The Regulation of Nucleic Acid Vaccine Responses by the Microbiome.

Nucleic acid vaccines, including both RNA and DNA platforms, are key technologies that have considerable promise in combating both infectious disease and cancer. However, little is known about the extrinsic factors that regulate nucleic acid vaccine responses and which may determine their effectiveness. The microbiome is recognized as a significant regulator of immune development and response, whose role in regulating some traditional vaccine platforms has recently been discovered. Using germ-free and specific pathogen-free mouse models in combination with different protein, DNA, and mRNA vaccine regimens, we demonstrate that the microbiome is a significant regulator of nucleic acid vaccine immunogenicity. Although the presence of the microbiome enhances CD8+ T cell responses to mRNA lipid nanoparticle immunization, the microbiome suppresses Ig and CD4+ T cell responses to DNA-prime, DNA-protein-boost immunization, indicating contrasting roles for the microbiome in the regulation of these different nucleic acid vaccine platforms. In the case of mRNA lipid nanoparticle vaccination, germ-free mice display reduced dendritic cell/macrophage activation that may underlie the deficient vaccine response. Our study identifies the microbiome as a relevant determinant of nucleic acid vaccine response with implications for continued therapeutic development and deployment of these vaccines.

Quantitative PCR analysis of functional genes in iron-rich microbial mats at an active hydrothermal vent system (Lo'ihi Seamount, Hawai'i).

The chemolithotrophic Zetaproteobacteria represent a novel class of Proteobacteria which oxidize Fe(II) to Fe(III) and are the dominant bacterial population in iron-rich microbial mats. Zetaproteobacteria were first discovered at Lo'ihi Seamount, located 35 km southeast off the big island of Hawai'i, which is characterized by low-temperature diffuse hydrothermal venting. Novel nondegenerate quantitative PCR (qPCR) assays for genes associated with microbial nitrogen fixation, denitrification, arsenic detoxification, Calvin-Benson-Bassham (CBB), and reductive tricarboxylic acid (rTCA) cycles were developed using selected microbial mat community-derived metagenomes. Nitrogen fixation genes were not detected, but all other functional genes were present. This suggests that arsenic detoxification and denitrification processes are likely cooccurring in addition to two modes of carbon fixation. Two groups of microbial mat community types were identified by terminal restriction fragment length polymorphism (T-RFLP) and were further described based on qPCR data for zetaproteobacterial abundance and carbon fixation mode preference. qPCR variance was associated with mat morphology but not with temperature or sample site. Geochemistry data were significantly associated with sample site and mat morphology. Together, these qPCR assays constitute a functional gene signature for iron microbial mat communities across a broad array of temperatures, mat types, chemistries, and sampling sites at Lo'ihi Seamount.

The Regulation of Nucleic Acid Vaccine Responses by the Microbiome.

Nucleic acid vaccines, including both RNA and DNA platforms, are key technologies that have considerable promise in combating both infectious disease and cancer. However, little is known about the extrinsic factors that regulate nucleic acid vaccine responses and which may determine their effectiveness. The microbiome is recognized as a significant regulator of immune development and response, whose role in regulating some traditional vaccine platforms has recently been discovered. Using germ-free and specific-pathogen-free mouse models in combination with different protein, DNA, and mRNA vaccine regimens, we demonstrate that the microbiome is a significant regulator of nucleic acid vaccine immunogenicity. While the presence of the microbiome enhances CD8+ T cell responses to mRNA lipid nanoparticle (LNP) immunization, the microbiome suppresses immunoglobulin and CD4+ T cell responses to DNA-prime, DNA-protein-boost immunization, indicating contrasting roles for the microbiome in the regulation of these different nucleic acid vaccine platforms. In the case of mRNA-LNP vaccination, germ-free mice display reduced dendritic cell/macrophage activation that may underlie the deficient vaccine response. Our study identifies the microbiome as a relevant determinant of nucleic acid vaccine response with implications for their continued therapeutic development and deployment.

Oral tolerance to systemic vaccination remains intact without RORγt expression in regulatory T cells.

