In this study, the chemical composition of the vapor and liquid phase of Pinus cembra L., Pinus mugo Turra, Picea abies L., and Abies Alba M. needles essential oils (EOs) was investigated by Headspace-Gas Chromatography/Mass Spectrometry (HS-GC/MS). In the examined EOs, a total of twenty-eight components were identified, most of which belong to the monoterpenes family. α-Pinene (16.6–44.0%), β-pinene (7.5–44.7%), limonene (9.5–32.5%), and γ-terpinene (0.3–19.7%) were the most abundant components of the liquid phase. Such major compounds were also detected in the vapor phase of all EOs, and α-pinene reached higher relative percentages than in the liquid phase. Then, both the liquid and vapor phases were evaluated in terms of antibacterial activity against three Gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, and Acinetobacter bohemicus) and two Gram-positive bacteria (Kocuria marina and Bacillus cereus) using a microwell dilution assay, disc diffusion assay, and vapor phase test. The lowest Minimum Inhibitory Concentration (MIC) (13.28 mg/mL) and Minimal Bactericidal Concentration (MBC) (26.56 mg/mL) values, which correspond to the highest antibacterial activities, were reported for P. abies EO against A. bohemicus and for A. alba EO against A. bohemicus and B. cereus. The vapor phase of all the tested EOs was more active than liquid phase, showing the inhibition halos from 41.00 ± 10.15 mm to 80.00 ± 0.00 mm for three bacterial strains (A. bohemicus, K. marina, and B. cereus). Furthermore, antioxidant activities were also investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays, and a concentration-dependent antioxidant capacity for all EOs was found. P. mugo EO showed the best antioxidant activity than the other Pinaceae EOs. The four Pinaceae EOs could be further investigated for their promising antibacterial and antioxidant properties, and, in particular, α-pinene seems to have interesting possibilities for use as a novel natural antibacterial agent.

Shigellosis is the leading cause of diarrheal disease, especially in children of low- and middle-income countries, and is often associated with anti-microbial resistance. Currently, there are no licensed vaccines widely available against Shigella, but several candidates based on the O-antigen (OAg) portion of lipopolysaccharides are in development. We have proposed Generalized Modules for Membrane Antigens (GMMA) as an innovative delivery system for OAg, and a quadrivalent vaccine candidate containing GMMA from S. sonnei and three prevalent S. flexneri serotypes (1b, 2a and 3a) is moving to a phase II clinical trial, with the aim to elicit broad protection against Shigella. GMMA are able to induce anti-OAg-specific functional IgG responses in animal models and healthy adults. We have previously demonstrated that antibodies against protein antigens are also generated upon immunization with S. sonnei GMMA. In this work, we show that a quadrivalent Shigella GMMA-based vaccine is able to promote a humoral response against OAg and proteins of all GMMA types contained in the investigational vaccine. Proteins contained in GMMA provide T cell help as GMMA elicit a stronger anti-OAg IgG response in wild type than in T cell-deficient mice. Additionally, we observed that only the trigger of Toll-like Receptor (TLR) 4 and not of TLR2 contributed to GMMA immunogenicity. In conclusion, when tested in mice, GMMA of a quadrivalent Shigella vaccine candidate combine both adjuvant and carrier activities which allow an increase in the low immunogenic properties of carbohydrate antigens.

Shigellosis is one of the leading causes of diarrheal disease in low- and middle-income countries, particularly in young children, and is more often associated with antimicrobial resistance. Therefore, a preventive vaccine against shigellosis is an urgent medical need. We have proposed Generalised Modules for Membrane Antigens (GMMA) as an innovative delivery system for Shigella sonnei O-antigen, and an Alhydrogel formulation (1790GAHB) has been extensively tested in preclinical and clinical studies. Alhydrogel has been used as an adsorbent agent with the main purpose of reducing potential GMMA systemic reactogenicity. However, the immunogenicity and systemic reactogenicity of this GMMA-based vaccine formulated with or without Alhydrogel have never been compared. In this work, we investigated the potential adjuvant effect of aluminium salt-based adjuvants (Alhydrogel and AS37) on S. sonnei GMMA immunogenicity in mice and rabbits, and we found that S. sonnei GMMA alone resulted to be strongly immunogenic. The addition of neither Alhydrogel nor AS37 improved the magnitude or the functionality of vaccine-elicited antibodies. Interestingly, rabbits injected with either S. sonnei GMMA adsorbed on Alhydrogel or S. sonnei GMMA alone showed a limited and transient body temperature increase, returning to baseline values within 24 h after each vaccination. Overall, immunisation with unadsorbed GMMA did not raise any concern for animal health. We believe that these data support the clinical testing of GMMA formulated without Alhydrogel, which would allow for further simplification of GMMA-based vaccine manufacturing.

Shigellosis represents a significant global health concern particularly affecting children under 5 years in low- and middle-income countries (LMICs) and is associated with stunting and antimicrobial resistance. There is a critical need for an effective vaccine offering broad protection against the different Shigella serotypes. A correlate of protection has not yet been established but there is a general consensus about the relevant role of anti-O-Antigen-specific IgG and its functionality evaluated by the Serum Bactericidal Assay (SBA). This study aims to characterize a high-throughput luminescence-based SBA (L-SBA) against seven widespread Shigella serotypes. The assay was previously developed and characterized for S. sonnei and S. flexneri 1b, 2a, and 3a and has now been refined and extended to an additional five serotypes (S. flexneri 4a, 5b, 6, X, and Y). The characterization of the assay with human sera confirmed the repeatability, intermediate precision, and linearity of the assays; both homologous and heterologous specificity were verified as well; finally, limit of detection and quantification were established for all assays. Moreover, different sources of baby rabbit complement showed to have no impact on L-SBA output. The results obtained confirm the possibility of extending the L-SBA to multiple Shigella serotypes, thus enabling analysis of the functional response induced by natural exposure to Shigella in epidemiological studies and the ability of candidate vaccines to elicit cross-functional antibodies able to kill a broad panel of prevalent Shigella serotypes in a complement-mediated fashion.