Partial table of contents: PRINCIPLES OF SELF-ASSEMBLY. Polymorphism of Lipids, Nucleic Acids, and their Interactions (D. Lasic, et al.). Self-Assembly of Bacteriophage (M. Burbea P. Prevelige). NATURAL MECHANISMS FOR GENE-DELIVERY TO CELLS. Endocytosis: An Overview (V. Slepnev P. De Camilli). NOVEL SYSTEMS USING CATIONIC LIPOSOMES. Novel Supramolecular Assemblies for Gene Delivery (F.-P. Yang L. Huang). POLYELECTROLYTE DNA COMPLEXES. Cationic Block Copolymers as Self-Assembling Vectors for Gene Delivery (L. Seymour, et al.). Cellular Internalization and Fate of Polycation-DNA Complexes (C. Pouton, et al.). SYSTEMIC BIODISTRIBUTION OF DRUG DELIVERY SYSTEMS. In Vitro and In Vivo Availability of Liposomes (V. Torchilin). TARGETING OF CONJUGATES FOR GENE DELIVERY. Polylysine-Conjugate Based DNA Delivery (E. Wagner). Peptides and Fusion Proteins as Modular DNA Carriers (W. Wels J. Fominaya). NEW APPORACHES TO GENE DELIVERY. Semi-Synthetic Systems for Gene Delivery (C. Hodgson, et al.). CLINICAL EVALUATION. Cystic Fibrosis Clinical Trials (D. Geddes E. Alton). Index.
Heterogeneous immunoassays (HI) are an invaluable tool for biomarker detection and remain an ideal candidate for microfluidic point-of-care diagnostics. However, automating and controlling sustained fluid flow from benchtop to microfluidics for the HI reaction during the extended sample incubation step, remains difficult to implement; this leads to challenges for assay integration and assay result interpretation. To address these issues, we investigated the liquid reciprocation process on a microfluidic centrifugal disc (CD) to generate continuous, bidirectional fluid flow using only a rotating motor. Large volumetric flow rates (μL s-1) through the HI reaction chamber were sustained for extended durations (up to 1 h). The CD liquid reciprocation operating behavior was characterized experimentally and simulated to determine fluid flow shear rates through our HI reaction chamber. We demonstrated the continuous CD liquid reciprocation for target molecule incubation for a microarray HI and that higher fluid shear rates negatively influenced our fluorescence intensity. We highlight the importance of proper fluid flow considerations when integrating HIs with microfluidics.
Until recently, malaria vaccine development efforts have focused almost exclusively on a handful of well characterized Plasmodium falciparum antigens. Despite dedicated work by many researchers on different continents spanning more than half a century, a successful malaria vaccine remains elusive. Sequencing of the P. falciparum genome has revealed more than five thousand genes, providing the foundation for systematic approaches to discover candidate vaccine antigens. We are taking advantage of this wealth of information to discover new antigens that may be more effective vaccine targets. Herein, we describe different approaches to large-scale screening of the P. falciparum genome to identify targets of either antibody responses or T cell responses using human specimens collected in Controlled Human Malaria Infections (CHMI) or under conditions of natural exposure in the field. These genome, proteome and transcriptome based approaches offer enormous potential for the development of an efficacious malaria vaccine.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Leptospirosis is the leading zoonotic disease in terms of morbidity and mortality worldwide. Effective prevention is urgently needed as the drivers of disease transmission continue to intensify. The key challenge has been developing a widely applicable vaccine that protects against the >300 serovars that can cause leptospirosis. Live attenuated mutants are enticing vaccine candidates and poorly explored in the field. We evaluated a recently characterized motility-deficient mutant lacking the expression of a flagellar protein, FcpA. Although the fcpA- mutant has lost its ability to cause disease, transient bacteremia was observed. In two animal models, immunization with a single dose of the fcpA- mutant was sufficient to induce a robust anti-protein antibodies response that promoted protection against infection with different pathogenic Leptospira species. Furthermore, characterization of the immune response identified a small repertoire of biologically relevant proteins that are highly conserved among pathogenic Leptospira species and potential correlates of cross-protective immunity. eLife digest Leptospirosis is a life-threatening disease with flu-like symptoms that is caused by bacteria known as Leptospira. It is more common in warmer regions with high rainfall, especially in impoverished areas. The disease is spread in the urine of animals such as rodents, farm animals or dogs. Humans and other animals can get leptospirosis when they come in contact with urine-contaminated water and soil. Current measures to control