ABSTRACT
BACKGROUND Neuromyelitis optica (NMO) is a rare neurological disease characterized by attacks of transverse myelitis and optic neuritis, contiguous spinal cord lesions on more than 3 vertebral segments on magnetic resonance imaging (MRI), and seropositivity for AQP-4 Ab. The tissue destruction from NMO is immune mediated and results in demyelination and axonal damage. Optic and spinal nerve involvement can eventually lead to blindness, weakness, and altered consciousness, and bladder and bowel involvement in some cases. CASE REPORT A 54-year-old Black woman presented with chest pain, dysphagia, generalized weakness, diplopia, and paresthesias in her bilateral feet. A brain MRI revealed an area of hyperintensity in the cervical medullary junction. A diagnosis of NMO was made after the treatment response was poor for systemic lupus erythematous (SLE) myelitis. She eventually developed acute hypercapnic respiratory failure, became encephalopathic, and was emergently intubated. She was extubated but had poor recovery and was eventually discharged home. CONCLUSIONS NMO is a rare immune-mediated disease that is often delayed in diagnosis and treatment. Clinical suspicion is important since there is a tendency for the disease to overlap concomitant autoimmune diseases in 25% of cases. Progressive and permanent tissue damage can occur despite the use of high-dose steroids, long-term immunosuppressant agents, immunomodulators, exchange transfusions, and even autologous hematopoietic stem cell bone marrow transplantation.
Subject(s)
Myelitis, Transverse , Neuromyelitis Optica , Respiratory Insufficiency , Brain/pathology , Female , Humans , Magnetic Resonance Imaging , Middle Aged , Neuromyelitis Optica/complications , Neuromyelitis Optica/diagnosis , Neuromyelitis Optica/therapy , Respiratory Insufficiency/etiology , Respiratory Insufficiency/therapyABSTRACT
Burkholderia mallei are Gram-negative bacteria, responsible for the disease glanders. B. mallei has recently been classified as a Tier 1 agent owing to the fact that this bacterial species can be weaponised for aerosol release, has a high mortality rate and demonstrates multi-drug resistance. Furthermore, there is no licensed vaccine available against this pathogen. Lipopolysaccharide (LPS) has previously been identified as playing an important role in generating host protection against Burkholderia infection. In this study, we present gold nanoparticles (AuNPs) functionalised with a glycoconjugate vaccine against glanders. AuNPs were covalently coupled with one of three different protein carriers (TetHc, Hcp1 and FliC) followed by conjugation to LPS purified from a non-virulent clonal relative, B. thailandensis. Glycoconjugated LPS generated significantly higher antibody titres compared with LPS alone. Further, they improved protection against a lethal inhalation challenge of B. mallei in the murine model of infection. FROM THE CLINICAL EDITOR: Burkholderia mallei is associated with multi-drug resistance, high mortality and potentials for weaponization through aerosol inhalation. The authors of this study present gold nanoparticles (AuNPs) functionalized with a glycoconjugate vaccine against this Gram negative bacterium demonstrating promising results in a murine model even with the aerosolized form of B. Mallei.
Subject(s)
Bacterial Vaccines/administration & dosage , Burkholderia mallei/drug effects , Glanders/drug therapy , Metal Nanoparticles/administration & dosage , Administration, Inhalation , Animals , Bacterial Vaccines/chemistry , Burkholderia mallei/pathogenicity , Disease Models, Animal , Glanders/immunology , Glanders/microbiology , Glycoconjugates/administration & dosage , Glycoconjugates/chemistry , Gold/chemistry , Humans , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/immunology , Metal Nanoparticles/chemistry , MiceABSTRACT
Burkholderia are highly evolved Gram-negative bacteria that primarily infect solipeds but are transmitted to humans by ingestion and cutaneous or aerosol exposures. Heightened concern over human infections of Burkholderia mallei and the very closely related species B. pseudomallei is due to the pathogens' proven effectiveness as bioweapons, and to the increased potential for natural opportunistic infections in the growing diabetic and immuno-compromised populations. These Burkholderia species are nearly impervious to antibiotic treatments and no vaccine exists. In this study, the genome of the highly virulent B. mallei ATCC23344 strain was examined by expression library immunization for gene-encoded protective antigens. This protocol for genomic-scale functional screening was customized to accommodate the unusually large complexity of Burkholderia, and yielded 12 new putative vaccine candidates. Five of the candidates were individually tested as protein immunogens and three were found to confer significant partial protection against a lethal pulmonary infection in a murine model of disease. Determinations of peripheral blood cytokine and chemokine profiles following individual protein immunizations show that interleukin-2 (IL-2) and IL-4 are elicited by the three confirmed candidates, but unexpectedly interferon-γ and tumor necrosis factor-α are not. We suggest that these pathogen components, discovered using genetic immunization and confirmed in a conventional protein format, will be useful toward the development of a safe and effective glanders vaccine.
