A new class of recombinant human albumin with multiple surface thiols exhibits stable conjugation and enhanced FcRn binding and blood circulation.

Human serum albumin is an endogenous ligand transport protein whose long circulatory half-life is facilitated by engagement with the human cellular recycling neonatal Fc receptor (hFcRn). The single free thiol located at Cys-34 in domain I of albumin has been exploited for monoconjugation of drugs. In this work, we increased the drug-to-albumin ratio potential by engineering recombinant human albumin (rHSA) variants with varying hFcRn affinity to contain three free, conjugation-competent cysteines. Structural analysis was used to identify positions for cysteine introduction to maximize rHSA stability and formation of the conjugated product without affecting hFcRn binding. The thiol rHSA variants exhibited up to 95% monomeric stability over 24 months and retained hFcRn engagement compared with a WT unconjugated control demonstrated by Biolayer Interferometry. The additional cysteines were further introduced into a panel of rHSA variants engineered with different affinities for hFcRn. After conjugation with three Alexa Fluor 680 (AF680) fluorophores, hFcRn binding was similar to that of the original triple-thiol nonconjugated rHSA variants (0.88 and 0.25 µm for WT albumin with or without 3xAF680 respectively, and 0.04 and 0.02 µm for a high hFcRn-binding variant with or without 3xAF680, respectively). We also observed a 1.3-fold increase in the blood circulatory half-life of a high hFcRn-binding triple-thiol variant conjugated with AF680 (t½ = 22.4 h) compared with its WT counterpart (t½ = 17.3 h) in mice. Potential high drug-to-albumin ratios combined with high hFcRn engagement are attractive features of this new class of albumins that offer a paradigm shift for albumin-based drug delivery.

Cellular recycling-driven in vivo half-life extension using recombinant albumin fusions tuned for neonatal Fc receptor (FcRn) engagement.

Recombinant albumin-drug genetic fusions are an effective technology to prolong the serum half-life of therapeutics that has resulted in marketed products. Indirect evidence suggests albumin fusions' long circulation is controlled by engagement with the cellular recycling neonatal Fc receptor (FcRn) in addition to reduced kidney filtration. In this work, we have used a panel of recombinant fusions, engineered with different human FcRn (hFcRn) affinity, including a novel high binding albumin variant (HBII), to directly define and importantly, control the intracellular mechanism as a half-life extension tuning method. mNeonGreen or mCherry fusion to the N-terminal of the recombinant human albumin (rHA) variants null-binder (rHA NB), wild-type (rHA WT), high-binder I (rHA HBI), and high-binder II (rHA HBII) did not generally interfere with hFcRn interaction determined by Biolayer Interferometry. Co-localisation of the albumins with endosomal, but not lysosomal, markers was shown by confocal microscopy for high, but not low, hFcRn binders in a human microvascular endothelial hFcRn overexpressing cell line (HMEC-1 FcRn) suggestive of endosomal compartmentalisation. Furthermore, a cellular recycling assay revealed increased recycling of albumin fusions for the high binding variants (mNeonGreen WT; ~1, mNeonGreen HBI; 5.26-fold higher, and mNeonGreen HBII; 5.77-fold higher) in the hFcRn overexpressing cell line. In vivo experiments demonstrated a direct in vitro recycling/in vivo half-life correlation with a longer circulation for the mCherry fusions engineered with high hFcRn affinity that was highest with the HBII variant of 30.1 h compared to 18.2 h for the mCherry WT. This work gives the first direct evidence for an FcRn-driven endosomal cellular recycling pathway for recombinant albumin fusions that correlates with half-life extension controlled by the affinity to hFcRn; promoting a versatile method to tune the pharmacokinetics of albumin fusion-based therapeutics not met by current technologies.

Development of a fluorescent in situ method for visualization of enteric viruses.