leptospirosis are largely ineffective. Although a vaccine is available for animals, it only protects against a few types of the 300 disease-causing Leptospira bacteria. It also fails to stop the bacteria from colonizing the kidneys of the infected animals, which means that vaccinated animals can still spread disease. Previous research has shown that inactivating a protein called FcpA, which is necessary for Leptospira bacteria to move, can stop them from infecting hamsters. Moreover, when these animals were exposed to the mutant bacteria, they did not get sick nor developed the disease. Here, Wunder et al. tested whether bacteria lacking the FcpA protein could be used as an attenuated vaccine. This form of vaccine contains live bacteria that have been modified to become harmless but are able to train the immune system to produce a long-lasting immune response against the invaders. The results showed that a single dose of the vaccine was enough to prevent hamsters and mice from dying of leptospirosis. It also worked against several types of Leptospira and could stop them from colonizing mice kidneys. Moreover, Wunder et al. found that the immune system targeted specific proteins that were common to various types of Leptospira, which may explain the broad spectrum of protection the vaccine offered. Rapid urbanization and climate change are among the main drivers of leptospirosis. An effective vaccine for this disease would reduce the public health burden by providing protection against leptospirosis and by reducing the spread of the disease. A next step will be to ensure the mutant Leptospira are safe to use in animals and potentially humans. Introduction Leptospirosis is caused by a genetically and antigenically diverse group of spirochetes of the Leptospira genus (Picardeau, 2017; Ko et al., 2009; Adler, 2015). Currently the Leptospira genus comprises 64 species with more than 300 serovars, with 17 of those species containing strains that can potentially cause severe disease in humans and animals (Casanovas-Massana et al., 2020; Vincent et al., 2019). A broad range of mammalian reservoirs harbor the spirochete in their renal tubules shedding the bacteria in their urine for long periods of time (Thibeaux et al., 2017; Xu et al., 2016). Leptospirosis is an environmentally transmitted disease with great health and economic impact in both humans and animals, for which the primary mode of transmission to humans is through contact with contaminated water or soil (Casanovas-Massana et al., 2018a). Although Leptospira has a worldwide distribution, the large majority of the burden occurs in the world’s most impoverished populations (Costa et al., 2015), where the rapid growth of urban slums worldwide has created conditions for rat-borne transmission. The disease causes life-threatening manifestations such as Weil’s disease (Ko et al., 1999; Adler, 2015) and leptospiral pulmonary hemorrhage syndrome (LPHS) (Gouveia et al., 2008). A recent study estimated that leptospirosis causes 1.03 million cases and 58,900 deaths each year (Costa et al., 2015). Case fatality for Weil’s disease and LPHS is >10 and >50%, respectively, despite aggressive supportive care (Gouveia et al., 2008). These estimates place leptospirosis as a leading zoonotic cause of morbidity and mortality worldwide. The burden of leptospirosis will increase as climate and land use change continues to evolve and the world’s slum population doubles to 2 billion by 2025 (UN-HABITAT, 2003). The public health priority is therefore prevention of leptospirosis before severe complications develop. However, there is no effective control for leptospirosis and safe and efficacious vaccines are not available for human use (Ko et al., 2009; Picardeau, 2017). China and Cuba use whole-cell vaccines in humans (Yan et al., 2003; Martínez et al., 2004), but they are not licensed to be used elsewhere. Whole-cell vaccines are widely used for veterinary purposes but have significant limitations, since immunity is of short duration and predominantly humoral against LPS, which are serovar-specific moieties. Multivalent vaccines are unable to achieve sufficient coverage against the spectrum of serovars that are important for animal and human health (Adler, 2015). Research has thus focused on characterizing surface-associated and host-expressed proteins as sub-unit vaccine candidates (Adler, 2015; Ko et al., 2009). To date, these conventional approaches have not yielded candidates and attempts have failed to identify a universal, widely applicable vaccine. Current control measures have been uniformly ineffective in addressing the large human and animal global health burden due to leptospirosis, especially in developing countries. Given the limitations of the whole-cell vaccines available and the ineffective attempts to identify protein vaccine candidates (Adler, 2015), an