House Dust Mite-Induced Allergic Lung Inflammation Is Not Exacerbated in Sickle Cell Disease Mice.

AIM: Asthma appears to be a common comorbid condition in children with sickle cell disease (SCD), and such individuals may be at a higher risk for increased morbidity and mortality. However, several reports have indicated that asthma severity is not particularly high in those with SCD, and airway hyperreactivity and wheeze may be independently associated with SCD. In SCD mice, exacerbated allergic airway disease (AAD) has been observed in response to the model antigen ovalbumin (OVA). We sought to determine if allergic lung inflammation is also exacerbated in SCD mice when they are exposed to the human allergen, house dust mite (HDM). METHODS AND RESULTS: Eosinophil counts in bronchoalveolar lavage fluid were determined by cytocentrifugation and increased in both wild-type (WT) and SCD mice after acute exposure to a high dose (25 µg) of HDM, which then decreased in chronically exposed mice. WT mice exposed to a low dose of HDM (1 µg) followed the same pattern of eosinophil flux, but SCD mice did not induce much eosinophilia after acute exposure to HDM. As was observed in previous studies, lung lesions similarly increased in severity in both WT and SCD mice after acute exposure to HDM, which remained elevated after chronic exposure. Furthermore, serum HDM-specific IgE titers similarly increased and selected serum cytokines were similar in both WT and SCD mice. CONCLUSION: These results contrast with previous reports of exacerbated AAD in SCD mice exposed to OVA and support the alternative hypothesis that asthmatic responses are normal in those with SCD.

Long-Term Exposure to House Dust Mite Leads to the Suppression of Allergic Airway Disease Despite Persistent Lung Inflammation.

BACKGROUND: Allergic asthma is a major cause of worldwide morbidity and results from inadequate immune regulation in response to innocuous, environmental antigens. The need exists to understand the mechanisms that promote nonreactivity to human-relevant allergens such as house dust mite (HDM) in order to develop curative therapies for asthma. The aim of our study was to compare the effects of short-, intermediate- and long-term HDM administration in a murine asthma model and determine the ability of long-term HDM exposure to suppress allergic inflammation. METHODS: C57BL/6 mice were intranasally instilled with HDM for short-term (2 weeks), intermediate-term (5 weeks) and long-term (11 weeks) periods to induce allergic airway disease (AAD). The severity of AAD was compared across all stages of the model via both immunological and pulmonary parameters. RESULTS: Short- and intermediate-term HDM exposure stimulated the development of AAD that included eosinophilia in the bronchoalveolar lavage fluid (BALF), pronounced airway hyperreactivity (AHR) and evidence of lung inflammation. Long-term HDM exposure promoted the suppression of AAD, with a loss of BALF eosinophilia and AHR despite persistent mononuclear inflammation in the lungs. Suppression of AAD with long-term HDM exposure was associated with an increase in both Foxp3+ regulatory T cells and IL-10-positive alveolar macrophages at the site of inflammation. CONCLUSIONS: This model recapitulates the key features of human asthma and may facilitate investigation into the mechanisms that promote immunological tolerance against clinically relevant aeroallergens.

Semi-automated curation of metabolic models via flux balance analysis: a case study with Mycoplasma gallisepticum.

Primarily used for metabolic engineering and synthetic biology, genome-scale metabolic modeling shows tremendous potential as a tool for fundamental research and curation of metabolism. Through a novel integration of flux balance analysis and genetic algorithms, a strategy to curate metabolic networks and facilitate identification of metabolic pathways that may not be directly inferable solely from genome annotation was developed. Specifically, metabolites involved in unknown reactions can be determined, and potentially erroneous pathways can be identified. The procedure developed allows for new fundamental insight into metabolism, as well as acting as a semi-automated curation methodology for genome-scale metabolic modeling. To validate the methodology, a genome-scale metabolic model for the bacterium Mycoplasma gallisepticum was created. Several reactions not predicted by the genome annotation were postulated and validated via the literature. The model predicted an average growth rate of 0.358±0.12[Formula: see text], closely matching the experimentally determined growth rate of M. gallisepticum of 0.244±0.03[Formula: see text]. This work presents a powerful algorithm for facilitating the identification and curation of previously known and new metabolic pathways, as well as presenting the first genome-scale reconstruction of M. gallisepticum.