Studying the interactions between enteric pathogens and their environment is important to improving our understanding of their persistence and transmission. However, this remains challenging in large part because of difficulties associated with tracking pathogens in their natural environment(s). In this study, we report a fluorescent labeling strategy which was applied to murine norovirus (MNV-1), a human norovirus surrogate, and hepatitis A virus (HAV). Specifically, streptavidin-labeled Quantum dots (Q-Dots) were bound to biotinylated capsids of MNV-1 and HAV (bio-MNV-1 and bio-HAV); the process was confirmed by using a sandwich-type approach in which streptavidin-bound plates were reacted with biotinylated virus followed by a secondary binding to Q-Dots with an emission range of 635 to 675 nm (Q-Dots 655). The assay demonstrated a relative fluorescence of 528 +/- 48.1 and 112 +/- 8.6 for bio-MNV-1 and control MNV-1, respectively. The biotinylation process did not impact virus infectivity, nor did it interfere with the interactions between the virus and host cells or model produce items. Using fluorescent microscopy, it was possible to visualize both bio-HAV and bio-MNV-1 attached to the surfaces of permissive mammalian cells and green onion tissue. The method provides a powerful tool for the labeling and detection of enteric viruses (and their surrogates) which can be used to track virus behavior in situ.

Detection of viable Zygosaccharomyces bailii in fruit juices using ethidium monoazide bromide and real-time PCR.

In this study, we use ethidium monoazide (EMA) a dye commonly used to differentiate viable and nonviable populations of bacteria in real-time PCR (QPCR) assays to eliminate the nonviable cells from the Z. bailii population. Thus we are able to determine the viable Z. bailii population using QPCR plus EMA. To do this we first, optimized the EMA exposure conditions; EMA concentration of 50 microg/ml with an incubation at 30 degrees C in the dark for 5 min. Followed by light exposure on ice, for 5 min using a 500 W halogen lamp at a distance of 12 cm. Using these optimized conditions, we determined that the assay could detect as few as 12.5 viable Z. bailii cells in the presence of 10(5) CFU/ml of heat killed-cells. The EMA assay was also more consistent at determining viable cell counts when compared to plating than fluorescent microscopy viable cell counts. Finally, we used the assay to determine the viable population in heat-treated (72 degrees C, 2 min), ethanol-treated and raspberry cranberry juice Z. bailii cultures. When examining Z. bailii cells treated with 70% ethanol the QPCR assay with EMA (1.22 x 10(2)) showed a better correlation with plating (4.5 x 10(1) CFU/ml) compared to the QPCR assay without EMA (5.31 x 10(6) CFU/ml) and this was also seen in the other two injured populations. Thus we feel that we have designed an assay which will be useful for the detection of viable spoilage yeasts in various fruit juices.

Characterization of the lactococcal group II intron target site in its native host.

The Lactococcus lactis group II intron (Ll.ltrB) retrohomes into the ltrB gene at high efficiency. To date, the critical DNA bases recognized in vivo by the Ll.ltrB ribonucleoprotein (RNP) have been exclusively elucidated in Escherichia coli. However, recent evidence indicates host-dependant differences in Ll.ltrB mobility, raising the possibility of limitations of the current model for RNP-homing site recognition in the native L. lactis host. In this work, intron retargeting experiments in L. lactis have demonstrated that adherence to specific target site critical bases is not sufficient to predict success or failure of chromosomal invasion, as in E. coli. Accordingly, a quantitative real-time PCR (QPCR) assay was developed to test target site nucleotides previously demonstrated as critical for homing in E. coli, for relevance in its native host. This two-plasmid QPCR homing assay is highly sensitive and, unlike previous E. coli-based assays, resolves differential homing efficiencies in the absence of selection. As in E. coli, deviation from wild type at target site positions -23, -21, -20, -19, and +5 resulted in lower homing efficiencies in L. lactis. Furthermore, the same trends are observed when assaying select variants in Enterococcus faecalis. Our results suggest that these target site positions are critical in both E. coli and L. lactis.

Optimization of fed-batch production of the model recombinant protein GFP in Lactococcus lactis.