attenuated-vaccine approach remains a feasible strategy. Attenuation of Leptospira virulence has been long-recognized yet poorly understood phenomenon (Adler, 2015; Srikram et al., 2011). Until recently, the inability to produce well-defined mutants has preempted efforts to identify a safe and efficacious attenuated-vaccine. However, current advances in genomic tools and whole-genome sequencing data for Leptospira (Thibeaux et al., 2017; Picardeau, 2017) have circumvented this limitation and some promising results have been shown (Srikram et al., 2011; Murray et al., 2018). Recently, our group identified and characterized a novel flagellar protein in the Leptospira genus involved in the composition of the sheath of the leptospiral flagella, Flagella-coil protein A (FcpA). A mutant deficient in the fcpA gene lost its ability to produce translational motility and to penetrate mucous membranes, resulting in loss of kidney colonization and lethality in the hamster model of leptospirosis. Although highly attenuated in the hamster model, a needle inoculation of the mutant produced a transient bacteremia prior to clearance by the host immune response (Wunder et al., 2016a). In the present study, we evaluated the fcpA- motility-deficient mutant as a potential candidate for a live attenuated-vaccine that could provide a major public health benefit and opportunity to leverage One Health approaches. Results A motility-deficient strain as an attenuated-vaccine candidate We characterized a previously unidentified flagellar sheath protein (FcpA) that was essential for translational motility and thus for virulence (Wunder et al., 2016a). Despite the phenotype of complete attenuation, we observed that the L1-130 fcpA- mutant caused a transient systemic infection, which was cleared 7 days after intraperitoneal inoculation of 108 leptospires in hamsters (Wunder et al., 2016a). In this study, after inoculation of 107 leptospires using the subcutaneous route of infection in hamsters, we detected the presence of DNA of the mutant by qPCR in all the tissues tested, with the exception of the brain (Figure 1A). These results were similar to those observed previously, with the wild-type reaching higher number of leptospires in all tissues analyzed, leading to the death or euthanasia of the animals due to clinical signs of disease 5–7 days after infection. In comparison, the signal for the fcpA- mutant strain was undetectable after 7 days with all inoculated animals surviving with no detectable leptospires in either kidney or blood, measured by qPCR and culture. Similarly, no detectable signal was observed for the animals immunized with the L1-130 heat-killed strain (Figure 1A). We also tested the fcpA- mutant in the mouse model using different doses of infection (Figure 1B). Although the dose of the wild-type strain was not enough to produce disease and lethality on infected mice, all animals were colonized and the presence of the leptospiral DNA in blood was detectable until the fifteenth day after infection (Figure 1B). Furthermore, no dose of the fcpA- mutant caused colonization (data not shown) and there was a significant difference in the magnitude of dissemination of the mutant in the blood compared to the wild type (Figure 1B). DNA signal of the fcpA- mutant was only observed in the blood of animals infected with doses of 107 and 105 until the 13th and 8th day after infection, respectively. Taken together, these results indicate that although the fcpA- mutant is attenuated in both the hamster and mouse model, there is a hematogenous dissemination of this mutant, identified by detection of its DNA. The mutant appears to be cleared by the immune system before it results in observable disease or death of the animals. Furthermore, we observed in the mouse model that the dissemination of the mutant is dose dependent. However, it is important to notice that although we do not see any signal of the mutant in doses equal or lower to 103 leptospires, the theoretic limit of detection of the qPCR assay used here (Wunder et al., 2016a; Stoddard et al., 2009) is 100 leptospires/mL of blood which can result in false negative results. Figure 1 Download asset Open asset Dissemination of L1-130 fcpA- mutant in animal tissues. (A) Kinetics of infection of L1-130 WT, L1-130 fcpA- vaccine, and L1-130 heat-killed vaccine in blood, kidney, liver, and brain of hamsters after inoculation with 107 bacteria. All animals infected with WT strain died between 5 and 6 days post-infection; (B) Kinetics of infection of L1-130 WT (107 leptospires) and L1-130 fcpA- attenuated-vaccine (dose range from 107 to 101 leptospires) in blood of mouse. Results are expressed by logarithmic genome equivalent per gram or milliliter of tissues with mean and standard deviation. All doses were inoculated by subcutaneous route in both models. Figure 1—source data 1 Raw data for