ABSTRACT
Burkholderia pseudomallei, and other members of the Burkholderia, are among the most antibiotic-resistant bacterial species encountered in human infection. Mortality rates associated with severe B. pseudomallei infection approach 50% despite therapeutic treatment. A protective vaccine against B. pseudomallei would dramatically reduce morbidity and mortality in endemic areas and provide a safeguard for the U.S. and other countries against biological attack with this organism. In this study, we investigated the immunogenicity and protective efficacy of B. pseudomallei-derived outer membrane vesicles (OMVs). Vesicles are produced by Gram-negative and Gram-positive bacteria and contain many of the bacterial products recognized by the host immune system during infection. We demonstrate that subcutaneous (SC) immunization with OMVs provides significant protection against an otherwise lethal B. pseudomallei aerosol challenge in BALB/c mice. Mice immunized with B. pseudomallei OMVs displayed OMV-specific serum antibody and T-cell memory responses. Furthermore, OMV-mediated immunity appears species-specific as cross-reactive antibody and T cells were not generated in mice immunized with Escherichia coli-derived OMVs. These results provide the first compelling evidence that OMVs represent a non-living vaccine formulation that is able to produce protective humoral and cellular immunity against an aerosolized intracellular bacterium. This vaccine platform constitutes a safe and inexpensive immunization strategy against B. pseudomallei that can be exploited for other intracellular respiratory pathogens, including other Burkholderia and bacteria capable of establishing persistent infection.
Subject(s)
Bacterial Vaccines/immunology , Burkholderia pseudomallei/immunology , Exosomes/immunology , Melioidosis/prevention & control , Pneumonia, Bacterial/prevention & control , Animals , Antibodies, Bacterial/blood , Bacterial Vaccines/administration & dosage , Cross Reactions , Female , Immunologic Memory , Injections, Subcutaneous , Melioidosis/immunology , Melioidosis/mortality , Mice , Mice, Inbred BALB C , Pneumonia, Bacterial/immunology , Pneumonia, Bacterial/mortality , Survival Analysis , T-Lymphocytes/immunologyABSTRACT
Rapid detection of the category B biothreat agents Burkholderia pseudomallei and Burkholderia mallei in acute infections is critical to ensure that appropriate treatment is administered quickly to reduce an otherwise high probability of mortality (ca. 40% for B. pseudomallei). We are developing assays that can be used in clinical laboratories or security applications for the direct detection of surface-localized and secreted macromolecules produced by these organisms. We present our current medium-throughout approach for target selection and production of Burkholderia macromolecules and describe the generation of a Fab molecule targeted to the B. mallei BimA protein. We also present development of prototype assays for detecting Burkholderia species using anti-lipopolysaccharide antibodies.
Subject(s)
Burkholderia mallei/isolation & purification , Burkholderia pseudomallei/isolation & purification , Enzyme-Linked Immunosorbent Assay/methods , Glanders/microbiology , Melioidosis/microbiology , Animals , Burkholderia mallei/metabolism , Burkholderia pseudomallei/metabolism , Chaperonin 60/chemistry , Chaperonin 60/metabolism , Glanders/diagnosis , Humans , Melioidosis/diagnosis , Microfilament Proteins/chemistry , Microfilament Proteins/metabolismABSTRACT
Burkholderia thailandensis is a less virulent close relative of Burkholderia pseudomallei, a CDC category B biothreat agent. We have previously shown that lipopolysaccharide (LPS) extracted from B. pseudomallei can provide protection against a lethal challenge of B. pseudomallei in a mouse model of melioidosis. Sugar analysis on LPS from B. thailandensis strain E264 confirmed that this polysaccharide has a similar structure to LPS from B. pseudomallei. Mice were immunised with LPS from B. thailandensis or B. pseudomallei and challenged with a lethal dose of B. pseudomallei strain K96243. Similar protection levels were observed when either LPS was used as the immunogen. This data suggests that B. thailandensis LPS has the potential to be used as part of a subunit based vaccine against pathogenic B. pseudomallei.
