Search Results (28)

Vs30, the average shear wave velocity in the uppermost 30 m, is a critical parameter in seismic hazard analysis. In the Philippines, the Refraction Microtremor (ReMi) survey is the standard method for Vs30 Estimation. This study evaluates the efficiency of using an elitist Genetic Algorithm (GA) to invert Horizontal-to-Vertical Spectral Ratio (HVSR) data as an alternative approach. Unlike ReMi surveys, which require geophone arrays, HVSR surveys use a single-unit three-component microtremor seismograph, enabling faster and broader data collection. Analysis of 174 HVSR and 52 ReMi datasets from the Greater Metro Manila Area (GMMA) and Leyte Province revealed strong correlations between estimated and measured Vs30 values. The overall match rates for soil profile classification under the National Structural Code of the Philippines (NSCP 2015) were 76% in GMMA and 81% in Leyte, with R-squared values of 0.885 and 0.806, respectively. Additionally, the relationship between the fundamental site period and estimated Vs30 values was explored. The R-squared values of 0.772 for GMMA and 0.707 for Leyte indicate a strong correlation and demonstrate the expected inverse relationship between the two variables. Given the Philippines’ high seismic activity, this method provides an efficient means to enhance seismic hazard mapping, improving earthquake preparedness and mitigation. Full article

Background: An emerging trend of mutual convergence between drug-resistant and highly virulent strains of K. pneumoniae has been identified, highlighting the urgent need for the development of novel vaccines. Methods: To delete the target genes and eliminate the plasmids carrying antibiotic resistance genes, CRISPR-Cas9 technology was employed to perform genome editing on a clinically isolated O2 serotype of K. pneumoniae. Subsequently, this strain was utilized as a host to express genes associated with the synthesis of O1 serotype LPSs to construct the recombinant strain capable of simultaneously expressing LPSs of both O1 and O2 serotypes. This recombinant strain was then used as the production strain for the preparation of vaccines based on GMMAs (Generalized Modules for Membrane Antigens), and its biological characteristics were characterized. Finally, the safety and immunogenicity of the vaccine were evaluated using mice as the model animals. Result: a GMMA vaccine characterized by a high yield and low toxicity was gained. Importantly, the lipopolysaccharides (LPSs) of both O1 and O2 serotypes of K. pneumoniae were successfully expressed on the surface of the outer membrane vesicles. Following immunization with the GMMA vaccine, mice were capable of producing antibodies against the GMMA and demonstrated resistance to the invasion of both serotypes of clinically isolated K. pneumoniae. Conclusions: The GMMA vaccine showed significant promise as a bivalent vaccine against K. pneumoniae. Full article

The most widely known pyrogen impurity in vaccines is the Gram-negative bacterial endotoxin lipopolysaccharide (LPS). When administered at toxic doses, endotoxin triggers inflammatory responses, which lead to endotoxic shock. The literature on endotoxic content (EC) for preclinical vaccines’ formulations used in animal studies is very poor, and the recommended thresholds are solely based on commercial vaccine limits set for humans and are, therefore, not connected to the actual impact of EC on animal welfare for species used in preclinical research studies. An extensive study to evaluate the presence of a potential relationship between endotoxin content in formulations administered to mice (the most common species used in preclinical research studies) and their welfare was conducted to calculate an EC threshold for formulations of candidate vaccines. Three years of historical data, from more than 500 formulations of different antigen types (i.e., proteins, glycoconjugates, OMV/GMMA) injected into more than 5000 mice, was evaluated with two alternative statistical methodologies, both demonstrating that there is no significant relationship between actual endotoxin levels and mouse welfare. The calculation of thresholds was, therefore, performed by consistency versus formulations that demonstrated no impact on animal welfare. Full article

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. Full article

Shigellosis, an acute gastroenteritis infection caused by Shigella species, remains a public health burden in developing countries. Recently, many outbreaks due to Shigella sonnei multidrug-resistant strains have been reported in high-income countries, and the lack of an effective vaccine represents a major hurdle to counteract this bacterial pathogen. Vaccine candidates against Shigella sonnei are under clinical development, including a Generalized Modules for Membrane Antigens (GMMA)-based vaccine. The mechanisms by which GMMA-based vaccines interact and activate human immune cells remain elusive. Our previous study provided the first evidence that both adaptive and innate immune cells are targeted and functionally shaped by the GMMA-based vaccine. Here, flow cytometry and confocal microscopy analysis allowed us to identify monocytes as the main target population interacting with the S. sonnei 1790-GMMA vaccine on human peripheral blood. In addition, transcriptomic analysis of this cell population revealed a molecular signature induced by 1790-GMMA mostly correlated with the inflammatory response and cytokine-induced processes. This also impacts the expression of genes associated with macrophages’ differentiation and T cell regulation, suggesting a dual function for this vaccine platform both as an antigen carrier and as a regulator of immune cell activation and differentiation. Full article

