Protective RNA nanovaccines against Mycobacterium avium subspecies hominissuis

The induction of an effective immune response is critical for the success of mRNA-based therapeutics. Here, we developed a nanoadjuvant system compromised of Quil-A and DOTAP (dioleoyl 3 trimethylammonium propane), hence named QTAP, for the efficient delivery of mRNA vaccine constructs into cells. Electron microscopy indicated that the complexation of mRNA with QTAP forms nanoparticles with an average size of 75 nm and which have ~90% encapsulation efficiency. The incorporation of pseudouridine-modified mRNA resulted in higher transfection efficiency and protein translation with low cytotoxicity than unmodified mRNA. When QTAP-mRNA or QTAP alone transfected macrophages, pro-inflammatory pathways (e.g., NLRP3, NF-kb, and MyD88) were upregulated, an indication of macrophage activation. In C57Bl/6 mice, QTAP nanovaccines encoding Ag85B and Hsp70 transcripts (QTAP-85B+H70) were able to elicit robust IgG antibody and IFN- ɣ, TNF-α, IL-2, and IL-17 cytokines responses. Following aerosol challenge with a clinical isolate of M. avium ss. hominissuis (M.ah), a significant reduction of mycobacterial counts was observed in lungs and spleens of only immunized animals at both 4- and 8-weeks post-challenge. As expected, reduced levels of M. ah were associated with diminished histological lesions and robust cell-mediated immunity. Interestingly, polyfunctional T-cells expressing IFN- ɣ, IL-2, and TNF- α were detected at 8 but not 4 weeks post-challenge. Overall, our analysis indicated that QTAP is a highly efficient transfection agent and could improve the immunogenicity of mRNA vaccines against pulmonary M. ah, an infection of significant public health importance, especially to the elderly and to those who are immune compromised

Genome-Wide Sequence Variation among Mycobacterium avium Subspecies paratuberculosis Isolates: A Better Understanding of Johne’s Disease Transmission Dynamics

Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne’s disease, infects many farmed ruminants, wild-life animals, and recently isolated from humans. To better understand the molecular pathogenesis of these infections, we analyzed the whole-genome sequences of several M. ap and M. avium subspecies avium (M. avium) isolates to gain insights into genomic diversity associated with variable hosts and environments. Using Next-generation sequencing technology, all six M. ap isolates showed a high percentage of similarity (98%) to the reference genome sequence of M. ap K-10 isolated from cattle. However, two M. avium isolates (DT 78 and Env 77) showed significant sequence diversity (only 87 and 40% similarity, respectively) compared to the reference strain M. avium 104, a reflection of the wide environmental niches of this group of mycobacteria. Within the M. ap isolates, genomic rearrangements (insertions/deletions) were not detected, and only unique single nucleotide polymorphisms (SNPs) were observed among M. ap isolates. While more of the SNPs (~100) in M. ap genomes were non-synonymous, a total of ~6,000 SNPs were detected among M. avium genomes, most of them were synonymous suggesting a differential selective pressure between M. ap and M. avium isolates. In addition, SNPs-based phylo-genomics had a enough discriminatory power to differentiate between isolates from different hosts but yet suggesting a bovine source of infection to other animals examined in this study. Interestingly, the human isolate (M. ap 4B) was closely related to a M. ap isolate from a dairy facility, suggesting a common source of infection. Overall, the identified phylo-genomes further supported the idea of a common ancestor to both M. ap and M. avium isolates. Genome-wide analysis described here could provide a strong foundation for a population genetic structure that could be useful for the analysis of mycobacterial evolution and for the tracking of Johne’s disease transmission among animals

Transcriptional Profiling of Mycobacterium Tuberculosis During Infection: Lessons Learned

Infection with Mycobacterium tuberculosis, the causative agent of tuberculosis, is considered one of the biggest infectious disease killers worldwide. A significant amount of attention has been directed toward revealing genes involved in the virulence and pathogenesis of this air-born pathogen. With the advances in technologies for transcriptional profiling, several groups, including ours, took advantage of DNA microarrays to identify transcriptional units differentially regulated by M. tuberculosis within a host. The main idea behind this approach is that pathogens tend to regulate their gene expression levels depending on the host microenvironment, and preferentially express those needed for survival. Identifying this class of genes will improve our understanding of pathogenesis. In our case, we identified an in vivo expressed genomic island that was preferentially active in murine lungs during early infection, as well as groups of genes active during chronic tuberculosis. Other studies have identified additional gene groups that are active during macrophage infection and even in human lungs. Despite all of these findings, one of the lingering questions remaining was whether in vivo expressed transcripts are relevant to the virulence, pathogenesis, and persistence of the organism. The work of our group and others addressed this question by examining the contribution of in vivo expressed genes using a strategy based on gene deletions followed by animal infections. Overall, the analysis of most of the in vivo expressed genes supported a role of these genes in M. tuberculosis pathogenesis. Further, these data suggest that in vivo transcriptional profiling is a valid approach to identify genes required for bacterial pathogenesis