Subject(s)
Burkholderia pseudomallei/pathogenicity , Burkholderia/pathogenicity , Lipopolysaccharides/immunology , Melioidosis/prevention & control , Animals , Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Antigens, Bacterial/isolation & purification , Bacterial Vaccines/immunology , Burkholderia/immunology , Burkholderia pseudomallei/immunology , Disease Models, Animal , Female , Lipopolysaccharides/isolation & purification , Melioidosis/immunology , Mice , Mice, Inbred BALB CABSTRACT
An approach for enhancing antibody affinity is to engineer Chelating Recombinant Antibodies (CRAbs) which consist of two tandemly linked single-chain Fvs (scFvs) that bind to distinct non-overlapping epitopes on the antigen molecule leading to a synergistic decrease in K(D). In order to develop this technology, the aim of this present study was to identify scFvs which can simultaneously bind to the tetanus toxin heavy chain C-terminal sub-domain (H(c)), characterise their bio-physical properties and determine their functional efficacy. Over 50 antibodies specific for Hc were isolated from a human scFv phagemid library and found to bind specifically to the C-terminal sub-domain of H(c) (H(c)C clones), the N-terminal sub-domain (HcN clones) or junctional epitopes on the whole Hc fragment only (HcJ clones). Fifteen clones were assayed in a pairwise competition binding study. The revealed, with few exceptions, that H(c)C clones were able to simultaneously bind to the toxin with H(c)N or H(c)J clones. All other combinations competed for binding. Interestingly, we also observed cooperative binding with many non-competing scFv pairings which may impact upon the binding mechanism of CRAbs. We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity. This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells. For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency. The diverse array of clones characterised in this paper can be used to construct CRAbs and will prove useful in further characterisation of toxin biology and in measuring the effects of polyclonal antibody therapy.
Subject(s)
Antibodies, Bacterial/immunology , Antibody Affinity/immunology , Tetanus Toxin/immunology , Antibodies, Monoclonal/immunology , Binding, Competitive , Enzyme-Linked Immunosorbent Assay , Humans , Peptide Fragments/immunology , Peptide Library , Polymerase Chain Reaction , Recombinant Proteins/immunologyABSTRACT
Burkholderia mallei and B. pseudomallei are Gram-negative pathogenic bacteria, responsible for the diseases glanders and melioidosis, respectively. Furthermore, there is currently no vaccine available against these Burkholderia species. In this study, we aimed to identify protective proteins against these pathogens. Immunization with recombinant B. mallei Hcp1 (type VI secreted/structural protein), BimA (autotransporter protein), BopA (type III secreted protein), and B. pseudomallei LolC (ABC transporter protein) generated significant protection against lethal inhaled B. mallei ATCC23344 and B. pseudomallei 1026b challenge. Immunization with BopA elicited the greatest protective activity, resulting in 100% and 60% survival against B. mallei and B. pseudomallei challenge, respectively. Moreover, sera from recovered mice demonstrated reactivity with the recombinant proteins. Dendritic cells stimulated with each of the different recombinant proteins showed distinct cytokine patterns. In addition, T cells from immunized mice produced IFN-γ following in vitro re-stimulation. These results indicated therefore that it was possible to elicit cross-protective immunity against both B. mallei and B. pseudomallei by vaccinating animals with one or more novel recombinant proteins identified in B. mallei.
ABSTRACT
Clostridium difficile expresses a surface layer (S-layer) which coats the surface of the bacterium and acts as an adhesin facilitating interaction of the bacterium with host enteric cells. The S-layer contains a high-molecular-weight S-layer protein (HMW SLP) and its low-molecular-weight partner protein (LMW SLP). We show that these proteins form a tightly associated non-covalent complex, the H/L complex, and we identify the regions of both proteins responsible for complex formation. The 2.4 A X-ray crystal structure of a truncated derivative of the LMW SLP reveals two domains. Domain 1 has a two-layer sandwich architecture while domain 2, predicted to orientate towards the external environment, contains a novel fold. Small-angle X-ray scattering analysis of the H/L complex shows an elongated molecule, with the two SLPs arranged 'end-to-end' interacting with each other through a small contact area. Alignment of LMW SLPs, which exhibit high sequence diversity, reveals a core of conserved residues that could reflect functional conservation, while allowing for immune evasion through sequence variation. These structures are the first described for the S-layer of a bacterial pathogen, and provide insights into the assembly and biogenesis of the S-layer.