Splenic morphological changes are accompanied by altered baseline immunity in a mouse model of sickle-cell disease.

Although functional asplenia from infarctions may be a major contributor to increased infectious mortality in sickle-cell disease (SCD), this relationship has not been fully defined. We used the transgenic Berkeley SCD mouse to define blood and splenic immunophenotypic differences in this model compared with C57BL/6 and hemizygous controls. In the serum of SCD mice, we found increased IgG2a and suppressed IgM, IgG2b, and IgA levels. Serum IL-6 levels in SCD mice were elevated, whereas IL-1α, CXCL10, and CCL5 levels were decreased. The blood of SCD mice had higher white blood cell counts, with an increased percentage of lymphocytes and decreases in other leukocytes. Immunophenotyping of lymphocytes revealed higher percentages of CD8(+) and T-regulatory cells and lower percentages of B cells. SCD mouse spleens exhibited histological disorganization, with reduction of defined lymphoid follicles and expansion of red pulp, a greater than fourfold increase in splenic mononuclear cells, marked expansion of the nucleated red blood cell fraction, and B-cell and CD8(+) T-cell lymphopenia. Within the splenic B-cell population, there was a significant decrease in B-1a B cells, with a corresponding decrease in IgA secreting plasma cells in the gut. Confocal microscopy of spleens demonstrated complete disruption of the normal lymphofollicular structure in the white pulp of SCD mice without distinct B, T, and marginal zones. Our findings suggest that altered SCD splenic morphological characteristics result in an impaired systemic immune response.

Transgenic sickle cell disease mice have high mortality and dysregulated immune responses after vaccination.

BACKGROUND: Children with sickle cell disease (SCD) are susceptible to recurrent infections, which are often life threatening and necessitate frequent vaccinations. Given the altered baseline immunity and proinflammatory state associated with SCD, we sought to determine the relative safety and efficacy of vaccination in transgenic SCD mice. METHODS: Eight-week-old SCD mice were vaccinated with ovalbumin and aluminum hydroxide weekly for 3 wk by the intraperitoneal or intramuscular route. One week after the third vaccination, serum cytokines/chemokines, immunoglobulins, and bronchoalveolar lavage fluid cytokines were measured. RESULTS: Only SCD mice were prone to mortality associated with vaccination, as 40% of the animals died after the intraperitoneal vaccinations and 50% died after the intramuscular vaccinations. Serum IgG2b and IgM were significantly lower in SCD mice than in C57BL/6 mice after vaccination, but ovalbumin-specific IgE was significantly higher. Serum interleukin (IL)-1α, IL-2, IL-5, macrophage inflammatory protein 1α, and granulocyte macrophage-colony stimulating factor were significantly lower in SCD mice than in C57BL/6 mice after vaccination, whereas bronchoalveolar lavage fluid IL-1ß and IL-6 were increased. CONCLUSION: Mice with SCD appear to have a dysregulated immune response to vaccination. Thus, the relative safety and immunogenicity of vaccination should be studied in greater detail in the context of SCD.

LC-MS/MS Identification of a Bromelain Peptide Biomarker from Ananas comosus Merr.

