Optimized Formulation of a Thermostable Spray-Dried Virus-Like Particle Vaccine against Human Papillomavirus.

Existing vaccines against human papillomavirus (HPV) require continuous cold-chain storage. Previously, we developed a bacteriophage virus-like particle (VLP)-based vaccine for HPV infection, which elicits broadly neutralizing antibodies against diverse HPV types. Here, we formulated these VLPs into a thermostable dry powder using a multicomponent excipient system and by optimizing the spray-drying parameters using a half-factorial design approach. Dry-powder VLPs were stable after spray drying and after long-term storage at elevated temperatures. Immunization of mice with a single dose of reconstituted dry-powder VLPs that were stored at 37 °C for more than a year elicited high anti-L2 IgG antibody titers. Spray-dried thermostable, broadly protective L2 bacteriophage VLPs vaccine could be accessible to remote regions of the world (where ∼84% of cervical cancer patients reside) by eliminating the cold-chain requirement during transportation and storage.

A stable live bacterial vaccine.

Formulating vaccines into a dry form enhances its thermal stability. This is critical to prevent administering damaged and ineffective vaccines, and to reduce its final cost. A number of vaccines in the market as well as those being evaluated in the clinical setting are in a dry solid state; yet none of these vaccines have achieved long-term stability at high temperatures. We used spray-drying to formulate a recombinant live attenuated Listeria monocytogenes (Lm; expressing Francisella tularensis immune protective antigen pathogenicity island protein IglC) bacterial vaccine into a thermostable dry powder using various sugars and an amino acid. Lm powder vaccine showed minimal loss in viability when stored for more than a year at ambient room temperature (∼23°C) or for 180days at 40°C. High temperature viability was achieved by maintaining an inert atmosphere in the storage container and removing oxygen free radicals that damage bacterial membranes. Further, in vitro antigenicity was confirmed by infecting a dendritic cell line with cultures derived from spray dried Lm and detection of an intracellularly expressed protective antigen. A combination of stabilizing excipients, a cost effective one-step drying process, and appropriate storage conditions could provide a viable option for producing, storing and transporting heat-sensitive vaccines, especially in regions of the world that require them the most.

Impacts of Long-Term Irrigation of Domestic Treated Wastewater on Soil Biogeochemistry and Bacterial Community Structure.

Freshwater scarcity and regulations on wastewater disposal have necessitated the reuse of treated wastewater (TWW) for soil irrigation, which has several environmental and economic benefits. However, TWW irrigation can cause nutrient loading to the receiving environments. We assessed bacterial community structure and associated biogeochemical changes in soil plots irrigated with nitrate-rich TWW (referred to as pivots) for periods ranging from 13 to 30 years. Soil cores (0 to 40 cm) were collected in summer and winter from five irrigated pivots and three adjacently located nonirrigated plots. Total bacterial and denitrifier gene abundances were estimated by quantitative PCR (qPCR), and community structure was assessed by 454 massively parallel tag sequencing (MPTS) of small-subunit (SSU) rRNA genes along with terminal restriction fragment length polymorphism (T-RFLP) analysis of nirK, nirS, and nosZ functional genes responsible for denitrification of the TWW-associated nitrate. Soil physicochemical analyses showed that, regardless of the seasons, pH and moisture contents (MC) were higher in the irrigated (IR) pivots than in the nonirrigated (NIR) plots; organic matter (OM) and microbial biomass carbon (MBC) were higher as a function of season but not of irrigation treatment. MPTS analysis showed that TWW loading resulted in the following: (i) an increase in the relative abundance of Proteobacteria, especially Betaproteobacteria and Gammaproteobacteria; (ii) a decrease in the relative abundance of Actinobacteria; (iii) shifts in the communities of acidobacterial groups, along with a shift in the nirK and nirS denitrifier guilds as shown by T-RFLP analysis. Additionally, bacterial biomass estimated by genus/group-specific real-time qPCR analyses revealed that higher numbers of total bacteria, Acidobacteria, Actinobacteria, Alphaproteobacteria, and the nirS denitrifier guilds were present in the IR pivots than in the NIR plots. Identification of the nirK-containing microbiota as a proxy for the denitrifier community indicated that bacteria belonged to alphaproteobacteria from the Rhizobiaceae family within the agroecosystem studied. Multivariate statistical analyses further confirmed some of the above soil physicochemical and bacterial community structure changes as a function of long-term TWW application within this agroecosystem.

