Immunogenic profile of a plant-produced nonavalent African horse sickness viral protein 2 (VP2) vaccine in IFNAR-/- mice.

A safe, highly immunogenic multivalent vaccine to protect against all nine serotypes of African horse sickness virus (AHSV), will revolutionise the AHS vaccine industry in endemic countries and beyond. Plant-produced AHS virus-like particles (VLPs) and soluble viral protein 2 (VP2) vaccine candidates were developed that have the potential to protect against all nine serotypes but can equally well be formulated as mono- and bi-valent formulations for localised outbreaks of specific serotypes. In the first interferon α/ß receptor knock-out (IFNAR-/-) mice trial conducted, a nine-serotype (nonavalent) vaccine administered as two pentavalent (5 µg per serotype) vaccines (VLP/VP2 combination or exclusively VP2), were directly compared to the commercially available AHS live attenuated vaccine. In a follow up trial, mice were vaccinated with an adjuvanted nine-serotype multivalent VP2 vaccine in a prime boost strategy and resulted in the desired neutralising antibody titres of 1:320, previously demonstrated to confer protective immunity in IFNAR-/- mice. In addition, the plant-produced VP2 vaccine performed favourably when compared to the commercial vaccine. Here we provide compelling data for a nonavalent VP2-based vaccine candidate, with the VP2 from each serotype being antigenically distinguishable based on LC-MS/MS and ELISA data. This is the first preclinical trial demonstrating the ability of an adjuvanted nonavalent cocktail of soluble, plant-expressed AHS VP2 proteins administered in a prime-boost strategy eliciting high antibody titres against all 9 AHSV serotypes. Furthermore, elevated T helper cells 2 (Th2) and Th1, indicative of humoral and cell-mediated memory T cell immune responses, respectively, were detected in mouse serum collected 14 days after the multivalent prime-boost vaccination. Both Th2 and Th1 may play a role to confer protective immunity. These preclinical immunogenicity studies paved the way to test the safety and protective efficacy of the plant-produced nonavalent VP2 vaccine candidate in the target animals, horses.

Immunogenicity of adjuvanted plant-produced SARS-CoV-2 Beta spike VLP vaccine in New Zealand white rabbits.

The outbreak of the SARS-CoV-2 global pandemic heightened the pace of vaccine development with various vaccines being approved for human use in a span of 24 months. The SARS-CoV-2 trimeric spike (S) surface glycoprotein, which mediates viral entry by binding to ACE2, is a key target for vaccines and therapeutic antibodies. Plant biopharming is recognized for its scalability, speed, versatility, and low production costs and is an increasingly promising molecular pharming vaccine platform for human health. We developed Nicotiana benthamiana-produced SARS-CoV-2 virus-like particle (VLP) vaccine candidates displaying the S-protein of the Beta (B.1.351) variant of concern (VOC), which triggered cross-reactive neutralising antibodies against Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs. In this study, immunogenicity of the VLPs (5 µg per dose) adjuvanted with three independent adjuvants i.e. oil-in-water based adjuvants SEPIVAC SWETM (Seppic, France) and "AS IS" (Afrigen, South Africa) as well as a slow-release synthetic oligodeoxynucleotide (ODN) adjuvant designated NADA (Disease Control Africa, South Africa) were evaluated in New Zealand white rabbits and resulted in robust neutralising antibody responses after booster vaccination, ranging from 1:5341 to as high as 1:18204. Serum neutralising antibodies elicited by the Beta variant VLP vaccine also showed cross-neutralisation against the Delta and Omicron variants with neutralising titres ranging from 1:1702 and 1:971, respectively. Collectively, these data provide support for the development of a plant-produced VLP based candidate vaccine against SARS-CoV-2 based on circulating variants of concern.

Protective immunity of plant-produced African horse sickness virus serotype 5 chimaeric virus-like particles (VLPs) and viral protein 2 (VP2) vaccines in IFNAR-/- mice.