dissemination experiments in hamster and mouse. https://cdn.elifesciences.org/articles/64166/elife-64166-fig1-data1-v1.xls Download elife-64166-fig1-data1-v1.xls Model for cross-protective immunity to leptospirosis We hypothesized that the transient infection produced by the fcpA- mutant induces cross-protective responses, given previous findings (Wunder et al., 2016a; Srikram et al., 2011). Immunization with a single dose of the fcpA- mutant (Figure 2A) conferred complete protection against mortality in hamsters from infection with homologous and heterologous serovars (Figure 2B and Supplementary file 2). In contrast, immunization with heat-killed leptospires conferred partial protection to the homologous but not against the heterologous serovar (Figure 2B and Supplementary file 2). Heat-killed bacterins can give a high protection level against an homologous challenge (Adler, 2015), but usually the protocol for vaccination includes at least a second dose of the vaccine. Our poor results here with the heat-killed vaccine, especially for the homologous challenge (Figure 2B and Supplementary file 2), might be due to the lack of a vaccine boost. For the purpose of evaluating the efficacy of the attenuated-vaccine after a single dose, we decided to keep a standard protocol for vaccination and thus using only one dose of the heat-killed vaccine as well. It is important to mention that the strain Hardjo 203 was described to cause only colonization in the hamster model infected by intraperitoneal route (Zuerner et al., 2012). However, in our LD50 experiments using the conjunctival route we reproducibly observed 25% death rate when using the conjunctival route (Supplementary file 1). Furthermore, in the non-vaccinated group we observed an overall death rate of 21.4% after challenge with the strain Hardjo 203, but no deaths in the vaccinated group, which explains the wide 95% CI range (Figure 2B and Table S2). Figure 2 Download asset Open asset Efficacy of L1-130 fcpA- attenuated-vaccine model. Animals were vaccinated with a dose of 107 leptospires (hamsters) or a range of doses from 107 to 101 leptospires (mice) by subcutaneous (SC) route. Animals were bled the day before immunization (day −1) and day 20 post-immunization (A). Hamsters were challenged by conjunctival route with either the homologous strain or different heterologous strains. Mice were challenged by intraperitoneal route with the heterologous serovar Manilae of L. interrogans. By combining all vaccine experiments performed, efficacy of the vaccine against death and colonization was evaluated for hamsters (B and D) and mice (C and E) and represented by percentage and 95% CI based on frequency of outcomes compared to PBS-immunized animals. Hamster experiment are showing the results after vaccination with the fcpA- attenuated-vaccine (red) and heat-killed vaccine (blue). Bacterial load in the kidney was measured by qPCR in hamsters (F) and mice (G) and compared between PBS-immunized animals (blue) and animals immunized with fcpA- attenuated-mutant (red). Results are expressed in logarithmic genome equivalents per gram of renal tissue with mean and standard deviation. Asterisk symbols represent statistical significance calculated by t-test: *p<0.01, ***p<0.0001. See also Supplementary files 2 and 3. Figure 2—source data 1 Raw data for qPCR experiments in hamster and mouse. https://cdn.elifesciences.org/articles/64166/elife-64166-fig2-data1-v1.xls Download elife-64166-fig2-data1-v1.xls Protection against renal colonization was only observed in 80% of the animals immunized with fcpA- mutant after homologous infection. Heterologous infection gave varying levels of protection, from 0% to 35.7% (Figure 2D and Supplementary file 2). Hamsters are highly susceptible to leptospirosis (Haake, 2006), so the finding that the attenuated strain conferred partial protection against colonization was not unexpected. To understand the efficacy of the fcpA- mutant vaccine to protect against colonization, we tested different doses of immunization using the mouse model against heterologous infection. Our results indicate that the protection conferred by the fcpA- mutant is dose dependent. Against death, the vaccine conferred 100% protection up to a dose of 103 leptospires of the fcpA- mutant (Figure 2C and Supplementary file 3), but a dose as high as 107 leptospires was necessary to obtain 100% protection against colonization (Figure 2E and Supplementary file 3). Furthermore, our quantitative analyzes of renal colonization showed that although the fcpA- mutant cannot promote complete protection, there is a significant reduction of the burden of the disease both in hamster after heterologous infection (Figure 2F and Supplementary file 2) and in lower doses of the vaccine in the mouse model, which also revealed a dose-dependent phenotype (Figure 2G and Supplementary file 