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

Endobronchial Ultrasound Transbronchial Needle Aspiration In Thoracic Diseases: Much More Than Mediastinal Staging

Background and Objective. EBUS-TBNA has revolutionized the diagnostic approach to thoracic diseases from a surgical to minimally invasive procedure. In non small-cell lung cancer (NCSLC) patients, EBUS-TBNA is able to dictate the consecutive therapy both for early and advanced stages, providing pathological diagnosis, mediastinal staging, and even adequate specimens for molecular analysis. This study reports on the ability of EBUS-TBNA to make different diagnoses and dictates the consecutive therapy in a large cohort of patients presenting different thoracic diseases. Methods. All procedures performed from January 2012 to September 2016 were reviewed. Five groups of patients were created according to the main indications for the procedure. Group 1: lung cancer staging; Group 2: pathological diagnosis in advanced stage lung cancer; Group 3: lymphadenopathy in previous malignancies; Group 4: pulmonary lesions; Group 5: unknown origin lymphadenopathy. In each group, the diagnostic yield of the procedure was analysed. Non malignant diagnosis at EBUS-TBNA was confirmed by a surgical procedure or clinical and radiological follow-up. Results. 1891 patients were included in the analysis. Sensitivity, negative predictive value, and diagnostic accuracy in each group were 90.7%, 79.4%, and 93.1% in Group 1; 98.5%, 50%, and 98.5% in Group 2; 92.4%, 85.1%, and 94.7% in Group 3; 90.9%, 51.0%, and 91.7% in Group 4; and 25%, 83.3%, and 84.2% in Group 5. Overall sensitivity, negative predictive value, and accuracy were 91.7%, 78.5%, and 93.6%, respectively. Conclusions. EBUS-TBNA is the best approach for invasive mediastinal investigation, confirming its strategic role and high accuracy in thoracic oncology

Proper Selection of In Vitro Cell Model Affects the Characterization of the Neutralizing Antibody Response against SARS-CoV-2

(1) Background: Our aim is the evaluation of the neutralizing activity of BNT162b2 mRNA vaccine-induced antibodies in different in vitro cellular models, as this still represents one of the surrogates of protection against SARS-CoV-2 viral variants. (2) Methods: The entry mechanisms of SARS-CoV-2 in three cell lines (Vero E6, Vero E6/TMPRSS2 and Calu-3) were evaluated with both pseudoviruses and whole virus particles. The neutralizing capability of sera collected from vaccinated subjects was characterized through cytopathic effects and Real-Time RT PCR. (3) Results: In contrast to Vero E6 and Vero E6/TMPRSS2, Calu-3 allowed the evaluation of both viral entry mechanisms, resembling what occurs during natural infection. The choice of an appropriate cellular model can decisively influence the determination of the neutralizing activity of antibodies against SARS-CoV-2 variants. Indeed, the lack of correlation between neutralizing data in Calu-3 and Vero E6 demonstrated that testing the antibody inhibitory activity by using a single cell model possibly results in an inaccurate characterization. (4) Conclusions: Cellular systems allowing only one of the two viral entry pathways may not fully reflect the neutralizing activity of vaccine-induced antibodies moving increasingly further away from possible correlates of protection from SARS-CoV-2 infection

Biochemical analysis of human ovarian cancer-associated antigens defined by murine monoclonal antibodies.