Optical mapping of the Mycobacterium avium subspecies paratuberculosis genome

<p>Abstract</p> <p>Background</p> <p>Infection of cattle with <it>Mycobacterium avium </it>subspecies <it>paratuberculosis </it>(<it>M. ap</it>) causes severe economic losses to the dairy industry in the USA and worldwide. In an effort to better examine diversity among <it>M. ap </it>strains, we used optical mapping to profile genomic variations between strains of <it>M. ap </it>K-10 (sequenced strain) and <it>M. ap </it>ATCC 19698 (type strain).</p> <p>Results</p> <p>The assembled physical restriction map of <it>M. ap </it>ATCC 19698 showed a genome size of 4,839 kb compared to the sequenced K-10 genome of 4,830 kb. Interestingly, alignment of the optical map of the <it>M. ap </it>ATCC 19698 genome to the complete <it>M. ap </it>K-10 genome sequence revealed a 648-kb inversion around the origin of replication. However, Southern blotting, PCR amplification and sequencing analyses of the inverted region revealed that the genome of <it>M. ap </it>K-10 differs from the published sequence in the region starting from 4,197,080 bp to 11,150 bp, spanning the origin of replication. Additionally, two new copies of the coding sequences > 99.8% were identified, identical to the MAP0849c and MAP0850c genes located immediately downstream of the MAP3758c gene.</p> <p>Conclusion</p> <p>The optical map of <it>M. ap </it>ATCC 19698 clearly indicated the miss-assembly of the sequenced genome of <it>M. ap </it>K-10. Moreover, it identified 2 new genes in <it>M. ap </it>K-10 genome. This analysis strongly advocates for the utility of physical mapping protocols to complement genome sequencing projects.</p

Biomarkers for Early Stages of Johne’s Disease Infection and Immunization in Goats

Background:Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne’s disease, a chronic enteric infection of ruminants. Infection occurs within the first few months of life but remains subclinical for an average of 2–5 years. Current diagnostics to detect early subclinical infections lack diagnostic sensitivity, which hinders disease control resulting in significant economic losses to the dairy industry worldwide. The pathophysiology of early infection with M. paratuberculosis is still not well understood and represents a key hurdle toward the development of better diagnostics.Methods: The present study employed a large-scale RNA-Sequencing technology to better understand early stages of M. paratuberculosis infection and immunization. Specifically, gene expression profiles of peripheral blood mononuclear cells (PBMCs) from infected or vaccinated goats were compared to controls.Results: When compared to the naïve control goats, we identified a large number of transcripts (N = 226, 1018, 1714) that were differentially expressed in the M. paratuberculosis-infected goats, goats vaccinated with live attenuated or inactivated vaccines. There were also 1133 differentially expressed (DE) transcripts between vaccinated goats and infected ones. Bioinformatics evaluation of the DE genes indicated the regulation of a large number of genes with immunity and inflammatory functions including IL-18BP, IFN-γ, IL-17A, NOS2, LIPG, and IL-22. Interestingly, a large number of goat genes (N = 667) were regulated whether live or inactivated vaccine were used. Some of the regulated genes (e.g., IL-17A, IFN-γ) continued its unique transcriptional profile up to 12 months post-challenge.Conclusion: Overall, transcriptome analysis of infected and/or immunized goats identified potential targets for developing early diagnostics for Johne’s disease and a potential approach to differentiate infected from vaccinated animals. A similar approach could be used to analyze later stages of Johne’s disease or other chronic infections

Screening of Mycobacterium avium subsp. paratuberculosis mutants for attenuation in a bovine monocyte-derived macrophage model

Vaccination remains a major tool for prevention and progression of Johne’s disease, a chronic enteritis of ruminants worldwide. Currently there is only one licensed vaccine within the United States and two vaccines licensed internationally against Johne’s disease. All licensed vaccines reduce fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP) and delay disease progression. However, there are no available vaccines that prevent disease onset. A joint effort by the Johne’s Disease Integrated Program (JDIP), a USDA-funded consortium, and USDA—APHIS/VS sought to identify transposon insertion mutant strains as vaccine candidates in part of a three phase study. The focus of the Phase I study was to evaluate MAP mutant attenuation in a well-defined in vitro bovine monocyte-derived macrophage (MDM) model. Attenuation was determined by colony forming unit (CFUs) counts and slope estimates. Based on CFU counts alone, the MDM model did not identify any mutant that significantly differed from the wild-type control, MAP K-10. Slope estimates using mixed models approach identified six mutants as being attenuated. These were enrolled in protection studies involving murine and baby goat vaccination-challenge models. MDM based approach identified trends in attenuation but this did not correlate with protection in a natural host model. These results suggest the need for alternative strategies for Johne’s disease vaccine candidate screening and evaluation

Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association

<p>Abstract</p> <p>Background</p> <p>The genome of <it>Mycobacterium avium </it>subspecies <it>paratuberculosis </it>(<it>MAP</it>) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map.</p> <p>Results</p> <p>Our analysis of one of the isolates, <it>MAP </it>S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the <it>MAP </it>S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human <it>M. avium </it>subsp. <it>hominissuis </it>strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb). Whole-genome sequencing of 2 additional sheep strains of <it>MAP </it>(JTC1074 and JTC7565) further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level.</p> <p>Conclusions</p> <p>Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the <it>M. avium </it>complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of <it>MAP</it>. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for <it>M. avium </it>complex strains based on these genome sequences.</p

Evaluation of eight live attenuated vaccine candidates for protection against challenge with virulent Mycobacterium avium subspecies paratuberclosis in mice

Johne\u27s disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep, and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne\u27s disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistaence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321, and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues,indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP 1566::Tn 5370) was the most protective where as strain 318 (intergenic Tn 5367 insertion between MAP 0282c and MAP 0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward in to a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats

A rational framework for evaluating the next generation of vaccines against Mycobacterium avium subspecies paratuberculosis

Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne\u27s disease in cattle and sheep. These studies used whole-cell inactived vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transcriptional mutation of virulence and metablolic genes in MAP. In order to accelerate the process of identification and comparative elvaluation of he most promising modified live MAP vaccine candidates, members of a multi-institutional USDA- funded research consortium, the Johne\u27s disease integrated program (JDIP), met to established a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johne\u27s disease and other animals pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development