3). These findings indicate that a single dose of a live attenuated-vaccine elicits cross-protective immunity against serovars belonging to L. interrogans, L. kirschneri, and L. borgpetersenii, the species which encompasses the majority of serovars of human and animal health importance. Antibodies against Leptospira proteins as a correlate for the cross-protective immunity The fcpA- attenuated-vaccine induced a weak agglutinating antibodies response to the homologous serovar, Copenhageni, and undetectable microscopic agglutination test (MAT) titers against heterologous serovars, both in hamsters (Figure 3A) and mice (Figure 3C). Furthermore, in the mouse model, agglutinating antibodies were only measurable with a dose of at least 105 leptospires (Figure 3C). In contrast, a single dose of the fcpA- mutant was able to induce a robust immune response against leptospiral proteins, recognizing proteins across the different species of Leptospira used in the hamster model (Figure 3B) and the heterologous strain used in the mouse model with a dose of at least 103 leptospires (Figure 3D). In addition, the presence of detectable antibodies measured by ELISA correlates with the highest dose that induced 100% protection against death in the mouse model (103 leptospires), and there is a decrease on the OD for all doses when the Manilae antigen was treated with proteinase K (Figure 3E). Furthermore, our passive transfer experiments using hamster-immune sera against fcpA- attenuated-vaccine conferred 100% protection against heterologous lethal infection in hamsters (Figure 3F) and mice (Figure 3G). Taken together, these results indicate that anti-Leptospira protein antibodies, and not agglutinating antibodies, are the correlate of vaccine-mediated cross-protective immunity. Figure 3 Download asset Open asset Immunogenicity and correlates of immunity for L1-130 fcpA- attenuated-vaccine model. Individual sera of hamsters and mice were obtained after 20 days post-vaccination by a subcutaneous (SC) dose of 107 leptospires (hamsters) or a range of doses from 107 to 101 leptospires (mice) of the attenuated-vaccine. Microscopic agglutination test (MAT) (A and C) and western blot (B and D) were performed adopting as antigen all the strains used for challenged in both hamster and mice, respectively. Mice sera was additionally tested using an ELISA assay (E) adopting whole-cell extract of serovar Manilae with (red) and without (blue) Proteinase K treatment as antigen. Furthermore, a pool of hamster immune-sera vaccinated with a dose of 107 leptospires of fcpA- attenuated-vaccine was used for passive transfer experiments. 2 mL or 0.5 mL of sera was passively transfer to naïve hamsters (F) or mice (G), respectively, followed by challenge with a dose of 108 leptospires of heterologous serovar Manilae by conjunctival (CJ) or intraperitoneal (IP) route, respectively. Results are expressed in a survival curve of animals passively transferred with fcpA- anti-sera (red) and control hamster sera (blue). Figure 3—source data 1 Raw data for microscopic agglutination test (MAT) and passive transfer experiments in hamster and mouse, and ELISA data in mouse. https://cdn.elifesciences.org/articles/64166/elife-64166-fig3-data1-v1.xls Download elife-64166-fig3-data1-v1.xls Highly conserved seroreactive proteins as potential targets for eliciting cross-protective responses We characterized the antibody response to the attenuated-vaccine using a downsized proteome array of 660 and 330 ORFs for hamster and mouse sera, respectively. We identified a total of 133 (Figure 4A) and 56 (Figure 4B) protein targets on our analysis of hamsters (Hamster 107) and mice (Mouse 107) respectively, immunized with a dose of 107 leptospires and a total of 13 protein targets (Figure 4C) on our analysis of mouse immunized with different doses of the attenuated-vaccine (Mouse all). The reason to analyze the mouse results separately was based on the fact that a dose of 107 leptospires of the attenuated-vaccine was able to give 100% cross-protection against lethality and colonization (Figure 2C and E). When combined, these three different analyses resulted in a total of 154 unique protein targets (Figure 4D and Supplementary file 4). Of those, 55% (85) have no prediction of localization and 23% (36), 14% (21), and 8% (12) have a prediction to be cytoplasmic membrane-associated, outer membrane proteins (OMP), and cytoplasmic, respectively (Figure 4—figure supplement 1A). Enrichment analysis showed a 5.0-fold (p=4.51E-10) and 1.8-fold (p=2.92E-04) enrichment for OMP and cytoplasmic membrane-associated, respectively (Figure 4—figure supplement 1B). In contrast, cytoplasmic proteins were 0.3-fold (p=2.91E-10) underrepresented in reactive antigens groups (Figure 4—figure supplement 1B). Figure 4 with 2 supplements see all Download asset Open asset Proteome array analysis of