Next generation vaccines have the capability to contribute to and revolutionise the veterinary vaccine industry. African horse sickness (AHS) is caused by an arbovirus infection and is characterised by respiratory distress and/or cardiovascular failure and is lethal to horses. Mandatory annual vaccination in endemic areas curtails disease occurrence and severity. However, development of a next generation AHSV vaccine, which is both safe and efficacious, has been an objective globally for years. In this study, both AHSV serotype 5 chimaeric virus-like particles (VLPs) and soluble viral protein 2 (VP2) were successfully produced in Nicotiana benthamiana ΔXT/FT plants, partially purified and validated by gel electrophoresis, transmission electron microscopy and liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing before vaccine formulation. IFNAR-/- mice vaccinated with the adjuvanted VLPs or VP2 antigens in a 10 µg prime-boost regime resulted in high titres of antibodies confirmed by both serum neutralising tests (SNTs) and enzyme-linked immunosorbent assays (ELISA). Although previous studies reported high titres of antibodies in horses when vaccinated with plant-produced AHS homogenous VLPs, this is the first study demonstrating the protective efficacy of both AHSV serotype 5 chimaeric VLPs and soluble AHSV-5 VP2 as vaccine candidates. Complementary to this, coating ELISA plates with the soluble VP2 has the potential to underpin serotype-specific serological assays.

Low Oxygen Storage Improves Tomato Postharvest Cold Tolerance, Especially for Tomatoes Cultivated with Far-Red LED Light.

We investigated the effects of low oxygen storage on chilling injury development, colour development, respiration and H2O2 levels of 'Merlice' tomatoes cultivated with and without far red (FR) LED lighting during 20 days of shelf-life. Mature green (MG) and red (R) tomatoes were stored at 2 °C in combination with 0.5, 2.5, 5 and 21 kPa O2 for 15 days (experiment 1). MG tomatoes cultivated under either white LED or white LED light with FR LED light were stored at 2 °C in combination with 1, 5 and 21 O2 kPa for 14 days (experiment 2). Chilled MG and R tomatoes from experiment 1 showed decay, firmness loss and higher weight loss during shelf-life which were reduced under low oxygen conditions. FR during cultivation improved chilling tolerance of MG tomatoes. Fastest colour development and lowest respiration rate during shelf-life were observed for MG fruit cultivated with FR lighting prior to storage at 1 kPa O2/0 kPa CO2. H2O2 levels during the shelf-life were not affected during cold storage. The improved cold tolerance of MG tomatoes cultivated with FR lighting is likely due to lower oxygen uptake that led to both higher lycopene synthesis and less softening.

Spray-Dried, Nanoencapsulated, Multi-Drug Anti-Tuberculosis Therapy Aimed at Once Weekly Administration for the Duration of Treatment.

Aiming to improve the treatment outcomes of current daily tuberculosis (TB) chemotherapy over several months, we investigated whether nanoencapsulation of existing drugs would allow decreasing the treatment frequency to weekly, thereby ultimately improving patient compliance. Nanoencapsulation of three first-line anti-TB drugs was achieved by a unique, scalable spray-drying technology forming free-flowing powders in the nanometer range with encapsulation efficiencies of 82, 75, and 62% respectively for rifampicin, pyrazinamide, and isoniazid. In a pre-clinical study on TB infected mice, we demonstrate that the encapsulated drugs, administered once weekly for nine weeks, showed comparable efficacy to daily treatment with free drugs over the same experimental period. Both treatment approaches had equivalent outcomes for resolution of inflammation associated with the infection of lungs and spleens. These results demonstrate how scalable technology could be used to manufacture nanoencapsulated drugs. The formulations may be used to reduce the oral dose frequency from daily to once weekly in order to treat uncomplicated TB.

Mycolic acids, a promising mycobacterial ligand for targeting of nanoencapsulated drugs in tuberculosis.