Many promising vaccine candidates and licensed vaccines lead to variable immune responses within humans. Studies suggest that environmental exposures in the gastrointestinal tract could contribute to a reduction in vaccine efficacy via immune tolerance at this site; this is partly achieved by a high abundance of regulatory T cells (Tregs). It is unclear if Treg subsets regulate systemic vaccine responses following oral antigen pre-exposure. Here, we implemented a conditional knock-out mouse model of RORγt+ Tregs to examine the role of these cells in mediating this process. Following oral exposure to the model antigen ovalbumin (OVA) prior to immunization, we found similar induction of vaccine-induced antibody responses in mice lacking RORγt expression in Tregs compared to sufficient controls. Use of various adjuvants led to distinct findings. Our data suggest that expression of RORγt+ within Tregs is not required to regulate tolerance to systemic vaccination following oral antigen exposure.

Fine-Scale Biogeography and the Inference of Ecological Interactions Among Neutrophilic Iron-Oxidizing Zetaproteobacteria as Determined by a Rule-Based Microbial Network.

Hydrothermal vents, such as those at Lo'ihi Seamount and the Mariana Arc and back-arc, release iron required to support life from the Earth's crust. In these ecosystems, bacteria and archaea can oxidize the released iron and therefore play an important role in the biogeochemical cycles of essential nutrients. These organisms often form microbial mats, and the primary producers in these communities can support diverse higher trophic levels. One such class of bacteria are the Zetaproteobacteria. This class of bacteria oxidize iron and commonly produce extracellular iron oxyhydroxide matrices that provide architecture to the microbial mats, so they are considered foundational members of the community and ecosystem engineers. Zetaproteobacteria are responsible for the majority of iron-oxidation in circumneutral, marine, low-oxygen environments. To study the composition of these communities, microbial mats were collected using a biomat sampler, which allows for fine-scale collection of microbial mats. DNA was then extracted and amplified for analysis of the SSU rRNA gene. After quality control and filtering, the SSU rRNA genes from Mariana Arc and Lo'ihi Seamount microbial mat communities were compared pairwise to determine which site exhibits a greater microbial diversity and how much community overlap exists between the two sites. In-depth analysis was performed with the rule-based microbial network (RMN) algorithm, which identified a possible competitive relationship across oligotypes of a cosmopolitan Zetaproteobacteria operational taxonomic unit (OTU). This result demonstrated the ecological relevance of oligotypes, or fine-scale OTU variants. The oligotype distributions of the cosmopolitan ZetaOTUs varied greatly across the Pacific Ocean. The competitive relationship between dominant oligotypes at Lo'ihi Seamount and the Mariana Arc and back-arc may be driving their differential distributions across the two regions and may result in species divergence within a cosmopolitan ZetaOTU. This implementation of the RMN algorithm can both predict directional relationships within a community and provide insight to the level at which evolution is occurring across ecosystems.

Utilizing gnotobiotic models to inform the role of the microbiome in vaccine response heterogeneity.

PURPOSE OF REVIEW: Gnotobiotic models have the potential to provide substantial insight into how the microbiome shapes its host's response to vaccines. This review aims to summarize literature about the role of the microbiome in shaping the immune system and vaccine response heterogeneity, summarize gnotobiotic and other murine models that help us understand the immune system and vaccine response, and suggest novel ways that these models could be used to further understand vaccine response heterogeneity. RECENT FINDINGS: Clinical data have suggested that numerous vaccines' effectiveness are regulated by the microbiome and often correlate with the abundance of specific taxa. Gnotobiotic and other animal models are beginning to illuminate the complex effects induced by the presence of particular microbial groups and communities. Such models have identified microbial groups that improve vaccine response to rotavirus vaccine and identified pathways by which the microbiome influences response to influenza and other vaccines. SUMMARY: By applying a range of vaccines across gnotobiotic mouse models, researchers may be able to identify the effects of single microorganisms as well as interacting communities of microorganisms on the immune response.

Hidden diversity revealed by genome-resolved metagenomics of iron-oxidizing microbial mats from Lo'ihi Seamount, Hawai'i.