immune-sera against L1-130 fcpA- attenuated-vaccine. Using statistical modeling we calculated the t-statistics value for each individual antigen used in the proteome array (660 for hamster and 330 for mice) based on three groups: the contrast between vaccinated and unvaccinated hamsters (A) or mice (B) using a vaccine dose of 107 leptospires; the dose–response relationship for each antigen on mice (C) vaccinated with a range of doses from 107 to 101 leptospires of the attenuated-vaccine. Results are ranked based on individual t-statistics values for each antigen, and the dashed line represents the selection point for the antigens based on Bhp-test. The Venn-diagram (D) shows the relationship of all the 154 antigens identified in the three groups. The subgroups of antigens selected for further characterization are highlighted in color. See Figure 5. Figure 4—source data 1 Raw data of proteome array experiments in hamster and mouse. https://cdn.elifesciences.org/articles/64166/elife-64166-fig4-data1-v1.xls Download elife-64166-fig4-data1-v1.xls Clusters of orthologous groups (COGs) of proteins were widely represented in those targets (Supplementary file 4), with at least one protein in each of the 18 functional categories. The COGs with higher representation were general function prediction only (R), cell wall/membrane/envelope biogenesis (M), intracellular trafficking, secretion, and vesicular transport (U), and cell motility (N) with 19, 17, 16, and 14 proteins, respectively. However, in addition to the 11 protein targets assigned as function unknown (S), the vast majority of the proteins had no COG assigned (59) (Figure 4—figure supplement 1C). Enrichment analysis identified proteins with predicted COG-U, COG-N, and COG-M function as highly enriched among the reactive antigens, by 4.9-fold (p=2.27E-07), 3.1-fold (p=8.35E-05), and 1.6-fold (p<0.05), respectively (Figure 4—figure supplement 1D). Furthermore, proteins predicted to be involved in signal transduction mechanisms (COG-T) and in amino acid transport and metabolism (COG-E) were significantly underrepresented in reactive antigens, by 0.4-fold (p=0.016) and 0.3-fold (0.02), respectively (Figure 4—figure supplement 1D). Taken together, the enrichment analysis validates our approach to identify biologically relevant protein candidates for a cross-protective vaccine. We were able to narrow down the identified 154 proteins to 41 protein targets based on their relationship among the three different groups of the proteome array’s analysis (Figure 5 and Figure 5—figure supplement 1). Seven proteins were identified in all groups (Figures 4D and 5 and Supplementary file 4, red) and 31 proteins were identified in both hamster and mouse vaccinated with a dose of 107 leptospires of the attenuated-vaccine (Figures 4D and 5 and Supplementary file 4, yellow). Furthermore, we identified three extra proteins identified in the group of mice immunized with different doses, two between the group of mice immunized with a dose of 107 leptospires (Figures 4D and 5 and Supplementary file 4, green) and one extra protein between the group of hamsters immunized with a dose of 107 leptospires (Figures 4D and 5 and Supplementary file 4, blue). Hamster and mice immune sera were highly reactive to the majority of the 41 proteins (Figure 5), in contrast to the low reactivity for the control sera and animals vaccinated with the heat-killed vaccine (Figure 5—figure supplement 1), indicating the ability of the attenuated-vaccine to induce immunity against leptospiral proteins. We identified plausible vaccine candidates among these 41 seroreactive proteins (Figure 5), which included six OMPs and known putative virulence factors such as LipL32, LipL41, and Lig proteins (Ko et al., 2009; Picardeau, 2017), providing supportive evidence for using proteome arrays to identify such proteins. Not surprisingly, 40% of those targets are identified as hypothetical proteins with no described function. However, the majority (70%) have high amino acid sequence identity (>80%) among their respective orthologs in all the 13 pathogenic Leptospira species analyzed (Figure 5), and therefore may be targeted for eliciting cross-protective responses. Moreover, sera from confirmed patients with acute leptospirosis reacted with 17 of the 41 Leptospira proteins recognized by sera from animals immunized with the attenuated-vaccine (Figure 5—figure supplement 1). Figure 5 with 1 supplement see all Download asset Open asset Heat-map of 41 seroreactive proteins recognized by hamsters and mice immunized with attenuated L1-130 fcpA- attenuated-vaccine. Proteins were selected based on the groups depicted on Figure 4 and Supplementary file 4: present in all three groups of analysis (red), present in both hamster and mice immunized with 107 leptospires (yellow), present in both hamsters immunized with 107 leptospires