Evaluation of Volumetric Absorptive Microsampling and Mass Spectrometry Data-Independent Acquisition of Hemoglobin-Related Clinical Markers.

Data-independent acquisition (DIA) allows comprehensive proteome coverage, while it also potentially works as a unified protocol to determine a multitude of proteins found in blood. Because of its high specificity, mass spectrometry may greatly reduce the interference observed in other assays to evaluate blood markers. Here, we combined DIA with volumetric absorptive microsampling (VAMS) and automated proteomics sample processing in a platform to assess clinical markers. As a proof of concept, we evaluated two hemoglobin-related biomarkers: the glycated hemoglobin (HbA1c) and hemoglobin (Hb) variants. HbA1c by DIA showed good correlation with the reference method, but method imprecision did not meet the quality requirement for this biomarker. We developed a strategy to identify Hb variants based on a customized database combined with a workflow for DIA data extraction and rigorous peptide evaluation. Data are available via ProteomeXchange with identifier PXD029918.

Reverse Vaccinology: An Approach for Identifying Leptospiral Vaccine Candidates.

Leptospirosis is a major public health problem with an incidence of over one million human cases each year. It is a globally distributed, zoonotic disease and is associated with significant economic losses in farm animals. Leptospirosis is caused by pathogenic Leptospira spp. that can infect a wide range of domestic and wild animals. Given the inability to control the cycle of transmission among animals and humans, there is an urgent demand for a new vaccine. Inactivated whole-cell vaccines (bacterins) are routinely used in livestock and domestic animals, however, protection is serovar-restricted and short-term only. To overcome these limitations, efforts have focused on the development of recombinant vaccines, with partial success. Reverse vaccinology (RV) has been successfully applied to many infectious diseases. A growing number of leptospiral genome sequences are now available in public databases, providing an opportunity to search for prospective vaccine antigens using RV. Several promising leptospiral antigens were identified using this approach, although only a few have been characterized and evaluated in animal models. In this review, we summarize the use of RV for leptospirosis and discuss the need for potential improvements for the successful development of a new vaccine towards reducing the burden of human and animal leptospirosis.

Immune response in hamsters immunised with a recombinant fragment of LigA from Leptospira interrogans, associated with carrier molecules.

Immunisation with the C-terminal region of leptospiral immunoglobulin-like A protein (LigANI) has shown promising results against leptospirosis. We evaluated the humoral immune response and protection induced by LigANI associated with carboxyl multi-walled carbon nanotubes (COOH-MWCNTs), CpG oligodeoxynucleotides (CpG ODNs), or Alhydrogel. Animals immunised with CpG ODNs were unable to develop a humoral immune response, whereas immunisation with LigANI and COOH-MWCNTs produced a high level of IgG antibodies, similar to that with LigANI and Alhydrogel, but it was not protective. The use of carbon nanotubes as an adjuvant in subunit vaccines against leptospirosis is a novel approach for improving specific IgG production.

DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis.

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the Leptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.

The use of halloysite clay and carboxyl-functionalised multi-walled carbon nanotubes for recombinant LipL32 antigen delivery enhanced the IgG response.

We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.

Mass Spectrometry Multiplexed Detection of SARS-CoV-2.

Testing of large populations for virus infection is now a reality worldwide due to the coronavirus (SARS-CoV-2) pandemic. The demand for SARS-CoV-2 testing using alternatives other than PCR led to the development of mass spectrometry (MS)-based assays. However, MS for SARS-CoV-2 large-scale testing have some downsides, including complex sample preparation and slow data analysis. Here, we describe a high-throughput targeted proteomics method to detect SARS-CoV-2 directly from nasopharyngeal and oropharyngeal swabs. This strategy employs fully automated sample preparation mediated by magnetic particles, followed by detection of SARS-CoV-2 nucleoprotein peptides by turbulent flow chromatography coupled with tandem mass spectrometry.

