Martina Monti

Among the common strategies to design next-generation COVID-19 vaccines is broadening the antigenic repertoire thereby aiming to increase efficacy against emerging variants of concern (VoC). This study describes a new Orf virus-based vector (ORFV) platform to design a multiantigenic vaccine targeting SARS-CoV-2 spike and nucleocapsid antigens. Vaccine candidates were engineered, either expressing spike protein (ORFV-S) alone or co-expressing nucleocapsid protein (ORFV-S/N). Mono- and multiantigenic vaccines elicited comparable levels of spike-specific antibodies and virus neutralization in mice. Results from a SARS-CoV-2 challenge model in hamsters suggest cross-protective properties of the multiantigenic vaccine against VoC, indicating improved viral clearance with ORFV-S/N, as compared to equal doses of ORFV-S. In a nonhuman primate challenge model, vaccination with the ORFV-S/N vaccine resulted in long-term protection against SARS-CoV-2 infection. These results demonstrate the potential of the ORFV platform for prophylactic vaccination and represent a preclinical development program supporting first-in-man studies with the multiantigenic ORFV vaccine.

The next generation of COVID-19 vaccines needs to broaden the antigenic repertoire to improve breadth of immune response and efficacy against emerging variants of concern. This study describes a new parapoxvirus-based vector (ORFV) as a platform to design a multi-antigenic vaccine targeting SARS-CoV-2 spike and nucleocapsid antigens. Two vaccine candidates were engineered, one expressing spike protein alone (ORFV-S) and the other co-expressing the more conserved nucleocapsid protein (ORFV-S/N). Both vaccines elicited comparable levels of spike-specific antibodies and virus neutralization in mice. In a SARS-CoV-2 challenge model in hamsters, the multi-antigenic ORFV-S/N vaccine conferred protection in the upper and lower respiratory tract, while the ORFV-S-vaccinated animals showed protection restricted to the lungs. Similarly, in a non-human primates challenge model, vaccination with the ORFV-S/N vaccine resulted in rapid onset and long-term protection against SARS-CoV-2 infection. These results demonstrate the potential of ORFV as a platform for prophylactic vaccination and support ongoing first-in-man studies with the multi-antigenic ORFV vaccine.

Background: Vaccine effectiveness relies on various serological tests, whose aim is the measurement of antibody titer in serum samples collected during clinical trials before and after vaccination. Among the serological assays required by the regulatory authorities to grant influenza vaccine release there are: Hemagglutination inhibition (HAI), microneutralization (MN), and Single Radial Hemolysis (SRH). Although antibodies are regarded to be relatively stable, limited evidences on the effect of multiple freeze–thaw cycles on the stability of antibodies in frozen serum samples are available so far. In view of this, the present paper aimed to evaluate the impact of multiple freeze–thaw cycles on influenza antibody stability, performing HAI, MN and SRH assays. Methods: Ten serum samples were divided into 14 aliquots each, stored at −20 °C and taken through a total of 14 freeze–thaw cycles to assess influenza antibody stability. Each assay measurement was carried out following internal procedures based on World Health Organization (WHO) guidelines. Results: No statistically significant effect of 14 freeze–thaw cycles on antibody stability, measured through three different assays, was observed. Conclusions: Collectively, these data demonstrated that specific influenza antibody present in serum samples are stable up to 14 freeze–thaw cycles.

Recent studies have suggested that the CCR5 antagonist maraviroc (MVC) may exert an HIV-1 latency reversal effect. This study aimed at defining MVC-mediated induction of HIV-1 in three cell line latency models and inex vivoCD4 T cells from six patients with suppressed viraemia. HIV-1 induction was evaluated in TZM-bl cells by measuring HIV-1 LTR-driven luciferase expression, and in ACH-2 and U1 latently infected cell lines by measuring cell-free (CFR) and cell-associated (CAR) HIV-1 RNA by qPCR. NF-κB p65 was quantified in nuclear extracts by immunodetection. Inex vivoCD4 T cells, CAR, CFR and cell-associated DNA (CAD) were quantified at baseline and 1–7–14 days post-induction (T1, T7, T14). At T7 and T14, the infectivity of the CD4 T cells co-cultured with MOLT-4/CCR5 target cells was evaluated in the TZM-bl assay (TZA). Results were expressed as fold activation (FA) with respect to untreated cells. No LTR activation was observed in TZM-bl cells at any MVC concentration. NF-κB activation was only modestly upregulated (1.6±0.4) in TZM-bl cells with 5 µM MVC. Significant FA of HIV-1 expression was only detected at 80 µM MVC, namely on HIV-1 CFR in U1 (3.1±0.9;P=0.034) and ACH-2 cells (3.9±1.4;P=0.037). CFR was only weakly stimulated at 20 µM in ACH-2 (1.7±1.0 FA) cells and at 5 µM in U1 cells (1.9±0.5 FA). Although no consistent pattern of MVC-mediated activation was observed inex vivoexperiments, substantial FA values were detected sparsely on individual samples with different parameters. Notably, in one sample, MVC stimulated all parameters at T7 (2.3±0.2 CAD, 6.8±3.7 CAR, 18.7±16.7 CFR, 7.3±0.2 TZA). In conclusion, MVC variably induces HIV-1 production in some cell line models not previously used to test its latency reversal potential. Inex vivoCD4 T cells, MVC may exert patient-specific HIV-1 induction; however, clinically relevant patterns, if any, remain to be defined.

In the absence of effective drugs or vaccines for the treatment of the five Dengue Virus serotypes, the search for novel antiviral drugs is of primary importance for the scientific community. In this context, drug repurposing represents the most used strategy; however, the study of host targets is now attracting attention since it allows identification of broad-spectrum drugs endowed with high genetic barrier. In the last ten years our research group identified several small molecules DDX3X inhibitors and proved their efficacy against different viruses including novel emerging ones. Herein, starting from a screening of our compounds, we designed and synthesized novel derivatives with potent activity and high selectivity. Finally, we synthesized a fluorescent inhibitor that allowed us to study DDX3X cellular localization during DENV infection in vitro. Immunofluorescence analysis showed that our inhibitor colocalized with DDX3X, promoting the reduction of infected cells and recovering the number of viable cells.

Measles in pregnancy may lead to serious sequelae for newborns and mothers. In Italy assessment of immunity against measles is not recommended as pregnancy screening. This study aimed to assess the immunity against measles in pregnant women from Apulia Region in Southern Italy between 2016 and 2017. Overall, 96.9% of pregnant women were positive for anti-measles IgG, younger women aged 19–29 years had a seroprevalence below 90%. No samples were positive for anti-measles IgM. In conclusion, younger pregnant women showed to be at higher risk of contracting measles during pregnancy. These findings have implication for measles vaccination policy and highlight the need for measles antibody testing in pregnancy screening in Italy.

Recently, hydrogen sulfide (H₂S) has been characterized as an endogenous mediator able to control a series of cellular and tissue functions relevant for tissue homeostasis and repair such as angiogenesis. This chapter describes the tools and their use in a set of angiogenesis assays performed by using cultured endothelial cells in order to study the relevance of exogenous or endogenous H₂S production and release during the occurrence of angiogenesis.

The metal-nonoate Ni(SalPipNONO) inhibits in vitro tumor growth, invasiveness and angiogenesis The metal-nonoate Ni(SalPipNONO) inhibits in vitro tumor growth, invasiveness and angiogenesis