and mice immunized with a dose range (blue), and present in both mice immunized with 107 leptospires and mice immunized with a dose range (green). The proteins are identified by their L. interrogans serovar Copenhageni ORF number and the heat-map shows the signal intensity of antibody response (based on log-fold change) in all animals vaccinated with the fcpA- mutant used for this analysis (37 hamsters and 34 mice). Right panel shows amino acid sequence identity of respective ORFs among a representative of all pathogenic Leptospira species. Figure 5—source data 1 Raw data of amino acid identity of 41 selected protein targets among different Leptospira species. https://cdn.elifesciences.org/articles/64166/elife-64166-fig5-data1-v1.xls Download elife-64166-fig5-data1-v1.xls Discussion In this proof-of concept study, we showed the efficacy of the fcpA- attenuate-vaccine in preventing both death and renal colonization in animal models. Live attenuated-vaccines are one of the most universal vaccination technologies used for the prevention of important bacterial diseases like tuberculosis, cholera, and salmonellosis and viruses like yellow fever, influenza, and zoster (Minor, 2015; Detmer and Glenting, 2006). Despite the risks and the hurdles to identify the best candidates, including the best balance between attenuation and immunogenicity, research in this field continues to increase (Detmer and Glenting, 2006; Loessner et al., 2008). An effective vaccine against leptospirosis would provide interdependent health and societal benefits by preventing transmission and disease in livestock and domestic animals and reducing the risk of spill-over infections in humans. The transient bacteremia produced by needle inoculation of a single dose of the fcpA- mutant was sufficient to induce robust and cross-protective immunity in two different animal models of leptospirosis. The first live attenuated-vaccine for leptospirosis with a defined mutation has been recently described (Srikram et al., 2011; Murray et al., 2018) showing cross-protective immunity in hamsters and the potential for this approach. These authors made the important finding that immunization with an attenuated, LPS-deficient strain of serovar Manilae conferred varied levels of cross-protection against different species after one or two doses of the attenuated-vaccine. In our experiments, immunization with a single dose of the live fcpA- mutant conferred complete protection against death after heterologous infection on both hamster and mouse models. This result is highlighted by the poor performance of the heat-killed vaccine when given in a single dose. Given the promising results obtained with a single dose of the attenuated-vaccine, a boost dose was not evaluated. However, it should be considered for future experiments. Furthermore, we observed that the cross-protection against death after vaccination with the fcpA- mutant was dose dependent in the mouse model. In another spirochete, a similar approach has been successfully explored when a single dose of a flagella-less mutant in Borrelia burgdorferi induced homologous protection in mouse (Sadziene et al., 1996). More recently, a target mutant in B. burgdorferi induced cross immunity, but four immunizations were necessary for full protection (Hahn et al., 2016). Previous work with attenuated-vaccine in Leptospira showed evidence of protection against homologous challenge in guinea-pigs, cattle, hamsters, and swine, but only partial cross-protection in hamsters and gerbi
Variant surface antigens (VSAs) play a critical role in severe malaria pathogenesis. Defining gaps, or "lacunae", in immunity to these Plasmodium falciparum antigens in children with severe malaria would improve our understanding of vulnerability to severe malaria and how protective immunity develops. Using a protein microarray with 179 antigen variants from three VSA families as well as more than 300 variants of three other blood stage P. falciparum antigens, reactivity was measured in sera from Malian children with cerebral malaria or severe malarial anaemia and age-matched controls. Sera from children with severe malaria recognized fewer extracellular PfEMP1 fragments and were less reactive to specific fragments compared to controls. Following recovery from severe malaria, convalescent sera had increased reactivity to certain non-CD36 binding PfEMP1s, but not other malaria antigens. Sera from children with severe malarial anaemia reacted to fewer VSAs than did sera from children with cerebral malaria, and both of these groups had lacunae in their seroreactivity profiles in common with children who had both cerebral malaria and severe malarial anaemia. This microarray-based approach may identify a subset of VSAs that could inform the development of a vaccine to prevent severe disease or a diagnostic test to predict at-risk children.