The appearance of drug-resistant strains of Mycobacterium tuberculosis (Mtb) poses a great challenge to the development of novel treatment programmes to combat tuberculosis. Since innovative nanotechnologies might alleviate the limitations of current therapies, we have designed a new nanoformulation for use as an anti-TB drug delivery system. It consists of incorporating mycobacterial cell wall mycolic acids (MA) as targeting ligands into a drug-encapsulating Poly dl-lactic-co-glycolic acid polymer (PLGA), via a double emulsion solvent evaporation technique. Bone marrow-derived mouse macrophages, either uninfected or infected with different mycobacterial strains (Mycobacterium avium, Mycobacterium bovis BCG or Mtb), were exposed to encapsulated isoniazid-PLGA nanoparticles (NPs) using MA as a targeting ligand. The fate of the NPs was monitored by electron microscopy. Our study showed that i) the inclusion of MA in the nanoformulations resulted in their expression on the outer surface and a significant increase in phagocytic uptake of the NPs; ii) nanoparticle-containing phagosomes were rapidly processed into phagolysosomes, whether MA had been included or not; and iii) nanoparticle-containing phagolysosomes did not fuse with non-matured mycobacterium-containing phagosomes, but fusion events with mycobacterium-containing phagolysosomes were clearly observed.

Differential spontaneous folding of mycolic acids from Mycobacterium tuberculosis.

Mycolic acids are structural components of the mycobacterial cell wall that have been implicated in the pathogenicity and drug resistance of certain mycobacterial species. They also offer potential in areas such as rapid serodiagnosis of human and animal tuberculosis. It is increasingly recognized that conformational behavior of mycolic acids is very important in understanding all aspects of their function. Atomistic molecular dynamics simulations, in vacuo, of stereochemically defined Mycobacterium tuberculosis mycolic acids show that they fold spontaneously into reproducible conformational groupings. One of the three characteristic mycolate types, the keto-mycolic acids, behaves very differently from either α-mycolic acids or methoxy-mycolic acids, suggesting a distinct biological role. However, subtle conformational behavioral differences between all the three mycolic acid types indicate that cooperative interplay of individual mycolic acids may be important in the biophysical properties of the mycobacterial cell envelope and therefore in pathogenicity.

Thiol modified mycolic acids.

Patient serum antibodies to mycolic acids have the potential to be surrogate markers of active tuberculosis (TB) when they can be distinguished from the ubiquitously present cross-reactive antibodies to cholesterol. Mycolic acids are known to interact more strongly with antibodies present in the serum of patients with active TB than in patients with latent TB or no TB. Examples of single stereoisomers of mycolic acids with chain lengths corresponding to major homologues of those present in Mycobacterium tuberculosis have now been synthesised with a sulfur substituent on the terminal position of the α-chain; initial studies have established that one of these binds to a gold electrode surface, offering the potential to develop second generation sensors for diagnostic patient antibody detection.

Towards understanding the functional diversity of cell wall mycolic acids of Mycobacterium tuberculosis.

Mycolic acids constitute the waxy layer of the outer cell wall of Mycobacterium spp. and a few other genera. They are diverse in structure, providing a unique chromatographic foot-print for almost each of the more than 70 Mycobacterium species. Although mainly esterified to cell wall arabinogalactan, trehalose or glucose, some free mycolic acid is secreted during in vitro growth of Mycobacterium tuberculosis. In M. tuberculosis, α-, keto- and methoxy-mycolic acids are the main classes, each differing in their ability to attract neutrophils, induce foamy macrophages or adopt an antigenic structure for antibody recognition. Of interest is their particular relationship to cholesterol, discovered by their ability to attract cholesterol, to bind Amphotericin B or to be recognised by monoclonal antibodies that cross-react with cholesterol. The structural elements that determine this diverse functionality include the carboxylic acid in the mycolic motif, as well as the nature and stereochemistry of the two functional groups in the merochain. The functional diversity of mycolic acid classes implies that much information may be contained in the selective expression and secretion of mycolic acids to establish tuberculosis after infection of the host. Their cholesteroid nature may relate to how they utilize host cholesterol for their persistent survival.

Structure-function relationships of the antigenicity of mycolic acids in tuberculosis patients.

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.

Electron transfer dynamics across self-assembled N-(2-mercaptoethyl) octadecanamide/mycolic acid layers: impedimetric insights into the structural integrity and interaction with anti-mycolic acid antibodies.