Bromelain (Br) is a cysteine peptidase (GenBank AEH26024.1) from pineapple, with over 40 years of clinical use. The constituents mediating its anti-inflammatory activity are not thoroughly characterized and no peptide biomarker exists. Our objective is to characterize Br raw material and identify peptides in the plasma of Br treated mice. After SDS-PAGE in-gel digestion, Br (VN#3507; Middletown, CT, USA) peptides were analyzed via LC/MS/MS using 95% protein probability, 95% peptide probability, and a minimum peptide number = 5. Br spiked mouse plasma (1 ug/ul) and plasma from i.p. treated mice (12 mg/kg) were assessed using SRM. In Br raw material, we identified seven proteins: four proteases, one jacalin-like lectin, and two protease inhibitors. In Br spiked mouse plasma, six proteins (ananain, bromelain inhibitor, cysteine proteinase AN11, FB1035 precursor, FBSB precursor, and jacalin-like lectin) were identified. Using LC/MS/MS, we identified the unique peptide, DYGAVNEVK, derived from FB1035, in the plasma of i.p. Br treated mice. The spectral count of this peptide peaked at 6 hrs and was undetectable by 24 hrs. In this study, a novel Br peptide was identified in the plasma of treated mice for the first time. This Br peptide could serve as a biomarker to standardize the therapeutic dose and maximize clinical utility.

Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease.

Bacterial lipoproteins are an often-underappreciated class of microbe-associated molecular patterns with potent immunomodulatory activity. We previously reported that vaccination of BALB/c mice with Mycoplasma pneumoniae (Mp) lipid-associated membrane proteins (LAMPs) resulted in lipoprotein-dependent vaccine enhanced disease after challenge with virulent Mp, though the immune responses underpinning this phenomenon remain poorly understood. Herein, we report that lipoprotein-induced VED in a mouse model is associated with elevated inflammatory cytokines TNF-α, IL-1ß, IL-6, IL-17A, and KC in lung lavage fluid and with suppurative pneumonia marked by exuberant neutrophilia in the pulmonary parenchyma. Whole-lung-digest flow cytometry and RNAScope analysis identified multiple cellular sources for IL-17A, and the numbers of IL-17A producing cells were increased in LAMPs-vaccinated/Mp-challenged animals compared to controls. Specific IL-17A or neutrophil depletion reduced disease severity in our VED model-indicating that Mp lipoproteins induce VED in an IL-17A-dependent manner and through exuberant neutrophil recruitment. IL-17A neutralization reduced levels of TNF-α, IL-1ß, IL-6, and KC, indicating that IL-17A preceded other inflammatory cytokines. Surprisingly, we found that IL-17A neutralization impaired bacterial clearance, while neutrophil depletion improved it-indicating that, while IL-17A appears to confer both maladaptive and protective responses, neutrophils play an entirely maladaptive role in VED. Given that lipoproteins are found in virtually all bacteria, the potential for lipoprotein-mediated maladaptive inflammatory responses should be taken into consideration when developing vaccines against bacterial pathogens.

A Single-Administration Microneedle Skin Patch for Multi-Burst Release of Vaccine against SARS-CoV-2.

The necessity for multiple injections and cold-chain storage has contributed to suboptimal vaccine utilization, especially in pandemic situations. Thermally-stable and single-administration vaccines hold a great potential to revolutionize the global immunization process. Here, a new approach to thermally stabilize protein-based antigens is presented and a new high-throughput antigen-loading process is devised to create a single-administration, pulsatile-release microneedle (MN) patch which can deliver a recombinant SARS-CoV-2 S1-RBD protein-a model for the COVID-19 vaccine. Nearly 100% of the protein antigen could be stabilized at temperatures up to 100 °C for at least 1 h and at an average human body temperature (37 °C) for up to 4 months. Arrays of the stabilized S1-RBD formulations can be loaded into the MN shells via a single-alignment assembly step. The fabricated MNs are administered at a single time into the skin of rats and induce antibody response which could neutralize authentic SARS-CoV-2 viruses, providing similar immunogenic effect to that induced by multiple bolus injections of the same antigen stored in conventional cold-chain conditions. The MN system presented herein could offer the key solution to global immunization campaigns by avoiding low patient compliance, the requirement for cold-chain storage, and the need for multiple booster injections.