Subject(s)
Bacterial Proteins/metabolism , Clostridioides difficile/metabolism , Membrane Glycoproteins/metabolism , Amino Acid Sequence , Cell Wall/metabolism , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Transcutaneous immunization is a promising vaccination delivery strategy which targets potent immune cells residing in the outer layer of the skin. In this study, the immunogenicity and neutralizing potency of the non-toxic Hc fragment of tetanus toxin (HcWT) and a mutant of Hc lacking ganglioside binding activity were compared with that of tetanus toxoid (TTxd) following transcutaneous immunization (TCI) of mice. Mice immunized with HcWT in the absence of an adjuvant induced highest anti-toxoid and anti-Hc antibody titres, with a significant increase in the toxin neutralizing antibody response compared with TTxd. These results are in contrast to previous studies employing subcutaneous delivery, where TTxd was found to be a more potent immunogen than the Hc fragment of the toxin. We conclude that the HcWT protein is more immunogenic than TTxd when given via the transcutaneous route. Our results suggest that TCI may provide an opportunity for effective delivery of toxin-like antigens which harbor protective epitopes and that traditional toxoid proteins may not be optimal antigens for skin immunization.
Subject(s)
Antitoxins/blood , Peptide Fragments/administration & dosage , Peptide Fragments/immunology , Tetanus Toxin/immunology , Tetanus Toxoid/administration & dosage , Tetanus Toxoid/immunology , Administration, Cutaneous , Animals , Female , Immunoglobulin G/blood , Mice , Mice, Inbred BALB C , Tetanus Toxin/administration & dosage , Tetanus Toxin/antagonists & inhibitorsABSTRACT
Like many other bacterial cell surfaces, the cell wall of Clostridium difficile is also encapsulated by a proteinaceous paracrystalline layer, the surface (S)-layer. In many bacterial species, the S-layer proteins (SLPs) have been shown to be glycosylated, whereas in other species glycosylation is absent. Unusually, the S-layer of C. difficile is composed of two distinct proteins, the high-molecular weight (HMW) and low-molecular-weight (LMW) SLPs. Previous investigations have reported that one or both of these SLPs are glycosylated, though no definitive study has been conducted. We have used a variety of mass spectrometric approaches to analyse SLPs from a number of strains of C. difficile for the presence of associated glycans. Analysis of intact SLPs by matrix assisted laser desorption/ionisation time of flight (MALDI-ToF) mass spectrometry demonstrated that the observed molecular masses matched the predicted masses of the LMW and HMW SLPs. Furthermore, analysis of Cyanogen bromide (CNBr) and tryptic peptides displayed no evidence of post-translational modification. In the first in-depth study of its kind, we unequivocally demonstrate that the S-layer proteins from the C. difficile strains investigated are not glycosylated.
Subject(s)
Bacterial Proteins/chemistry , Clostridioides difficile/chemistry , Membrane Glycoproteins/chemistry , Recombinant Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Clostridioides difficile/genetics , Cyanogen Bromide/chemistry , Escherichia coli/genetics , Glycosylation , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Molecular Weight , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Trypsin/metabolismABSTRACT
BACKGROUND: Single chain Fvs (scFvs) are widely applied in research, diagnostics and therapeutic settings. Display and selection from combinatorial libraries is the main route to their discovery and many factors influence the success of this process. They exhibit low thermodynamic stability, resulting in low levels of premature cytosolic folding or aggregation which facilitates sec YEG-mediated translocation and phage in E. coli. However, there is little data analysing how this is related to and influenced by scFv protein expression. RESULTS: We characterised the relationship between overall scFv expression and display propensity for a panel of 15 anti-tetanus toxin scFvs and found a strong positive correlation (Rho = 0.88, p < 0.005) between the two parameters. Display propensity, overall expression and soluble localisation to the periplasm and extracellular fractions were clone specific characteristics which varied despite high levels of sequence homology. There was no correlation between display of scFv or its expression in non-fused (free) form with soluble scFv localisation to the periplasm or culture supernatant. This suggests that divergence in the fate of scFv-pIII and non-fused scFv after translocation to the periplasm accounts for the observed disparity. Differential degrees of periplasmic aggregation of non-fused scFv between clones may affect the partitioning of scFv in the periplasm and culture supernatant abrogating any correlation. We suggest that these factors do not apply to the scFv-pIII fusion since it remains anchored to the bacterial inner membrane as part of the innate phage packaging and budding process. CONCLUSION: We conclude that in the absence of premature cytosolic aggregation or folding, the propensity of a scFv to be displayed on phage is directly related to its overall expression level and is thus indirectly influenced by factors such as codon bias, mRNA abundance or putative DNA motifs affecting expression. This suggests that scFvs capable of high overall expression and display levels may not produce high yields of non phage-fused soluble protein in either the periplasmic or extracellular fractions of E. coli. This should be considered when screening clones selected from combinatorial libraries for further study.