Optimization of recombinant protein production using lactic acid bacteria (LAB) remains an important obstacle on the road to realizing LAB as oral vaccine delivery vehicles. Despite this, there have been few published investigations to explore the higher limits of LAB recombinant protein expression in fed-batch fermentations. In this study, results from response surface experiments suggested an optimal set of conditions for expression of green fluorescent protein (GFP), a model recombinant protein, in bench-scale, fed-batch Lactococcus lactis IL1403 fermentations. The 48 4-L fed-batch fermentations in this set of experiments, along with preliminary studies, investigated the effects of pH, temperature, hemin concentration, concentration of the nisin inducer per cell, and time of induction. Cell densities in this data set ranged from 2.9 to 7.4 g/L and maximum GFP expression per cell ranged from 0.1 to 4.4 relative fluorescence units (RFU)/g. The optimal 4-L, fed-batch fermentation process found here yields growth and protein expression values that dramatically improve upon results from traditional test tube and flask processes. Relative to the traditional process, the experimental optimum conditions yield 4.9 times the cell density, 1.6 times the protein per cell mass, and 8 times the total protein concentration. Unexpectedly, experiments also revealed that the compound hemin, known previously to improve growth and survival of Lactococcus lactis (L. lactis), negatively impacted recombinant protein production when added in concentrations from 5 to 20 microg/mL with this strain. The improvement in protein expression over traditional processes demonstrated here is an important step toward commercial development of LAB for oral delivery of recombinant vaccines and therapeutic proteins.

A real-time PCR assay for the enumeration and detection of Zygosaccharomyces bailii from wine and fruit juices.

Zygosaccharomyces bailii is a major food and beverage spoilage organism. Existing methods for its detection involve lengthy enrichment techniques and then the result does not always differentiate between Z. bailii and Saccharomyces cerevisiae. In this work, we developed a quantitative real-time PCR assay for the rapid detection of Z. bailii from fruit juices and wine even in the presence of non-target DNA. Primers were designed to the gene coding for the D1/D2 loop of the 26S ribosomal RNA subunit producing a single PCR product with a melting temperature of 83.5 degrees C. As few as 2 cells per ml could be detected by the assay in cranberry raspberry and apple juices and 22 cells per ml from grape juice. The assay was equally efficient in wine, detecting 6 cells per ml even in the presence of 10(7)S. cerevisiae cells. The CFU/ml as determined by plating on YM media showed excellent correlation with the CFU/ml established by the QPCR assay for all the beverages examined. Unknown samples of Z. bailii were prepared in the juices and wine and examined by QPCR. The QPCR estimated cell number was in good agreement with the cell counts obtained by plating, the exception being the cranberry raspberry juice sample. It was determined by live/dead cell counts that the Z. bailii cells were less viable in this juice thus leading to an overestimation of CFU/ml by QPCR. However, the correlation was high between QPCR and total cell count as determined by fluorescent microscopy. This assay provides a rapid and accurate method to establish the levels of the total Z. bailii population which consists of both viable and nonviable cells.

Multicopy integration of heterologous genes, using the lactococcal group II intron targeted to bacterial insertion sequences.

Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.

Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking.

Oenococcus oeni is an acidophilic member of the Leuconostoc branch of lactic acid bacteria indigenous to wine and similar environments. O. oeni is commonly responsible for the malolactic fermentation in wine and due to its positive contribution is frequently used as a starter culture to promote malolactic fermentation. In collaboration with the Lactic Acid Bacteria Genome Consortium the genome sequence of O. oeni PSU-1 has been determined. The complete genome is 1,780,517 nt with a GC content of 38%. 1701 ORFs could be predicted from the sequence of which 75% were functionally classified. Consistent with its classification as an obligately heterofermentative lactic acid bacterium the PSU-1 genome encodes all the enzymes for the phosphoketolase pathway. Moreover, genes related to flavor modification in wine, such as malolactic fermentation capacity and citrate utilization were readily identified. The completion of the O. oeni genome marks a significant new phase for wine-related research on lactic acid bacteria in which the physiology, genetic diversity and performance of O. oeni starter cultures can be more rigorously examined.