The integrity and properties of mycolic acid (MA) antigens integrated into a self-assembled monolayer (SAM) of N-(2-mercaptoethyl)octadecanamide, (MEODA), on a gold electrode have been interrogated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). EIS data showed that Au-MEODA and Au-MEODA-MA behave as microelectrode arrays, with pinholes acting as the microelectrodes that permit electron transport between a redox-active probe in solution and the underlying gold surface. The average radii of the pinholes (r(a)) and half the distance between the centers of the neighbouring pinholes (r(b)), were estimated from EIS using the pore size model, and discussed. Anti-MA antibodies present in a tuberculosis (TB)-infected patient (co-infected with HIV) strongly interact with Au-MEODA-MA showing a rather compact and stable bio-complex structure that is virtually defect-free. The electrochemical impedimetric properties associated with the ability of the Au-MEODA-MA to discriminate between TB positive and negative human sera are also discussed. We prove that the Au-MEODA and Au-MEODA-MA electrodes, as well as the MA-anti-MA antibody interactions, are characterized with time-constant dispersion, typical of microstructures with grain/grain boundary phases. These crucial physico-electrochemical insights into the behaviour of surface-confined MA should provide a useful basis for the design and development of a potential impedimetric immunosensing platform for active tuberculosis.

Recognition of anti-mycolic acid antibody at self-assembled mycolic acid antigens on a gold electrode: a potential impedimetric immunosensing platform for active tuberculosis.

Electrochemical impedimetric recognition by anti-mycolic acid antibodies, present in tuberculosis (TB)-positive human serum co-infected with human immunodeficiency virus (HIV), of mycolic acids (MA) integrated into a self-assembled monolayer of N-(2-mercaptoethyl)octadecanamide on a gold electrode is described, proving that the MA-based electrode can satisfactorily discriminate between a TB-positive and a TB-negative serum, thus offering promise as a potential impedimetric immunosensing platform for active tuberculosis.

Cholesteroid nature of free mycolic acids from M. tuberculosis.

Mycolic acids (MAs) are a major component of the cell walls of Mycobacterium tuberculosis and related organisms. These alpha-alkyl beta-hydroxy long fatty acids have been the subject of numerous studies for their immunological properties. We previously reported that an interaction between cholesterol and mycolic acids could be responsible for the low accuracy in the serodiagnosis of TB when using free mycolic acid in an ELISA assay. The aim of this work was to investigate if this interaction could be due to a similarity in the structural properties between mycolic acids and cholesterol. The investigation revealed that patient sera cross-reacted with mycolic acids and cholesterol in an ELISA experiment suggesting that both molecules may present related functionality in a similar structural orientation. This relation was further supported by the interaction of mycolic acids with Amphotericin B (AmB), a known binding agent to ergosterol and cholesterol. Using a resonant mirror biosensor, we observed that AmB recognised both cholesterol and mycolic acids. In addition, a specific attraction was observed between mycolic acid and cholesterol by the accumulation of cholesterol from liposomes in suspension onto immobilized mycolic acids containing liposomes, detected with a biosensor technique. Combined, these results suggest that mycolic acids can assume a three-dimensional conformation similar to a sterol. This requires that mycolic acid exposes its hydroxyl group and assumes rigidity in its chain structure to generate a hydrophobic surface topology matching that of cholesterol. A particular folded conformation would be required for this, of which a few different types have already been proven to exist in monolayers of mycolic acids.

A novel application of affinity biosensor technology to detect antibodies to mycolic acid in tuberculosis patients.