Establishing a mass spectrometry-based system for rapid detection of SARS-CoV-2 in large clinical sample cohorts.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Here, we develop a high-throughput targeted proteomics assay to detect SARS-CoV-2 nucleoprotein peptides directly from nasopharyngeal and oropharyngeal swabs. A modified magnetic particle-based proteomics approach implemented on a robotic liquid handler enables fully automated preparation of 96 samples within 4 hours. A TFC-MS system allows multiplexed analysis of 4 samples within 10 min, enabling the processing of more than 500 samples per day. We validate this method qualitatively (Tier 3) and quantitatively (Tier 1) using 985 specimens previously analyzed by real-time RT-PCR, and detect up to 84% of the positive cases with up to 97% specificity. The presented strategy has high sample stability and should be considered as an option for SARS-CoV-2 testing in large populations.

LemA and Erp Y-like recombinant proteins from Leptospira interrogans protect hamsters from challenge using AddaVax™ as adjuvant.

BACKGROUND: Recombinant subunit vaccines have been extensively evaluated as promising alternatives against leptospirosis. Here, we evaluated two proteins in formulations containing the adjuvant AddaVax™ as vaccine candidates for prevention and control of leptospirosis. METHODS: Recombinant proteins rErp Y-like and rLemA were characterized by ELISA to assess their ability to bind extracellular matrix (ECM) components and fibrinogen. Groups of eight hamsters were immunized intramuscularly with rErp Y-like or rLemA mixed with a squalene-based adjuvant (AddaVax), and then vaccine efficacy was determined in terms of protection against a lethal challenge. The humoral immune response was determined by ELISA, and the evidence of sub-lethal infection was evaluated by histopathology and kidney culture. RESULTS: rLemA protein binds laminin, fibrinogen, and collagen type IV, while rErp Y-like interacts with fibrinogen. Significant protection was achieved for rLemA and rErp Y-like vaccines, which showed 87.5% and 62.5% survivals, respectively. On day 28, the humoral immune response was significantly greater in the vaccine groups as compared to that in the control group, and the response was predominantly based on IgG2/3. The surviving animals showed negative results in culture isolation but presented with tissue lesions in the lungs and kidneys. CONCLUSION: Cumulatively, our findings suggest that LemA and Erp Y-like proteins act as adhesins and are able to protect against mortality, but not against tissue lesions. Moreover, AddaVax is a novel adjuvant with potential for improving the immunogenicity of leptospiral vaccines.

DNA vaccines against leptospirosis: A literature review.

Leptospirosis is an infectious disease caused by pathogenic Leptospira species. The vaccines that are currently available for leptospirosis are composed of whole-cell preparations and suffer from limitations such as low efficacy, multiple side-effects, poor immunological memory and lack of cross-protection against different serovars of Leptospira spp. In light of the global prevalence of this disease, the development of a more effective vaccine against leptospirosis is of paramount importance. Genetic immunization is a promising alternative to conventional vaccine development. In the last 25years, several novel strategies have been developed for increasing the efficacy of DNA vaccines. Examples of such strategies include the introduction of novel plasmid vectors, adjuvants, alternate delivery routes, and prime-boost regimens. Herein we discuss the latest and most promising advances that have been made in developing DNA vaccines against leptospirosis. We also deliberate over the future directions that must be undertaken in order to improve results in this field.

The Use of Xanthan Gum as Vaccine Adjuvant: An Evaluation of Immunostimulatory Potential in BALB/c Mice and Cytotoxicity In Vitro.

The successful production of new, safe, and effective vaccines that generate immunological memory is directly related to adjuvant feature, which is responsible for increasing and/or modulating the immune response. Several compounds display adjuvant activity, including carbohydrates. These compounds play important roles in the immune response, as well as having biocompatible properties in vaccine formulations. One such carbohydrate is xanthan gum, a polysaccharide that is produced by the plant-pathogenic bacterium Xanthomonas spp., which has adjuvant attributes. This study evaluated the immune response induced by xanthan gum associated with ovalbumin in BALB/c mice, which were subcutaneously immunized, in terms of antibody production (IgG1, IgG2a, IgG2b, and IgG3), and assessed the levels of IFN-γ in the splenocyte culture using indirect ELISA. Furthermore, we investigated in vitro cytotoxicity of xanthan in the embryo fibroblasts cell line of the NIH/3T3 mouse by MTT assay and propidium iodide uptake assay. The mice immunized with ovalbumin plus xanthan gum exhibited higher antibody IgG1 responses than control groups. Furthermore, the xanthan polysaccharide was capable of increasing the immunogenicity of antigens by producing IFN-γ and did not exhibit cytotoxicity effects in NIH/3T3 mouse fibroblast cells, considered a promising candidate for vaccine adjuvant.

