Surface Localization Determinants of Borrelia burgdorferi Lipoproteins

Borrelia spirochetes are the causative agents of Lyme disease and relapsing fever, two common vector-borne diseases. Early experimental evidence, gained from development of genetic tools in the Lyme disease spirochete, Borrelia burgdorferi, emphasized the importance of outer surface lipo-proteins (Osps) during the infectious cycle. Although the functions of these lipoproteins and the complex mechanism of differential regulation is known in increasing detail, it remains to be understood how these virulence factors reach the spirochetal surface. We observed in previous studies that monomeric red fluorescent protein 1 (mRFP1) fused to specifically mutated outer surface protein A (OspA) lipopeptides could be detected by epifluorescence microscopy in both the periplasm and on the bacterial surface. These findings supported the notion that Borrelia spirochetes do not adhere to the +2/+3/+4 sorting rules established in other eubacteria. Rather, borrelial lipoproteins seem to contain a disordered `tether' peptide located at the extreme N-terminus of the mature lipoprotein that influences sorting within the envelope. One facet of this study utilized an N-proximal tandem negative charge (Glu-Asp) that served as an inner membrane retention signal in OspA20:mRFP1 as a target for mutagenesis. A library of random mutants in the two codons was generated and expressed in B. burgdorferi. In situ surface proteolysis combined with fluorescence activated cell sorting (FACS) was then used to screen for viable spirochetes expressing subsurface OspA:mRFP1 fusions. We successfully recovered several mutants that mislocalized the lipo-mRFP1 fusions to the periplasm, adding to our database of peptide sequences that are not permissive for surface export. We then broadened our studies to include the structurally and functionally distinct dimeric OspC-Vsp family lipoproteins and identified their requirements for surface localization. As for OspA, tether sequences influence the localization of OspC-Vsp lipoproteins within the envelope. Interestingly, OspC-Vsp lipoproteins appear to be translocated across the outer membrane as monomers. This suggests that they assume their final oligomeric state only when reaching the spirochetal surface. Additionally, lower molecular weight variants of OspC and Vsp1 were detected indicating cleavage that was exacerbated upon addition of C-terminal epitope tags or mislocalization of the untagged proteins to the periplasm. C-terminal proteolysis of OspC was attributed to a carboxy-terminal protease, CtpA. To date, known substrates of CtpA include the 13-kDa outer membrane porin, P13, and a periplasmic lipoprotein BB0323. C-terminal proteolysis of OspC and Vsp1 suggests CtpA may also function as a periplasmic housekeeping protease. In turn, released C-terminal peptides may play a role in initiation of an envelope stress response. Another aspect of this work examined the subcellular localization pattern of Braun's lipoprotein (Lpp) from E. coli using B. burgdorferi as a surrogate expression host. Surprisingly, Lpp was localized to the B. burgdorferi inner membrane. On the other hand, B. burgdorferi OspA mutants were sorted by E. coli according to E. coli rules. This dataset confirmed that host factors are setting the rules for localization of lipoproteins within the bacterial envelope. Taken together, this work revealed several factors, such as the composition of the lipoprotein tether and the folding state of the lipoprotein, which influences trafficking within the spirochetal cell envelope, and also provided important insights into periplasmic lipoprotein processing of B. burgdorferi. These findings will broaden our understanding of spirochetal lipoprotein transport as well as cell envelope biogenesis. Ultimately, this work may lead to novel treatments and/or vaccination strategies that will be extremely helpful in combating Lyme disease and relapsing fever in the years and decades to come

Development and validation of a FACS-based lipoprotein localization screen in the Lyme disease spirochete Borrelia burgdorferi

<p>Abstract</p> <p>Background</p> <p>In our previous studies on lipoprotein secretion in the Lyme disease spirochete <it>Borrelia burgdorferi</it>, we used monomeric red fluorescent protein 1 (mRFP1) fused to specifically mutated outer surface protein A (OspA) N-terminal lipopeptides to gather first insights into lipoprotein sorting determinants. OspA:mRFP1 fusions could be detected by epifluorescence microscopy both in the periplasm and on the bacterial surface. To build on these findings and to complement the prior targeted mutagenesis approach, we set out to develop a screen to probe a random mutagenesis expression library for mutants expressing differentially localized lipoproteins.</p> <p>Results</p> <p>A Glu-Asp codon pair in the inner membrane-localized OspA20:mRFP1 fusion was chosen for mutagenesis since the two negative charges were previously shown to define the phenotype. A library of random mutants in the two codons was generated and expressed in <it>B. burgdorferi</it>. <it>In situ </it>surface proteolysis combined with fluorescence activated cell sorting (FACS) was then used to screen for viable spirochetes expressing alternative subsurface OspA:mRFP1 fusions. Analysis of 93 clones randomly picked from a sorted cell population identified a total of 43 distinct mutants. Protein localization assays indicated a significant enrichment in the selected subsurface phenotype. Interestingly, a majority of the subsurface mutant proteins localized to the outer membrane, indicating their impairment in "flipping" through the outer membrane to the spirochetal surface. OspA20:mRFP1 remained the protein most restricted to the inner membrane.</p> <p>Conclusions</p> <p>Together, these results validate this FACS-based screen for lipoprotein localization and suggest a rather specific inner membrane retention mechanism involving membrane anchor-proximal negative charge patches in this model <it>B. burgdorferi </it>lipoprotein system.</p

