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Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have emerged as a novel and flexible vaccine platform. OMVs can be decorated with foreign antigens and carry potent immunostimulatory components. Therefore, after their purification from the culture supernatant, they are ready to be formulated for vaccine use. It has been extensively demonstrated that immunization with engineered OMVs can elicit excellent antibody responses against the heterologous antigens. However, the definition of the conditions necessary to reach the optimal antibody titers still needs to be investigated. Here, we defined the protein concentrations required to induce antigen-specific antibodies, and the amount of antigen and OMVs necessary and sufficient to elicit saturating levels of antigen-specific antibodies. Since not all antigens can be expressed in OMVs, we also investigated the effectiveness of vaccines in which OMVs and purified antigens are mixed together without using any procedure for their physical association. Our data show that in most of the cases OMV–antigen mixtures are very effective in eliciting antigen-specific antibodies. This is probably due to the capacity of OMVs to “absorb” antigens, establishing sufficiently stable interactions that allow antigen–OMV co-presentation to the same antigen presenting cell. In those cases when antigen–OMV interaction is not sufficiently stable, the addition of alum to the formulation guarantees the elicitation of high titers of antigen-specific antibodies.

Human papillomaviruses (HPVs) are a large family of viruses with a capsid composed of the L1 and L2 proteins, which bind to receptors of the basal epithelial cells and promote virus entry. The majority of sexually active people become exposed to HPV and the virus is the most common cause of cervical cancer. Vaccines are available based on the L1 protein, which self-assembles and forms virus-like particles (VLPs) when expressed in yeast and insect cells. Although very effective, these vaccines are HPV type-restricted and their costs limit broad vaccination campaigns. Recently, vaccine candidates based on the conserved L2 epitope from serotypes 16, 18, 31, 33, 35, 6, 51, and 59 were shown to elicit broadly neutralizing anti-HPV antibodies. In this study, we tested whether E. coli outer membrane vesicles (OMVs) could be successfully decorated with L2 polytopes and whether the engineered OMVs could induce neutralizing antibodies. OMVs represent an attractive vaccine platform owing to their intrinsic adjuvanticity and their low production costs. We show that strings of L2 epitopes could be efficiently expressed on the surface of the OMVs and a polypeptide composed of the L2 epitopes from serotypes 18, 33, 35, and 59 provided a broad cross-protective activity against a large panel of HPV serotypes as determined using pseudovirus neutralization assay. Considering the simplicity of the OMV production process, our work provides a highly effective and inexpensive solution to produce universal anti-HPV vaccines.

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespective of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccines to induce potent immune responses against the associated proteins. Here, we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in vaccinated mice, resulting in the production of neutralizing antibodies (nAbs) with a titre higher than 1:300. The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induce nAbs against Omicron BA.1 and BA.5, as measured using the pseudovirus neutralization infectivity assay. Importantly, we show that the RBM438–509 ancestral-OMVs elicited antibodies which efficiently neutralize in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants with a neutralization titre ranging from 1:100 to 1:1500, suggesting its potential use as a vaccine targeting diverse SARS-CoV-2 variants. Altogether, given the convenience associated with the ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

In situ vaccination (ISV) is a promising cancer immunotherapy strategy that consists of the intratumoral administration of immunostimulatory molecules (adjuvants). The rationale is that tumor antigens are abundant at the tumor site, and therefore, to elicit an effective anti-tumor immune response, all that is needed is an adjuvant, which can turn the immunosuppressive environment into an immunologically active one. Bacterial outer membrane vesicles (OMVs) are potent adjuvants since they contain several microbe-associated molecular patterns (MAMPs) naturally present in the outer membrane and in the periplasmic space of Gram-negative bacteria. Therefore, they appear particularly indicted for ISV. In this work, we first show that the OMVs from E. coli BL21(DE3)Δ60 strain promote a strong anti-tumor activity when intratumorally injected into the tumors of three different mouse models. Tumor inhibition correlates with a rapid infiltration of DCs and NK cells. We also show that the addition of neo-epitopes to OMVs synergizes with the vesicle adjuvanticity, as judged by a two-tumor mouse model. Overall, our data support the use of the OMVs in ISV and indicate that ISV efficacy can benefit from the addition of properly selected tumor-specific neo-antigens.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have emerged as a novel and flexible vaccine platform. OMVs can be decorated with foreign antigens and carry potent immunostimulatory components. Therefore, after their purification from the culture supernatant, they are ready to be formulated for vaccine use. It has been extensively demonstrated that immunization with engineered OMVs can elicit excellent antibody responses against the heterologous antigens. However, the definition of the conditions necessary to reach the optimal antibody titers still needs to be investigated. Here, we defined the protein concentrations required to induce antigen-specific antibodies, and the amount of antigen and OMVs necessary and sufficient to elicit saturating levels of antigen-specific antibodies. Since not all antigens can be expressed in OMVs, we also investigated the effectiveness of vaccines in which OMVs and purified antigens are mixed together without using any procedure for their physical association. Our data show that in most of the cases OMV–antigen mixtures are very effective in eliciting antigen-specific antibodies. This is probably due to the capacity of OMVs to “absorb” antigens, establishing sufficiently stable interactions that allow antigen–OMV co-presentation to the same antigen presenting cell. In those cases when antigen–OMV interaction is not sufficiently stable, the addition of alum to the formulation guarantees the elicitation of high titers of antigen-specific antibodies.