Human and animal leptospirosis in Southern Brazil: A five-year retrospective study.

BACKGROUND: Leptospirosis is an emerging zoonosis attributed to multiple reservoirs. Climatic conditions influence the transmission of pathogenic leptospires, which require warm and humid conditions for survival. The influence of seasonality in human and animal leptospirosis in the subtropical region of Brazil remains poorly understood. METHODS: We performed a retrospective study to describe the patterns of human and animal exposure to leptospirosis and their association with precipitation events in Southern Brazil. Rainfall data were obtained from satellite images. Serum samples were tested using the microscopic agglutination test (MAT); samples with titer ≥ 100 were defined as seroreactive. Linear regression and Pearson's correlation were performed to assess whether there is a relationship between these variables. RESULTS: We found that precipitation events were not significantly associated with the exposure to leptospirosis in humans or animal species, except for dogs. The interspecies analysis revealed an association between canine and human exposure to leptospirosis. Leptospira kirschneri serovar Butembo (serogroup Autumnalis) presented the highest seroreactivity in humans. CONCLUSION: This study provides valuable insights in human and animal leptospirosis in Southern Brazil. These insights will be essential to design intervention measures directed to reduce disease dissemination.

Evaluation of the Leptospira interrogans Outer Membrane Protein OmpL37 as a Vaccine Candidate.

The identification of potential vaccine candidates against leptospirosis remains a challenge. However, one such candidate is OmpL37, a potentially surface-exposed antigen that has the highest elastin-binding ability described to date, suggesting that it plays an important role in host colonization. In order to evaluate OmpL37's ability to induce a protective immune response, prime-boost, DNA and subunit vaccine strategies were tested in the hamster model of lethal leptospirosis. The humoral immune response was evaluated using an indirect ELISA test, and the cytokine profile in whole blood was determined by quantitative real-time PCR. Unlike the DNA vaccine, the administration of recombinant OmpL37 induced a strong IgG antibody response. When individually administrated, both formulations stimulated a TNF-α mediated inflammatory response. However, none of the OmpL37 formulations or vaccination strategies induced protective immunity. Further studies are required towards the identification of new vaccine targets against leptospirosis.

Xanthan gum as an adjuvant in a subunit vaccine preparation against leptospirosis.

Leptospiral immunoglobulin-like (Lig) proteins are of great interest due to their ability to act as mediators of pathogenesis, serodiagnostic antigens, and immunogens. Purified recombinant LigA protein is the most promising subunit vaccine candidate against leptospirosis reported to date, however, as purified proteins are weak immunogens the use of a potent adjuvant is essential for the success of LigA as a subunit vaccine. In the present study, we compared xanthan pv. pruni (strain 106), aluminium hydroxide (alhydrogel), and CpG ODN as adjuvants in a LigA subunit vaccine preparation. Xanthan gum is a high molecular weight extracellular polysaccharide produced by fermentation of Xanthomonas spp., a plant-pathogenic bacterium genus. Preparations containing xanthan induced a strong antibody response comparable to that observed when alhydrogel was used. Upon challenge with a virulent strain of L. interrogans serovar Copenhageni, significant protection (Fisher test, P < 0.05) was observed in 100%, 100%, and 67% of hamsters immunized with rLigANI-xanthan, LigA-CpG-xanthan, and rLigANI-alhydrogel, respectively. Furthermore, xanthan did not cause cytotoxicity in Chinese hamster ovary (CHO) cells in vitro. The use of xanthan as an adjuvant is a novel alternative for enhancing the immunogenicity of vaccines against leptospirosis and possibly against other pathogens.

DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.

The use of halloysite clay and carboxyl-functionalised multi-walled carbon nanotubes for recombinant LipL32 antigen delivery enhanced the IgG response

We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.