Application of radar chart array analysis to visualize effects of formulation variables on IgG1 particle formation as measured by multiple analytical techniques

This study presents a novel method to visualize protein aggregate and particle formation data to rapidly evaluate the effect of solution and stress conditions on the physical stability of an IgG1 monoclonal antibody (mAb). Radar chart arrays were designed so that hundreds of Microflow Digital Imaging (MFI) solution measurements, evaluating different mAb formulations under varying stresses, could be presented in a single figure with minimal loss of data resolution. These MFI radar charts show measured changes in subvisible particle number, size and morphology distribution as a change in the shape of polygons. Radar charts were also created to visualize mAb aggregate and particle formation across a wide size range by combining data sets from size exclusion chromatography (SEC), Archimedes resonant mass measurements, and MFI. We found that the environmental/mechanical stress condition (e.g., heat vs. agitation) was the most important factor in influencing the particle size and morphology distribution with this IgG1 mAb. Additionally, the presence of NaCl exhibited a pH and stress dependent behavior resulting in promotion or inhibition mAb particle formation. This data visualization technique provides a comprehensive analysis of the aggregation tendencies of this IgG1 mAb in different formulations with varying stresses as measured by different analytical techniques

Structural Characterization of IgG1 mAb Aggregates and Particles Generated under Various Stress Conditions

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size exclusion chromatography (SEC), Nanosight Tracking Analysis (NTA), Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from TEM and MFI images. Shaking samples without NaCl generated the most fibrillar particles, while stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1 containing aggregates and particles with some non-native disulfide crosslinks, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions

Formulation development of a stable, orally delivered live human neonatal rotavirus(rv3-bb) vaccine candidate

Rotavirus is the most common cause of gastroenteritis among children under 5 years of age leading to ~200,000 deaths in 2013.1 Rotavirus-attributed mortality can be significantly reduced by promoting global implementation of rotavirus vaccination by vaccine dosage cost reduction and optimizing vaccine efficacy in low-resource countries. Furthermore, a rotavirus vaccine administered at birth could prevent neonatal mortality and reduce the risk of intussusception 2. An oral human neonatal rotavirus vaccine candidate (RV3-BB) has been developed from the human neonatal rotavirus strain RV3 (G3P[6]) 2 , and a recently published Phase IIb clinical trial showed RV3-BB was efficacious in preventing severe rotavirus gastroenteritis via a neonatal or infant schedule in Indonesia2. The overall goals of this project are to develop and implement commercially viable bulk and drug product manufacturing processes of a stable liquid formulation for oral delivery (without pre-neutralization) that is affordable in the developing world (Fig. 1). The consortium working on this program is sponsored by the Bill and Melinda Gates Foundation between Batavia Biosciences, Murdoch Children’s Research Institute, BioFarma, and The University of Kansas. Please click Additional Files below to see the full abstract

Analytical characterization and formulation assessment of model secretory- immunoglobulin-A (sIgAs) for their potential use as low cost, orally delivered sIgAs

Enterotoxigenic Escherichia coli (ETEC) is a major cause of bacterial diarrheal disease in developing countries, especially among children and infants. ETEC is estimated to cause 280-400 million diarrheal episodes per year in children \u3c5 years of age, resulting in 300,000 to 500,000 deaths.1 Despite the need for a vaccine, there are currently no licensed vaccines against ETEC. Alternatively, passive immunization by oral delivery of pathogen-specific immunoglobulins is another promising approach to provide “instant” protection against ETEC. The potential advantages of oral delivery are reduced cost, simplicity of administration and localized treatment within the GI tract. Secretory IgA (sIgA) is of particular interest because it is naturally found in the mucosal surfaces within the GI tract, relatively more resistant to proteolysis by digestive enzymes (vs. IgGs), and can protect against enteric bacteria by directly neutralizing virulence factors.2 One major challenge of this approach is the instability of protein molecules during oral delivery (in the digestive tract) as well as during long-term storage (in various formulations). In this study, two proteins, sIgA1 and sIgA2 against heat labile toxin (LT, one of the major virulence factors of ETEC), were used as model sIgA molecules for developing analytical techniques and assessing stability (physicochemical as well as in vitro binding) under various conditions. A combination of biochemical and biophysical methods were employed to comprehensively characterize the sIgA1 and sIgA2 model proteins including primary structure, post translational modifications (i.e., N-linked glycans), size, apparent solubility, higher order structure and conformational stability as well as in vitro antigen binding. Using these characterization and stability indicating methods, we are monitoring the stability of these two model sIgAs both in an in vitro digestion model (to mimic in vivo degradation conditions), and during accelerated stability studies (to assess storage stability). Our goal is to use the information gained by these aforementioned methods and stability studies to design stable, low-cost liquid formulations for oral delivery of sIgAs in the developing world. Please click Additional Files below to see the full abstract