Yellow Fever disease is caused by the Yellow Fever virus (YFV), an arbovirus from the Flaviviridae family. The re-emergence of Yellow Fever (YF) was facilitated by the increasing urbanization of sylvatic areas, the wide distribution of the mosquito vector, and the low percentage of people immunized in the Americas, which caused severe outbreaks in recent years, with a high mortality rate. Therefore, serological approaches capable of discerning antibodies generated from the wild-type (YFV-WT) strain between the vaccinal strain (YFV-17DD) could facilitate vaccine coverage surveillance, enabling the development of strategies to avoid new outbreaks. In this study, peptides were designed and subjected to microarray procedures with sera collected from individuals infected by WT-YFV and 17DD-YFV of YFV during the Brazilian outbreak of YFV in 2017/2018. From 222 screened peptides, around ten could potentially integrate serological approaches aiming to differentiate vaccinated individuals from naturally infected individuals. Among those peptides, one was synthesized and validated through ELISA.
Background. Heterozygous states of hemoglobin (Hb) A and HbS (HbAS, sickle-cell trait) or HbC (HbAC) protect against Plasmodium falciparum malaria by unclear mechanisms. Several studies suggest that HbAS and HbAC accelerate the acquisition of immunity to malaria, possibly by enhancing P. falciparum–specific antibody responses.
Routine serodiagnosis of herpes simplex virus (HSV) infections is currently performed using recombinant glycoprotein G (gG) antigens from herpes simplex virus 1 (HSV-1) and HSV-2. This is a single-antigen test and has only one diagnostic application. Relatively little is known about HSV antigenicity at the proteome-wide level, and the full potential of mining the antibody repertoire to identify antigens with other useful diagnostic properties and candidate vaccine antigens is yet to be realized. To this end we produced HSV-1 and -2 proteome microarrays in Escherichia coli and probed them against a panel of sera from patients serotyped using commercial gG-1 and gG-2 (gGs for HSV-1 and -2, respectively) enzyme-linked immunosorbent assays. We identified many reactive antigens in both HSV-1 and -2, some of which were type specific (i.e., recognized by HSV-1- or HSV-2-positive donors only) and others of which were nonspecific or cross-reactive (i.e., recognized by both HSV-1- and HSV-2-positive donors). Both membrane and nonmembrane virion proteins were antigenic, although type-specific antigens were enriched for membrane proteins, despite being expressed in E. coli.
High-throughput and rapid screening testing is highly desirable to effectively combat the rapidly evolving COVID-19 pandemic co-presents with influenza and seasonal common cold epidemics. Here, we present a general workflow for iterative development and validation of an antibody-based microarray assay for the detection of a respiratory viral panel: (a) antibody screening to quickly identify optimal reagents and assay conditions, (b) immunofluorescence assay design including signal amplification for low viral titers, (c) assay characterization with recombinant proteins, inactivated viral samples and clinical samples, and (d) multiplexing to detect a panel of common respiratory viruses. Using RT-PCR-confirmed SARS-CoV-2 positive and negative pharyngeal swab samples, we demonstrated that the antibody microarray assay exhibited a clinical sensitivity and specificity of 77.2% and 100%, respectively, which are comparable to existing FDA-authorized antigen tests. Moreover, the microarray assay is correlated with RT-PCR cycle threshold (Ct) values and is particularly effective in identifying high viral titers. The multiplexed assay can selectively detect SARS-CoV-2 and influenza virus, which can be used to discriminate these viral infections that share similar symptoms. Such protein microarray technology is amenable for scale-up and automation and can be broadly applied as a both diagnostic and research tool.
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