B cells oppose Mycoplasma pneumoniae vaccine enhanced disease and limit bacterial colonization of the lungs.

Development of an effective vaccine for Mycoplasma pneumoniae has been hindered by reports of Vaccine Enhanced Disease (VED) in test subjects vaccinated and challenged in studies conducted in the 1960s. The exact mechanism of disease exacerbation has yet to be fully described, but host immune responses to Lipid-Associated Membrane Proteins (LAMPs) lipoprotein lipid moieties have been implicated. LAMPs-induced exacerbation appears to involve helper T cell recall responses, due in part to their influence on neutrophil recruitment and subsequent inflammatory responses in the lung. Herein, we characterized the functions of host B cell responses to M. pneumoniae LAMPs and delipidated-LAMPs (dLAMPs) by conducting passive transfer and B cell depletion studies to assess their contribution to disease exacerbation or protection using a BALB/c mouse model. We found that antibody responses to M. pneumoniae LAMPs and dLAMPs differ in magnitude, but not in isotype or subclass. Passive transfer, dLAMP denaturation, and monoclonal antibody studies indicate that antibodies do not cause VED, but do appear to contribute to control of bacterial loads in the lungs. Depletion of B cells prior to LAMPs-vaccination results in significantly enhanced pathology in comparison to B cell competent controls, suggesting a possible regulatory role of B cells distinct from antibody secretion. Taken together, our findings suggest that B cell antibody responses to M. pneumoniae contribute to, but are insufficient for protection against challenge on their own, and that other functional properties of B cells are necessary to limit exacerbation of disease in LAMPs-vaccinated mice after infection.

A GATA3 Targeting Nucleic Acid Nanocapsule for In Vivo Gene Regulation in Asthma.

Allergic asthma is one of the leading chronic lung diseases of both children and adults worldwide, resulting in significant morbidity and mortality in affected individuals. Many patients have severe asthma, which is refractory to treatment, illustrating the need for the development of new therapeutics for this disease. Herein, we describe the use of a peptide cross-linked nucleic acid nanocapsule (NAN) for the delivery of a GATA3-specific DNAzyme to immune cells, with demonstration of modulated transcriptional activity and behavior of those cells. The NAN, built from peptide cross-linked surfactants, is chemically designed to degrade under inflammation conditions releasing individual DNAzyme-surfactant conjugates in response to proteolytic enzymes. Using the NAN, GATA3 DNAzymes were delivered efficiently to human peripheral blood mononuclear cells, with clear evidence of uptake by CD4+ helper T cells without the need for harsh transfection agents. Knockdown of GATA3 was achieved in vitro using human Jurkat T cells, which express GATA3 under homeostatic conditions. Additionally, mice treated with DNAzyme-NANs during house dust mite (HDM)-induced asthma developed less severe allergic lung inflammation than HDM-only control mice, as measured by pulmonary eosinophilia. This study suggests that peptide cross-linked GATA3 DNAzyme-NANs may have the potential to decrease the severity of asthma symptoms in human patients, and development of this technology for human use warrants further investigation.

Transdermal microneedles for the programmable burst release of multiple vaccine payloads.

Repeated bolus injections are associated with higher costs and poor compliance and can hinder the implementation of global immunization campaigns. Here, we report the development and preclinical testing of patches of transdermal core-shell microneedles-which were fabricated by the micromoulding and alignment of vaccine cores and shells made from poly(lactic-co-glycolic acid) with varying degradability kinetics-for the preprogrammed burst release of vaccine payloads over a period of a few days to more than a month from a single administration. In rats, microneedles loaded with a clinically available vaccine (Prevnar-13) against the bacterium Streptococcus pneumoniae induced immune responses that were similar to immune responses observed after multiple subcutaneous bolus injections, and led to immune protection against a lethal bacterial dose. Microneedle patches delivering preprogrammed doses may offer an alternative strategy to prophylactic and therapeutic protocols that require multiple injections.