Nontyphoidal Salmonella (NTS) is a leading cause of morbidity and mortality caused by enteric pathogens worldwide in both children and adults, and vaccines are not yet available. The measurement of antigen-specific antibodies in the sera of vaccinated or convalescent individuals is crucial to understand the incidence of disease and the immunogenicity of vaccine candidates. A solid and standardized assay used to determine the level of specific anti-antigens IgG is therefore of paramount importance. In this work, we presented the characterization of a customized enzyme-linked immunosorbent assay (ELISA) with continuous readouts and a standardized definition of EU/mL. We assessed various performance parameters: standard curve accuracy, dilutional linearity, intermediate precision, specificity, limits of blanks, and quantification. The simplicity of the assay, its high sensitivity and specificity coupled with its low cost and the use of basic consumables and instruments without the need of high automation makes it suitable for transfer and application to different laboratories, including resource-limiting settings where the disease is endemic. This ELISA is, therefore, fit for purpose to be used for quantification of antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-antigens in human samples, both for vaccine clinical trials and large sero-epidemiological studies. Full article

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria that can be used to design affordable subunit vaccines. GMMA have been observed to induce a potent humoral immune response in preclinical and clinical studies. In addition, in preclinical studies, it has been found that GMMA can be exploited as optimal antigen carriers for both protein and saccharide antigens, as they are able to promote the enhancement of the antigen-specific humoral immune response when the antigen is overexpressed or chemically conjugated to GMMA. Here we investigated the mechanism of this GMMA carrier effect by immunizing mice and using factor H binding protein and GMMA of Neisseria meningitidis B as an antigen–GMMA model. We confirmed that the antigen displayed on the GMMA surface increased the antigen-specific IgG production and, above all, the antibody functionality measured by the serum bactericidal activity. We found that the enhancement of the bactericidal capacity induced by GMMA carrying the antigen on the surface was associated with the increase in antibody affinity to the antigen, and with the switching toward IgG subclasses with more bactericidal potential. Thus, we conclude that the potent carrier effect of GMMA is due to their ability to promote a better quality of humoral immunity. Full article

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. Full article

Salmonella Typhimurium and Salmonella Enteritidis are leading causative agents of invasive nontyphoidal Salmonella (iNTS) disease, which represents one of the major causes of death and morbidity in sub-Saharan Africa, still partially underestimated. Large sero-epidemiological studies are necessary to unravel the burden of disease and guide the introduction of vaccines that are not yet available. Even if no correlate of protection has been determined so far for iNTS, the evaluation of complement-mediated functionality of antibodies generated towards natural infection or elicited upon vaccination may represent a big step towards this achievement. Here we present the setup and the intra-laboratory characterization in terms of repeatability, intermediate precision, linearity, and specificity of a high-throughput luminescence-based serum bactericidal assay (L-SBA). This method could be useful to perform sero-epidemiological studies across iNTS endemic countries and for evaluation of antibodies raised against iNTS vaccine candidates in upcoming clinical trials. Full article

The gamma mass (µ_m) and linear (µ) attenuation coefficients of polycarbonate-bismuth oxide composites (PC-Bi₂O₃) with different bismuth oxide weight factors were investigated theoretically using EpiXS and a Monte Carlo simulation-based toolkit and Geant4 within an energy range between 0.1 and 2 MeV. The wide energy ranges of gamma rays and neutrons were chosen to cover as many applications as possible. The attenuation coefficients were then used to compute the half-value layers. The effective atomic numbers and effective electron densities of the studied samples obtained by EpiXS were compared as well. In order to further evaluate the shielding effectiveness of the studied samples, the thicknesses of all the investigated samples equivalent to 0.5 mm lead at a gamma energy of 511 keV were compared using a Geant4 code simulating a female numerical phantom with a gamma source placed facing the chest and a cylinder-shaped shield wrapped around the trunk area. The fast neutron removal cross sections of the investigated samples were studied to evaluate the effect of the weight factor of nanocomposites on the neutron shielding capabilities of the polymer as well. Full article

Shigellosis represents a major public health problem worldwide. The morbidity of the disease, especially in children in developing countries, together with the increase of antimicrobial resistance make a vaccine against Shigella an urgent medical need. Several vaccines under development are targeting Shigella lipopolysaccharide (LPS), whose extreme diversity renders necessary the development of multivalent vaccines. Immunity against Shigella LPS can elicit antibodies capable of killing bacteria in a serotype-specific manner. Therefore, although a correlation of protection against shigellosis has not been established, demonstration of vaccine-elicited antibody bactericidal activity may provide one means of vaccine protection against Shigella. To facilitate Shigella vaccine development, we have set up a high-throughput serum bactericidal assay based on luminescence readout (L-SBA), which has been already used to determine the functionality of antibodies against S. sonnei in multiple clinical trials. Here we present the setup and intra-laboratory characterization of L-SBA against three epidemiologically relevant Shigella flexneri serotypes using human sera. We assessed the linearity, repeatability and reproducibility of the method, demonstrating high assay specificity to detect the activity of antibodies against each homologous strain without any heterologous aspecificity against species-related and non-species-related strains; this assay is ready to be used to determine bactericidal activity of clinical sera raised by multivalent vaccines and in sero-epidemiological studies. Full article