Subject(s)
Coliphages/metabolism , Gene Expression , Immunoglobulin Variable Region/metabolism , Peptide Fragments/immunology , Peptide Library , Tetanus Toxin/immunology , Amino Acid Sequence , Coliphages/genetics , Densitometry , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Escherichia coli/metabolism , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics , Molecular Sequence Data , Periplasm/immunology , Spheroplasts/immunologyABSTRACT
We report the successful purification of lipopolysaccharide (LPS) from Burkholderia thailandensis, a Gram-negative bacterium, closely related to the highly pathogenic organisms B. pseudomallei and B. mallei. Burkholderia thailandensis LPS is shown to cross-react with rabbit and mouse sera obtained from inoculation with B. pseudomallei or B. mallei, respectively. These data suggest that B. thailandensis LPS shares similar structural features with LPS molecules from highly pathogenic Burkholderia species. This information may prove useful in ongoing efforts to develop novel vaccines and/or diagnostic reagents.
Subject(s)
Burkholderia/chemistry , Lipopolysaccharides/isolation & purification , Animals , Lipopolysaccharides/chemistry , Mice , RNA, Bacterial/isolation & purification , Rabbits , Species SpecificityABSTRACT
Many bacterial species produce a paracrystalline layer, the surface layer, which completely surrounds the exterior of the cell. In some bacteria, the surface layer is implicated in pathogenesis. Two proteins present in cell wall extracts from Clostridium tetani have been investigated and identified one of these has been unambiguously as the surface-layer protein (SLP). The gene, slpA, has been located in the genome of C. tetani E88 that encodes the SLP. The molecular mass of the protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is considerably larger than that predicted from the gene; however the protein does not appear to be glycosylated. Furthermore, analysis of five C. tetani strains, including three recent clinical isolates, shows considerable variation in the sizes of the SLP.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Clostridium tetani/metabolism , Amino Acid Motifs , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Clostridium tetani/genetics , Electrophoresis, Polyacrylamide Gel , Genetic Variation , Genome, Bacterial , Glycosylation , Mass SpectrometryABSTRACT
Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.
Subject(s)
Protein Structure, Quaternary , Tetanus Toxin/chemistry , Tetanus Toxin/metabolism , Binding Sites , Chromatography, Gel , Cysteine/metabolism , Dimerization , Electrophoresis, Polyacrylamide Gel , Gangliosides , Models, Molecular , Mutagenesis , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , X-RaysABSTRACT
In this study, the immunogenicities of the nontoxic H(C) fragment of tetanus toxin and derivatives lacking ganglioside binding activity were compared with that of tetanus toxoid after subcutaneous immunization of mice. Wild-type H(C) (H(C)WT) protein and tetanus toxoid both elicited strong antibody responses against toxoid and H(C) antigens and provided complete protection against toxin challenge. Mutants of H(C) containing deletions essential for ganglioside binding elicited lower responses than H(C)WT. H(C)M115, containing two amino acid substitutions within the ganglioside binding site, provided reduced protection against tetanus toxin challenge compared with H(C)WT, consistent with lower anti-H(C) and anti-toxoid antibody titers. Circular-dichroism spectroscopy and intrinsic fluorescence spectroscopy showed minimal structural perturbation in H(C)M115. We conclude that the presence of the ganglioside binding site within H(C) may be essential for induction of a fully protective anti-tetanus response comparable to that induced by tetanus toxoid by subcutaneous injection.
Subject(s)
Antibodies, Bacterial/blood , Gangliosides/metabolism , Peptide Fragments/immunology , Tetanus Toxin/immunology , Tetanus Toxoid/immunology , Animals , Female , Gene Deletion , Immunization , Mice , Peptide Fragments/administration & dosage , Peptide Fragments/genetics , Peptide Fragments/metabolism , Tetanus/prevention & control , Tetanus Toxin/administration & dosage , Tetanus Toxin/genetics , Tetanus Toxin/metabolism , Tetanus Toxoid/administration & dosage , Tetanus Toxoid/genetics , Tetanus Toxoid/metabolism , Toxins, BiologicalABSTRACT
Plant-expressed vaccines may provide a unique opportunity for generating anti-pathogen immunity, especially in countries where cold storage is lacking. In the following study, we show that soluble protein from tobacco leaves expressing fragment C of tetanus toxin protected mice against a lethal tetanus toxin challenge. More importantly, we show that a single intranasal (i.n.) vaccination was as efficient as oral delivery, inducing high levels of activated CD4(+) T cells and anti-toxin antibody. Unlike the oral route, i.n. delivery did not require the presence of adjuvant (cholera toxin). Indeed, addition of cholera toxin induced bystander immune responses to plant proteins as well. This is the first study documenting protective immunity by a single i.n. dose of plant vaccine. Plant-based vaccines are promising because they are more heat stable, are easy to produce, cheap and do not require needles.