Lipid moieties of Mycoplasma pneumoniae lipoproteins are the causative factor of vaccine-enhanced disease.

Vaccine-enhanced disease (VED) occurs as a result of vaccination followed by infection with virulent Mycoplasma pneumoniae. To date VED has prevented development of an efficacious vaccine against this significant human respiratory pathogen. Herein we report that vaccination of BALB/c mice with M. pneumoniae lipid-associated membrane proteins (LAMPs) induces lung lesions consistent with exacerbated disease following challenge, without reducing bacterial loads. Removal of lipid moieties from LAMPs prior to vaccination eliminates VED and reduces bacterial loads after infection. Collectively, these data indicate that lipid moieties of lipoproteins are the causative factors of M. pneumoniae VED.

The VP1 G-H loop hypervariable epitope contributes to protective immunity against Foot and Mouth Disease Virus in swine.

Foot and Mouth Disease is a highly contagious and economically important disease of livestock. While vaccination is often effective at controlling viral spread, failures can occur due to strain mismatch or viral mutation. Foot and Mouth Disease Virus (FMDV) possesses a hypervariable region within the G-H Loop of VP1, a capsid protein commonly associated with virus neutralization. Here, we investigate the effect of replacement of the G-H loop hypervariable epitope with a xenoepitope from PRRS virus on the immunogenicity and efficacy of an adenovirus vectored FMDV vaccine (Ad5-FMD). Pigs were vaccinated with Ad5-FMD, the modified Ad5-FMDxeno, or PBS, followed by intradermal challenge with FDMV strain O1 Manisa at 21 days post-vaccination. While overall serum antibody titers were significantly higher in Ad5-FMDxeno vaccinated animals, neutralizing antibody titers were decreased in pigs that received Ad5-FMDxeno, when compared to those vaccinated with Ad5-FMD, prior to viral challenge, indicative of immune redirection away from VP1 towards non-neutralizing epitopes. As expected, animals vaccinated with unmodified Ad5-FMD were protected from lesions, fever, and viremia. In contrast, animals vaccinated with Ad5-FMDxeno developed clinical signs and viremia, but at lower levels than that observed in PBS-treated controls. No significant difference was found in nasal shedding of virions between the two Ad5-FMD vaccinated groups. This data suggests that the hypervariable epitope of the VP1 G-H loop contributes to protective immunity conferred by Ad5 vector-delivered FMD vaccines in swine, and cannot be substituted without a loss of immunogenicity.

Snake Fungal Disease in Colubridae Snakes in Connecticut, USA in 2015 and 2017.

Snake fungal disease (SFD), caused by the fungus Ophidiomyces ophiodiicola, is an emerging threat to wild snake populations in the US. Data regarding its distribution, prevalence, and population-level impacts are sparse, and more information is needed to better manage SFD in the wild. In this study, we captured 38 wild snakes of five species in Connecticut in the summers of 2015 and 2017. Skin lesions were biopsied and evaluated histologically for fungal dermatitis. At least one individual from each species was positive for SFD, and 48% of snakes sampled in 2015 and 39% of snakes sampled in 2017 were positive for SFD. A Dekay's brownsnake (Storeria dekayi dekayi) with SFD lesions, captured in the summer of 2017, extended the host range of the disease. Thus, SFD was present in wild Connecticut snakes in 2015 and 2017, which demonstrated a wide-spread distribution throughout the state.

Influenza Vaccination Protects Against Pandemic H1N1 Infection in Sickle Cell Disease Mice.