Two monoclonal antibodies (MOv1 and MOv2) raised against a membrane preparation of a human surgical specimen from a mucinous ovarian cystoadenocarcinoma were used to biochemically define their target antigens. The heating of peritumoral mucus-soluble extracts and the sialidase treatment of crude membrane preparations did not affect the binding capacity of MOv1 and MOv2, which, on the contrary, was significantly reduced by periodate oxidation of the same materials. Pronase digestion completely solubilized MOv1-defined antigens, whereas MOv2-defined antigens were only partially solubilized. This, however, did not affect antibody binding with digested products. These data suggest that carbohydrate residues of recognized molecules constitute the antigenic determinants and that sialic acid residues are not involved. Gel filtration on Sepharose 4B of the peritumoral mucus, solubilized either by 200 mM NaCl or Pronase, revealed that most of the antigenic activity eluted in the void-volume fractions with a high carbohydrate content and in the included fractions before the elution volume of the ferritin standard protein. When CsCl gradient equilibrium ultracentrifugation of the solubilized mucus was used, MOv1-recognized antigens sedimented with a density of 1.45 g/ml, while the MOv2-defined epitope was carried by molecules with a density of 1.52 g/ml as well as by molecules with a lower density. Using thin-layer chromatography of organic solvent extracts obtained from mucus and crude membrane preparations, only MOv2-positive molecules could be resolved as a single band of glycolipid. Altogether, these data suggest that the antigens detected by MOv1 are mainly mucins whereas the determinant recognized by MOv2 is carried by both mucins and a glycolipid. To analyze the diagnostic potential of MOv1- and MOv2-recognized molecules, we tested their presence, as soluble products, in supernatants of tumor cell lines and in peritoneal effusions from cancer patients. To this aim, we developed an immunoradiometric assay using the same monoclonal antibody in insolubilized and soluble form. Whereas MOv1-immunoradiometric assay was always negative, by MOv2-immunoradiometric assay it was possible to detect the relevant antigen in 8 of the 10 effusions from patients with well-differentiated ovarian tumors and in 5 of the 11 effusions from patients with poorly differentiated ovarian tumors, whereas the 10 control effusions from patients with various diseases were negative

In vivo protection conferrend by human monoclonal antibody directed against HSV-1 and HSV-2

BACKGROUND: The worldwide incidence of Herpes Simplex Virus (HSV) infections is becoming a significant threat due to the growing number of individuals that are mainly prone to develop severe HSV disease such as CNS involvement. This is the case of patients with an impaired immune system, as post-transplant patients, those infected by HIV or perinatal infected new-borns. In these patients, severe diseases can develop after therapeutic failure of the standard therapy consisting in first line drug Acyclovir (ACV) due to the presence of isolates not fully susceptible to ACV. Moreover the high toxicity of second line drugs often compromises their use in therapy. Finally, the presence of isolates resistant both to first and second line treatments completely abrogate the possibility to contain the infection. Given that, developing new antiviral molecules able to inhibit virus infection certainly represents a medical need. METHODS: A human monoclonal antibody endowed with strong neutralizing activity against HSV-1 and -2 previously characterised in vitro has been tested in C57BL/6 mice for its activity against HSV-1 and HSV-2 infections. More in details, HSV-2 strain MS and HSV-1 strain LV have been used to perform both vaginal and ocular challenges. The antibody has been administered both topically and systemically, and has been tested both for prophylaxis and therapy. RESULTS: The antibody proved to be extremely protective against HSV-1 and HSV-2 infections when administered systemically. Importantly, its antiviral activity has been observed both therapeutically and prophylactically even with a single boost administration of the mAb. CONCLUSION: These experiments showed that the human mAb able to recognise and neutralise both HSV-1 and 2 is able to protect animals challenged with lethal doses of both HSV types opening new perspectives to its possible clinical use

Molecular characterization of the human neutralizing response against hepatitis C virus and its role in the prediction of the infection outcome

The hepatitis C virus (HCV) adopts several escape mechanisms and is able to evade the host immune response in the majority of patients. During primary infection, HCV is not cleared in 80% of cases resulting in chronic infection. The current treatment for HCV infection is mainly represented by the administration of a combined therapy (IFN-α, ribavirin) and by the use of new anti-viral drugs (protease inhibitors). Unfortunately, only 50% of the infected patients respond completely to these therapies. It has been demonstrated how a neutralizing antibody response is correlated with lower HCV titer in acute infection. Moreover, it is also demonstrated how a rapid induction of neutralizing antibodies can be correlated with the viral clearance. Under these purposes, results clear how neutralizing antibodies can be important for the HCV infection control. In addition, they can represent good candidates for passive immunotherapy. They also can be applied both in diagnosis, as useful tools for the evaluation of the presence of cross-neutralizing antibodies in patients sera, and in research studies to better understand the virus–host interplay, an aspect that can be crucial in predicting the infection clinical outcome. In this study, we characterized the synergistic neutralization of HCV by two broadly neutralizing human monoclonal antibodies directed against HCV/E2 glycoprotein, named e20 and e137