Preclinical refinements of a broadly protective VLP-based HPV vaccine targeting the minor capsid protein, L2.

An ideal prophylactic human papillomavirus (HPV) vaccine would provide broadly protective and long-lasting immune responses against all high-risk HPV types, would be effective after a single dose, and would be formulated in such a manner to allow for long-term storage without the necessity for refrigeration. We have developed candidate HPV vaccines consisting of bacteriophage virus-like particles (VLPs) that display a broadly neutralizing epitope derived from the HPV16 minor capsid protein, L2. Immunization with 16L2 VLPs elicited high titer and broadly cross-reactive and cross-neutralizing antibodies against diverse HPV types. In this study we introduce two refinements for our candidate vaccines, with an eye towards enhancing efficacy and clinical applicability in the developing world. First, we assessed the role of antigen dose and boosting on immunogenicity. Mice immunized with 16L2-MS2 VLPs at doses ranging from 2 to 25 µg with or without alum were highly immunogenic at all doses; alum appeared to have an adjuvant effect at the lowest dose. Although boosting enhanced antibody titers, even a single immunization could elicit strong and long-lasting antibody responses. We also developed a method to enhance vaccine stability. Using a spray dry apparatus and a combination of sugars & an amino acid as protein stabilizers, we generated dry powder vaccine formulations of our L2 VLPs. Spray drying of our L2 VLPs did not affect the integrity or immunogenicity of VLPs upon reconstitution. Spray dried VLPs were stable at room temperature and at 37 °C for over one month and the VLPs were highly immunogenic. Taken together, these enhancements are designed to facilitate implementation of a next-generation VLP-based HPV vaccine which addresses U.S. and global disparities in vaccine affordability and access in rural/remote populations.

Metagenomic assessment of the eastern oyster-associated microbiota.

Bacteria associated with the Eastern oysters (Crassostrea virginica) native to Apalachicola Bay, FL, were investigated using 16S rRNA gene amplicon metagenomic sequencing which revealed that the oyster microbiome was predominated by Cyanobacteria and Proteobacteria. We also found that the oyster tissues were predominated by the pathogenic and symbiotic Photobacterium spp. (formerly known as Vibrio damselae).

A survey of deepwater horizon (DWH) oil-degrading bacteria from the Eastern oyster biome and its surrounding environment.

The deepwater horizon (DWH) accident led to the release of an estimated 794,936,474 L of crude oil into the northern Gulf of Mexico over an 85 day period in 2010, resulting in the contamination of the Gulf of Mexico waters, sediments, permeable beach sands, coastal wetlands, and marine life. This study examines the potential response of the Eastern oyster's microbiome to hydrocarbon contamination and compares it with the bacterial community responses observed from the overlaying water column (WC) and the oyster bed sediments. For this purpose, microcosms seeded with DWH crude oil were established and inoculated separately with oyster tissue (OT), mantle fluid (MF), overlaying WC, and sediments (S) collected from Apalachicola Bay, FL, USA. Shifts in the microbial community structure in the amended microcosms was monitored over a 3-month period using automated ribosomal intergenic spacer region analysis, which showed that the microbiome of the OT and MF were more similar to the sediment communities than those present in the overlaying WC. This pattern remained largely consistent, regardless of the concentration of crude oil or the enrichment period. Additionally, 72 oil-degrading bacteria were isolated from the microcosms containing OT, MF, WC, and S and identified using 16S ribosomal RNA gene sequencing and compared by principal component analysis, which clearly showed that the WC isolates were different to those identified from the sediment. Conversely, the OT and MF isolates clustered together; a strong indication that the oyster microbiome is uniquely structured relative to its surrounding environment. When selected isolates from the OT, MF, WC, and S were assessed for their oil-degrading potential, we found that the DWH oil was biodegraded between 12 and 42%, under the existing conditions.

Watershed-scale fungal community characterization along a pH gradient in a subsurface environment cocontaminated with uranium and nitrate.