Influenza infection is associated with enhanced pathology in individuals with sickle cell disease (SCD). Despite being a high priority group for annual influenza vaccination, little is known about long-term responses to influenza vaccination in this patient population. To model flu vaccination, we inoculated SCD and wild type (WT) littermate mice with the seasonal flu vaccine [containing pandemic H1N1 (pH1N1) antigen], bled the mice before and after vaccination, and intranasally challenged them with a high dose (400 PFU) of pH1N1 12 weeks later. Both WT and SCD mice were fully protected from infection, and anti-influenza immunoglobulin G titers were significantly elevated in both groups after vaccination. It appears that flu vaccination is effective in SCD mice and our data support the clinical practice of regular flu vaccination in SCD patients.

Peritoneal B-1b and B-2 B-cells confer long-term protection from pneumococcal serotype 3 infection after vaccination with Prevnar-13 and are defective in sickle cell disease mice.

Long-term immunity after inoculation with the pneumococcal conjugate vaccine (Prevnar-13) is impaired in sickle cell disease (SCD) mice. We sought to determine which B-cell subsets are defective in SCD mice after vaccination with Prevnar-13, yet confer long-term immunity in wild-type (WT) mice. We vaccinated WT and SCD mice three times at three week intervals with Prevnar-13. Fourteen weeks later, 5∗104 cells of isolated peritoneal B-1a, B-1b, and B-2 cells were harvested and intraperitoneally transferred to Rag -/- recipients. A week later recipients were intraperitoneally challenged with 103CFU of Streptococcus pneumoniae (serotype 3). Recipient mice that received either B-1b or B-2 B-cells from WT mice survived challenge, whereas mice that received B-1a cells died. Recipient mice that received B-1a, B-1b, or B-2 cells from SCD mice died after challenge. Both B-1b and B-2 cells appear to confer long-term immunity after Prevnar-13 vaccination, yet neither subset functions properly in SCD mice.

Poor Long-Term Efficacy of Prevnar-13 in Sickle Cell Disease Mice Is Associated with an Inability to Sustain Pneumococcal-Specific Antibody Titers.

BACKGROUND: One of the most common causes of morbidity and mortality in children with sickle cell disease (SCD) is infection with the pneumococcal bacterium (Streptococcus pneumoniae). Unfortunately, the polysaccharide-conjugate vaccine appears to be less effective in individuals with SCD when compared to the general population. We sought to better understand the relative efficacy of pneumococcal vaccination in a SCD mouse challenge model. METHODS: Transgenic control and SCD mice were monitored for mortality after intranasal pneumococcal infection or pneumococcal vaccination with Prevnar-13 and type-matched challenge. Anti-pneumococcal antibody titers were measured by ELISA and opsonophagocytosis was measured in vitro. RESULTS: Mortality after pneumococcal infection was similar between control and SCD mice. However, after three intramuscular polysaccharide-conjugate vaccinations, all control mice were protected following high-dose intranasal infection, whereas 60% of SCD mice died. Anti-pneumococcal antibody titers showed initial IgG and IgM responses in both groups, but waning titers were observed in the SCD group, even after boosting. When functionally assayed in vitro, serum from SCD mice 13 weeks after a second booster shot maintained little to no ability to opsonize pneumococci, while serum from control mice sustained a significantly higher capacity opsonization. Thus, it appears that SCD mice do not maintain antibody responses to pneumococcal polysaccharides after Prevnar-13 vaccination, thereby leaving them susceptible to mortality after type-matched infection. CONCLUSION: Our results emphasize the need to better understand the correlates of immune protection in SCD so that pneumococcal vaccines can be improved and mortality reduced in this susceptible population.

Bromelain Inhibits Allergic Sensitization and Murine Asthma via Modulation of Dendritic Cells.

