibody subpopulations.IMPORTANCE Despite its importance in the physiopathology of HSV-1 and -2 infections, the cell-to-cell spreading mechanism is still poorly understood. The data shown here suggest that infection-elicited neutralizing antibodies capable of inhibiting cell-to-cell virus spread can be underrepresented in most infected subjects. These observations can be of great help in better understanding the role of humoral immunity in controlling virus reactivation and in the perspective of developing novel therapeutic strategies, studying novel correlates of protection, and designing effective vaccines.

Entry inhibition of HSV-1 and -2 protects mice from viral lethal challenge.

The present study focused on inhibition of HSV-1 and -2 replication and pathogenesis in vitro and in vivo, through the selective targeting of the envelope glycoprotein D. Firstly, a human monoclonal antibody (Hu-mAb#33) was identified that could neutralise both HSV-1 and -2 at nM concentrations, including clinical isolates from patients affected by different clinical manifestations and featuring different susceptibility to acyclovir in vitro. Secondly, the potency of inhibition of both infection by cell-free viruses and cell-to-cell virus transmission was also assessed. Finally, mice receiving a single systemic injection of Hu-mAb#33 were protected from death and severe clinical manifestations following both ocular and vaginal HSV-1 and -2 lethal challenge. These results pave the way for further studies reassessing the importance of HSV entry as a novel target for therapeutic intervention and inhibition of cell-to-cell virus transmission.

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.

Epitope mapping by epitope excision, hydrogen/deuterium exchange, and peptide-panning techniques combined with in silico analysis.

The fine characterization of protective B cell epitopes plays a pivotal role in the development of novel vaccines. The development of epitope-based vaccines, in fact, cannot be possible without a clear definition of the antigenic regions involved in the binding between the protective antibody (Ab) and its molecular target. To achieve this result, different epitope-mapping approaches have been widely described (Clementi et al. Drug Discov Today 18(9-10):464-471, 2013). Nowadays, the best way to characterize an Ab bound region is still the resolution of Ab-antigen (Ag) co-crystal structure. Unfortunately, the crystallization approaches are not always feasible. However, different experimental strategies aimed to predict Ab-Ag interaction and followed by in silico analysis of the results may be good surrogate approaches to achieve this result. Here, we review few experimental techniques followed by the use of "basic" informatics tools for the analysis of the results.

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.