GMMA are outer membrane vesicles (OMVs) released from Gram-negative bacteria genetically modified to enhance OMVs formation that have been shown to be optimal systems to enhance immunogenicity of protein antigens. Here, we selected Neisseria meningitidis factor H binding protein (fHbp) and used the conjugation chemistry as a tool to alter antigen orientation on GMMA. Indeed, fHbp was randomly linked to GMMA or selectively attached via the N-terminus to mimic native presentation of the protein on the bacterial surface. Interestingly, protein and peptide array analyses confirmed that antibodies induced by the selective and the random conjugates showed a pattern very similar to fHbp natively expressed on bacterial surfaces or to the recombinant protein mixed with GMMA, respectively. However, the two conjugates elicited antibodies with similar serum bactericidal activity against meningococcal strains, superior to the protein alone or physically mixed with GMMA. Presentation of fHbp on GMMA strongly enhances the functional immune response elicited by the protein but its orientation on the bacterial surface does not have an impact. This study demonstrates the flexibility of the GMMA platform as a display and delivery system for enhancing antigen immunogenicity and further supports the use of such promising technology for the development of effective vaccines. Full article

Group A Streptococcus (GAS) causes about 500,000 annual deaths globally, and no vaccines are currently available. The Group A Carbohydrate (GAC), conserved across all GAS serotypes, conjugated to an appropriate carrier protein, represents a promising vaccine candidate. Here, we explored the possibility to use Generalized Modules for Membrane Antigens (GMMA) as an alternative carrier system for GAC, exploiting their intrinsic adjuvant properties. Immunogenicity of GAC-GMMA conjugate was evaluated in different animal species in comparison to GAC-CRM₁₉₇; and the two conjugates were also compared from a techno-economic point of view. GMMA proved to be a good alternative carrier for GAC, resulting in a higher immune response compared to CRM₁₉₇ in different mice strains, as verified by ELISA and FACS analyses. Differently from CRM₁₉₇, GMMA induced significant levels of anti-GAC IgG titers in mice also in the absence of Alhydrogel. In rabbits, a difference in the immune response could not be appreciated; however, antibodies from GAC-GMMA-immunized animals showed higher affinity toward purified GAC antigen compared to those elicited by GAC-CRM₁₉₇. In addition, the GAC-GMMA production process proved to be more cost-effective, making this conjugate particularly attractive for low- and middle-income countries, where this pathogen has a huge burden. Full article

Shigellosis remains a major public health problem around the world; it is one of the leading causes of diarrhoeal disease in low- and middle-income countries, particularly in young children. The increasing reports of Shigella cases associated with anti-microbial resistance are an additional element of concern. Currently, there are no licensed vaccines widely available against Shigella, but several vaccine candidates are in development. It has been demonstrated that the incidence of disease decreases following a prior Shigella infection and that serum and mucosal antibody responses are predominantly directed against the serotype-specific Shigella O-antigen portion of lipopolysaccharide membrane molecules. Many Shigella vaccine candidates are indeed O-antigen-based. Here we present the journey towards the development of a potential low-cost four-component Shigella vaccine, eliciting broad protection against the most prevalent Shigella serotypes, that makes use of the GMMA (Generalized Modules for Membrane Antigens) technology, a novel platform based on bacterial outer membranes for delivery of the O-antigen to the immune system. Full article

Shigellosis is a diarrheal disease caused prevalently by Shigella flexneri and S. sonnei and representing a major global health risk, particularly in developing countries. Bacterial O-antigen (OAg) is the primary target of the host immune response and modifications of its oligosaccharide units, including O-acetylation, are responsible for the variability among the circulating S. flexneri serotypes. No vaccines are widely available against shigellosis and the understanding of the immunogenicity induced by the OAg is fundamental for the design of a vaccine that could cover the most prevalent Shigella serotypes. To understand whether a different O-acetylation pattern could influence the immune response elicited by S. flexneri OAg, we employed as a vaccine technology GMMA purified from S. flexneri 2a and 1b strains that were easily engineered to obtain differently O-acetylated OAg. Resulting GMMA were tested in mice, demonstrating not only no major impact of O-acetyl decorations on the immune response elicited by the two OAg against the homologous strains, but also that the O-acetylation of the Rhamnose III residue (O-factor 9), shared among serotypes 1b, 2a and 6, does not induce cross-reactive antibodies against these serotypes. This work contributes to the optimization of vaccine design against Shigella, providing indication about the ability of shared epitopes to elicit broad protection against S. flexneri serotypes and supporting the identification of critical quality attributes of OAg-based vaccines. Full article