The incidence of atopic conditions has increased in industrialized countries. Persisting symptoms and concern for drug side-effects lead patients toward adjunctive treatments such as phytotherapy. Previously, we have shown that Bromelain (sBr), a mixture of cysteine proteases from pineapple, Ananas comosus, inhibits ovalbumin (OVA)-induced murine model of allergic airway disease (AAD). However, sBr's effect on development of AAD when treatment is administered throughout OVA-alum sensitization was unknown and is the aim of the present study. C57BL/6J mice were sensitized with OVA/alum and challenged with 7 days OVA aerosol. sBr 6 mg/kg/0.5 ml or PBS vehicle were administered throughout sensitization. Lung, bronchoalveolar lavage (BAL), spleen, and lymph nodes were processed for flow cytometry and OVA-specific IgE was determined via ELISA. sBr treatment throughout OVA-alum sensitization significantly reduced the development of AAD (BAL eosinophils and lymphocytes). OVA-specific IgE and OVA TET(+) cells were decreased. sBr reduced CD11c(+) dendritic cell subsets, and in vitro treatment of DCs significantly reduced CD44, a key receptor in both cell trafficking and activation. sBr was shown to reduce allergic sensitization and the generation of AAD upon antigen challenge. These results provide additional insight into sBr's anti-inflammatory and antiallergic properties and rationale for translation into the clinical arena.

Xenoepitope substitution avoids deceptive imprinting and broadens the immune response to foot-and-mouth disease virus.

Many RNA viruses encode error-prone polymerases which introduce mutations into B and T cell epitopes, providing a mechanism for immunological escape. When regions of hypervariability are found within immunodominant epitopes with no known function, they are referred to as "decoy epitopes," which often deceptively imprint the host's immune response. In this work, a decoy epitope was identified in the foot-and-mouth disease virus (FMDV) serotype O VP1 G-H loop after multiple sequence alignment of 118 isolates. A series of chimeric cyclic peptides resembling the type O G-H loop were prepared, each bearing a defined "B cell xenoepitope" from another virus in place of the native decoy epitope. These sequences were derived from porcine respiratory and reproductive syndrome virus (PRRSV), from HIV, or from a presumptively tolerogenic sequence from murine albumin and were subsequently used as immunogens in BALB/c mice. Cross-reactive antibody responses against all peptides were compared to a wild-type peptide and ovalbumin (OVA). A broadened antibody response was generated in animals inoculated with the PRRSV chimeric peptide, in which virus binding of serum antibodies was also observed. A B cell epitope mapping experiment did not reveal recognition of any contiguous linear epitopes, raising the possibility that the refocused response was directed to a conformational epitope. Taken together, these results indicate that xenoepitope substitution is a novel method for immune refocusing against decoy epitopes of RNA viruses such as FMDV as part of the rational design of next-generation vaccines.

Effects of sialidase knockout and complementation on virulence of Mycoplasma gallisepticum.

Reannotation of the pathogenic Mycoplasma gallisepticum strain R(low) genome identified the hypothetical gene MGA_0329 as a homolog of the sialidase gene MS53_0199 of Mycoplasma synoviae strain MS53. Potent sialidase activity was subsequently quantitated in several M. gallisepticum strains. Because sialidase activity levels correlate significantly with differing M. synoviae strain virulence, we hypothesized this enzyme may also influence the virulence of M. gallisepticum. MGA_0329 was disrupted in strain R(low) to create mutants 6, 358 and P1C5, which resulted in the loss of sialidase activity in all three mutants. Chickens infected with the knockout mutants had significantly less severe (P<0.05) tracheal lesions and tracheal mucosal thickening than chickens infected with equal doses of strain R(low). Significantly fewer (P<0.05) CCU especially of strains 6 and P1C5 were recovered at necropsy. Mini-Tn4001tet plasmid pTF20 carrying a wild-type copy of MGA_0329 with its native promoter was used to complement the genetic lesion in strain P1C5. Three clones derived from P1C5, each having one copy of MGA_0329 stably transposed into a different site in its genome, expressed sialidase restored to wild-type activity levels (1.58×10(-8)U/CFU). Complementation of P1C5 with MGA_0329 did not restore it to wild-type levels of virulence, indicating that the contribution of sialidase to M. gallisepticum virulence is not straightforward.