The Zetaproteobacteria are ubiquitous in marine environments, yet this class of Proteobacteria is only represented by a few closely-related cultured isolates. In high-iron environments, such as diffuse hydrothermal vents, the Zetaproteobacteria are important members of the community driving its structure. Biogeography of Zetaproteobacteria has shown two ubiquitous operational taxonomic units (OTUs), yet much is unknown about their genomic diversity. Genome-resolved metagenomics allows for the specific binning of microbial genomes based on genomic signatures present in composite metagenome assemblies. This resulted in the recovery of 93 genome bins, of which 34 were classified as Zetaproteobacteria. Form II ribulose 1,5-bisphosphate carboxylase genes were recovered from nearly all the Zetaproteobacteria genome bins. In addition, the Zetaproteobacteria genome bins contain genes for uptake and utilization of bioavailable nitrogen, detoxification of arsenic, and a terminal electron acceptor adapted for low oxygen concentration. Our results also support the hypothesis of a Cyc2-like protein as the site for iron oxidation, now detected across a majority of the Zetaproteobacteria genome bins. Whole genome comparisons showed a high genomic diversity across the Zetaproteobacteria OTUs and genome bins that were previously unidentified by SSU rRNA gene analysis. A single lineage of cosmopolitan Zetaproteobacteria (zOTU 2) was found to be monophyletic, based on cluster analysis of average nucleotide identity and average amino acid identity comparisons. From these data, we can begin to pinpoint genomic adaptations of the more ecologically ubiquitous Zetaproteobacteria, and further understand their environmental constraints and metabolic potential.

Community Structure of Lithotrophically-Driven Hydrothermal Microbial Mats from the Mariana Arc and Back-Arc.

The Mariana region exhibits a rich array of hydrothermal venting conditions in a complex geological setting, which provides a natural laboratory to study the influence of local environmental conditions on microbial community structure as well as large-scale patterns in microbial biogeography. We used high-throughput amplicon sequencing of the bacterial small subunit (SSU) rRNA gene from 22 microbial mats collected from four hydrothermally active locations along the Mariana Arc and back-arc to explore the structure of lithotrophically-based microbial mat communities. The vent effluent was classified as iron- or sulfur-rich corresponding with two distinct community types, dominated by either Zetaproteobacteria or Epsilonproteobacteria, respectively. The Zetaproteobacterial-based communities had the highest richness and diversity, which supports the hypothesis that Zetaproteobacteria function as ecosystem engineers creating a physical habitat within a chemical environment promoting enhanced microbial diversity. Gammaproteobacteria were also high in abundance within the iron-dominated mats and some likely contribute to primary production. In addition, we also compare sampling scale, showing that bulk sampling of microbial mats yields higher diversity than micro-scale sampling. We present a comprehensive analysis and offer new insights into the community structure and diversity of lithotrophically-driven microbial mats from a hydrothermal region associated with high microbial biodiversity. Our study indicates an important functional role of for the Zetaproteobacteria altering the mat habitat and enhancing community interactions and complexity.

Physiological and ecological implications of an iron- or hydrogen-oxidizing member of the Zetaproteobacteria, Ghiorsea bivora, gen. nov., sp. nov.

Chemosynthetic Fe-oxidizing communities are common at diffuse-flow hydrothermal vents throughout the world's oceans. The foundational members of these communities are the Zetaproteobacteria, a class of Proteobacteria that is primarily associated with ecosystems fueled by ferrous iron, Fe(II). We report here the discovery of two new isolates of Zetaproteobacteria isolated from the Mid-Atlantic Ridge (TAG-1), and the Mariana back-arc (SV-108), that are unique in that they can utilize either Fe(II) or molecular hydrogen (H2) as sole electron donor and oxygen as terminal electron acceptor for growth. Both strains precipitated Fe-oxyhydroxides as amorphous particulates. The cell doubling time on H2 vs Fe(II) for TAG-1 was 14.1 vs 21.8 h, and for SV-108 it was 16.3 vs 20 h, and it appeared both strains could use either H2 or Fe(II) simultaneously. The strains were close relatives, based on genomic analysis, and both possessed genes for the uptake NiFe-hydrogenase required for growth on H2. These two strains belong to Zetaproteobacteria operational taxonomic unit 9 (ZetaOTU9). A meta-analysis of public databases found ZetaOTU9 was only associated with Fe(II)-rich habitats, and not in other environments where known H2-oxidizers exist. These results expand the metabolic repertoire of the Zetaproteobacteria, yet confirm that Fe(II) metabolism is the primary driver of their physiology and ecology.

Draft Genome Sequence of Mariprofundus ferrooxydans Strain JV-1, Isolated from Loihi Seamount, Hawaii.

Mariprofundus ferrooxydans strain JV-1 was isolated in 1998 from Loihi Seamount, Hawaii. Here, we present the draft genome of strain JV-1, which shows similarity to other sequenced Mariprofundus isolates, strains PV-1 and M34.