In situ vaccination (ISV) is a promising cancer immunotherapy strategy, consists in the intratumoral administration of immunostimulatory molecules (adjuvants). The rationale is that tumor antigens are abundant at the tumor site and therefore to elicit an effective anti-tumor immune response all is needed is an adjuvant, which can turn the immunosuppressive environment into an immunologically active one. Bacterial Outer Membrane Vesicles (OMVs) are potent adjuvants since they contain a number of microbe-associated molecular patterns (MAMPs) naturally present in the outer membrane and in the periplasmic space of Gram-negative bacteria. Therefore, they appear particularly indicted for ISV. In this work we first show that the OMVs from E. coli BL21(DE3)Δ60 strain promote a strong anti-tumor activity when intratumorally injected into the tumors of two different mouse models. Tumor inhibition correlates with a rapid infiltration of DCs and NK cells. We also show that the addition of neo-epitopes to OMVs synergizes with the vesicle adjuvanticity, as judged by a two-tumor mouse models. Overall, our data support the use of the OMVs in ISV and suggests that ISV efficacy could benefit from the addition of properly selected tumor-specific neo-antigens.

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in immunized mice, resulting in the production of neutralizing antibodies. The immunity induced by the vaccine is sufficient to protect K18-hACE2 transgenic mice from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with RBM peptides derived from a different genetic variant of SARS-CoV-2, inducing a similarly potent neutralization activity in vaccinated mice. Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

Ancient undeciphered scripts present problems of different nature, not just tied to linguistic identification. The undeciphered Cypro-Minoan script from second millennium BCE Cyprus, for instance, currently does not have a standardized, definitive inventory of signs, and, in addition, stands divided into three separate subgroups (CM1, CM2, CM3), which have also been alleged to record different languages. However, this state of the art is not consensually accepted by the experts. In this article, we aim to apply a method that can aid to shed light on the tripartite division, to assess if it holds up against a multi-pronged, multi-disciplinary approach. This involves considerations linked to paleography (shapes of individual signs) and epigraphy (writing style tied to the support used), and crucially, deep learning-based strategies. These automatic methods, which are widely adopted in many fields such as computer vision and computational linguistics, allow us to look from an innovative perspective at the specific issues presented by ancient, poorly understood scripts in general, and Cypro-Minoan in particular. The usage of a state-of-the-art convolutional neural model that is unsupervised, and therefore does not use any prior knowledge of the script, is still underrepresented in the study of undeciphered writing systems, and helps to investigate the tripartite division from a fresh standpoint. The conclusions we reached show that: 1. the use of different media skews to a large extent the uniformity of the sign shapes; 2. the application of several neural techniques confirm this, since they highlight graphic proximity among signs inscribed on similar supports; 3. multi-stranded approaches prove to be a successful tool to investigate ancient scripts whose language is still unidentified. More crucially, these aspects, together, point in the same direction, namely the validation of a unitary, single Cypro-Minoan script, rather than the current division into three subgroups.

In this work, a flexible and extensive digital platform for Smart Homes is presented, exploiting the most advanced technologies of the Internet of Things, such as Radio Frequency Identification, wearable electronics, Wireless Sensor Networks, and Artificial Intelligence. Thus, the main novelty of the paper is the system-level description of the platform flexibility allowing the interoperability of different smart devices. This research was developed within the framework of the operative project HABITAT (Home Assistance Based on the Internet of Things for the Autonomy of Everybody), aiming at developing smart devices to support elderly people both in their own houses and in retirement homes, and embedding them in everyday life objects, thus reducing the expenses for healthcare due to the lower need for personal assistance, and providing a better life quality to the elderly users.