The objective of this study was to characterize fungal communities in a subsurface environment cocontaminated with uranium and nitrate at the watershed scale and to determine the potential contribution of fungi to contaminant transformation (nitrate attenuation). The abundance, distribution, and diversity of fungi in subsurface groundwater samples were determined using quantitative and semiquantitative molecular techniques, including quantitative PCR of eukaryotic small-subunit rRNA genes and pyrosequencing of fungal internal transcribed spacer (ITS) regions. Potential bacterial and fungal denitrification was assessed in sediment-groundwater slurries amended with antimicrobial compounds and in fungal pure cultures isolated from the subsurface. Our results demonstrate that subsurface fungal communities are dominated by members of the phylum Ascomycota, and a pronounced shift in fungal community composition occurs across the groundwater pH gradient at the field site, with lower diversity observed under acidic (pH <4.5) conditions. Fungal isolates recovered from subsurface sediments, including cultures of the genus Coniochaeta, which were detected in abundance in pyrosequence libraries of site groundwater samples, were shown to reduce nitrate to nitrous oxide. Denitrifying fungal isolates recovered from the site were classified and found to be distributed broadly within the phylum Ascomycota and within a single genus of the Basidiomycota. Potential denitrification rate assays with sediment-groundwater slurries showed the potential for subsurface fungi to reduce nitrate to nitrous oxide under in situ acidic pH conditions.

Microbial and geochemical assessment of bauxitic un-mined and post-mined chronosequence soils from Mocho Mountains, Jamaica.

Microorganisms are very sensitive to environmental change and can be used to gauge anthropogenic impacts and even predict restoration success of degraded environments. Here, we report assessment of bauxite mining activities on soil biogeochemistry and microbial community structure using un-mined and three post-mined sites in Jamaica. The post-mined soils represent a chronosequence, undergoing restoration since 1987, 1997, and 2007. Soils were collected during dry and wet seasons and analyzed for pH, organic matter (OM), total carbon (TC), nitrogen (TN), and phosphorus. The microbial community structure was assessed through quantitative PCR and massively parallel bacterial ribosomal RNA (rRNA) gene sequencing. Edaphic factors and microbial community composition were analyzed using multivariate statistical approaches and revealed a significant, negative impact of mining on soil that persisted even after greater than 20 years of restoration. Seasonal fluctuations contributed to variation in measured soil properties and community composition, but they were minor in comparison to long-term effects of mining. In both seasons, post-mined soils were higher in pH but OM, TC, and TN decreased. Bacterial rRNA gene analyses demonstrated a general decrease in diversity in post-mined soils and up to a 3-log decrease in rRNA gene abundance. Community composition analyses demonstrated that bacteria from the Proteobacteria (α, ß, γ, δ), Acidobacteria, and Firmicutes were abundant in all soils. The abundance of Firmicutes was elevated in newer post-mined soils relative to the un-mined soil, and this contrasted a decrease, relative to un-mined soils, in proteobacterial and acidobacterial rRNA gene abundances. Our study indicates long-lasting impacts of mining activities to soil biogeochemical and microbial properties with impending loss in soil productivity.

Soil functional diversity analysis of a bauxite-mined restoration chronosequence.

Soil microorganisms are sensitive to environmental perturbations such that changes in microbial community structure and function can provide early signs of anthropogenic disturbances and even predict restoration success. We evaluated the bacterial functional diversity of un-mined and three chronosequence sites at various stages of rehabilitation (0, 10, and 20 years old) located in the Mocho Mountains of Jamaica. Samples were collected during the dry and wet seasons and analyzed for metal concentrations, microbial biomass carbon, bacterial numbers, and functional responses of soil microbiota using community-level physiological profile (CLPP) assays. Regardless of the season, un-mined soils consisted of higher microbial biomass and numbers than any of the rehabilitated sites. Additionally, the number and rate of substrates utilized and substrate evenness (the distribution of color development between the substrates) were significantly greater in the un-mined soils with carbohydrates being preferentially utilized than amino acids, polymers, carboxylic acids, and esters. To some extent, functional responses varied with the seasons but the least physiological activity was shown by the site rehabilitated in 1987 indicating long-term perturbation to this ecosystem. Small subunit ribosomal DNA (SSUrDNA)-denaturing gradient-gel electrophoresis analyses on the microbiota collected from the most preferred CLPP substrates followed by taxonomic analyses showed Proteobacteria, specifically the gamma-proteobacteria, as the most functionally active phyla, indicating a propensity of this phyla to out-compete other groups under the prevailing conditions. Additionally, multivariate statistical analyses, Shannon's diversity, and evenness indices, principal component analysis, biplot and un-weighted-pair-group method with arithmetic averages dendrograms further confirmed that un-mined sites were distinctly different from the rehabilitated soils.