Tuberculosis has re-emerged as a global health problem due to co-infection with HIV and the emergence of drug-resistant strains of Mycobacterium tuberculosis. HIV co-infection introduced a 30% underestimation in TB diagnosis based on sputum analysis, calling for a reliable and fast serodiagnostic assay to assist in the management of TB in HIV-burdened populations. Serodiagnosis with mycobacterial lipid cell wall antigens gave promising results, in particular with LAM and cord factor. Free mycolic acids have also been considered because they are unique in structure to each species of Mycobacterium and can be economically extracted and purified. In a standard immunoassay such as ELISA, however, an unacceptable number of false positive and false negative test results were obtained. Here we report a much improved biosensor method to detect antibodies to mycolic acids in patient serum as surrogate markers of active tuberculosis. Mycolic acid (MA) liposomes were immobilized on a non-derivatized twin-celled biosensor cuvette and blocked with saponin. A high dilution of serum was used to calibrate the binding signal of the two cells, followed by contact with patient serum at a lesser dilution, but pre-incubated with either antigen-carrying, or empty liposomes. The serum, or the protein A purified IgG thereof, from sputum-positive tuberculosis patients could be inhibited from binding to the MA in the biosensor by prior incubation with MA-containing liposomes. The accuracy of the inhibition test was 84% if HIV-positive patients for whom a negative TB sputum analyses could not be relied upon to serve as a reference standard were excluded. If biosensor technology could be made suitable for high throughput screening, then it may provide the solution to the serodiagnosis of tuberculosis against a background of HIV.

Macrophage reprogramming by mycolic acid promotes a tolerogenic response in experimental asthma.

RATIONALE: Mycolic acid (MA) constitutes a major and distinguishing cell wall biolipid from Mycobacterium tuberculosis. MA interferes with the lipid homeostasis of alveolar macrophages, inducing differentiation into foamy macrophages exhibiting increased proinflammatory function. OBJECTIVES: We verified the interference of this altered macrophage function with inhaled antigen-triggered allergic airway inflammation and underlying Th2 lymphocyte reactivity. METHODS: Using ovalbumin (OVA) as model allergen, C57BL/6 or BALB/C mice were sensitized by OVA-alum immunization. Experimental asthma, triggered subsequently by repetitive nebulized OVA inhalation, was assessed, using as readout parameters eosinophilia, peribronchial inflammation, and Th2 cytokine function. MEASUREMENTS AND MAIN RESULTS: A single intratracheal treatment of sensitized mice with MA, inserted into liposomes as carriers, prevented the onset of OVA-triggered allergic airway inflammation and promoted unresponsiveness to a secondary set of allergen exposures. The development of this tolerant condition required an 8-d lapse after MA instillation, coinciding with the appearance of foamy alveolar macrophages. MA-conditioned CD11b(+)F4/80(+) macrophages, transferred to the airways, mimicked the tolerogenic function of instilled MA; however, without the 8-d lapse requirement. Indicative of a macrophage-mediated tolerogenic antigen-presenting function, major histocompatibility complex (MHC)-mismatched donor macrophages failed to promote tolerance. Furthermore, Treg markers were strongly increased and established tolerance was lost after in situ depletion of CD25(+) Treg cells. Contrary to the interleukin-10 dependence of tolerogenic dendritic cells, IFN-gamma deficiency but not interleukin-10 deficiency abrogated the tolerogenic capacity of MA-conditioned macrophages. CONCLUSIONS: These results document an innate-driven Mycobacterium tuberculosis MA-triggered immune regulatory mechanism in control of pulmonary allergic responses by converting macrophages into IFN-gamma-dependent tolerogenic antigen-presenting cells.

Prevalence of anti-mycolic acid antibodies in patients with pulmonary tuberculosis co-infected with HIV.

Isolation and purification of mycolic acids from Mycobacterium tuberculosis have allowed them to be applied as antigen in an ELISA-based assay to detect specific antibodies in human sera. Tuberculosis patients have previously been shown to contain antimycolic acids antibodies. The aim of this study was to determine whether human immunodeficiency virus (HIV) co-infection increases false-negative testing rate and whether other random diseases for which hospitalisation is normally required will contribute to false-positive results. Sera from 118 human subjects were tested for the presence of antibodies to mycolic acids; 59 were patients with proven pulmonary tuberculosis and 59 were control hospitalised patients without evidence of tuberculosis. Each group consisted of HIV-seropositive and HIV-seronegative subjects. The endpoint was the detection of specific antibodies to mycolic acids in the sera, before and after precipitation of immune complexes. The two groups of subjects were well matched for age, gender, race and HIV status. On average, humans infected with Mycobacterium tuberculosis showed a specific antibody response to mycolic acids that was not affected by low CD4 T-lymphocyte counts in HIV-seropositive patients but was compromised by various other serious diseases.