Formulation and stabilization of a recombinant human Cytomegalovirus vector for use as a candidate vaccine for HIV-1

Vaccination using Cytomegalovirus (CMV) vectors have recently shown promising results in conferring protection in non-human primates against SIV and Mycobacterium tuberculosis infection (1-3). Since CMV vectors can stimulate the production of high concentrations of systemic effector memory T-cells, CMV vectors (containing the appropriate insert) have the potential to clear SIV/HIV and Mycobacterium tuberculosis infection, provided administration occurs at the onset of infection (1, 3). Despite the promising animal data, CMV vectors are prone to potency loss (i.e., degradation) by freeze-thaw and storage at 2-8°C. In this study, we wished to develop formulations with increased freeze-thaw and liquid stability for a recombinant human CMV vector (rHCMV-1) for use in initial clinical trials including i) reduce vector potency loss to \u3c0.5 log after 1 freeze-thaw cycle and ii) reduce vector potency loss to \u3c0.5 log after 4 hours at 2-8°C storage. To achieve these goals, we screened a library of ~50 pharmaceutical excipients and evaluated their effect on vector potency after 3 freeze-thaw cycles or incubation at 4°C for several days. We found that certain additives completely protected rHCMV-1 against freeze-thaw mediated potency loss. With regards to liquid stability, we found certain additives slowed the rate of rHCMV-1 titer loss when stored at 4°C. After screening various excipient combinations, we evaluated three candidate formulations and benchmarked them against the bulk drug substance (BDS) formulation buffer and another published formulation (4). The candidate formulations were significantly more stable than the formulations used for benchmarking in terms reducing rHCMV-1 titer loss due to freeze-thaw and incubation at 4°C for up to 30 days. Despite providing greater stability than the current BDS formulation buffer, rHCMV-1 titer loss was still observed at 4°C as a function of incubation time, which suggests further stabilization (i.e., lyophilization) is likely necessary for longer term development. This study highlights the utility of empirical design of a liquid formulation of a live viral vector where freeze-thaw and short-term liquid storage are necessary. References S. G. Hansen et al., Prevention of tuberculosis in rhesus macaques by a cytomegalovirus-based vaccine. Nat Med 24, 130-143 (2018). S. G. Hansen et al., Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine. Nature 473, 523-527 (2011). S. G. Hansen et al., Immune clearance of highly pathogenic SIV infection. Nature 502, 100-104 (2013). T.-M. Fu, D. Wang, M. B. Medi, M. S. D. Corp, Ed. (2013). Acknowledgements: This work was funded by the Bill and Melinda Gates Foundation. *Current affiliation: Bill and Melinda Gates Foundation

Preformulation Characterization and Stability Assessments of Secretory IgA Monoclonal Antibodies as Potential Candidates for Passive Immunization by Oral Administration

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease in children in developing countries, and there are no licensed vaccines to protect against ETEC. Passive immunization by oral delivery of ETEC-specific secretory IgAs (sIgAs) could potentially provide an alternative approach for protection in targeted populations. In this study, a series of physiochemical techniques and an in vitro gastric digestion model were used to characterize and compare key structural attributes and stability profiles of three anti-heat labile enterotoxin monoclonal antibodies (sIgA1, sIgA2 and IgG1 produced in CHO cells). The mAbs were evaluated in terms of primary structure, N-linked glycan profiles, size and aggregate content, relative apparent solubility, conformational stability, and in vitro antigen binding. Compared to IgG1 mAb, sIgA1 and sIgA2 mAbs showed increased sample heterogeneity, especially in terms of N-glycan composition and the presence of higher molecular weight species. The sIgA mAbs showed overall better physical stability and were more resistant to loss of antigen binding activity during incubation at low pH, 37 degrees C with pepsin. These results are discussed in terms of future challenges to design stable, low-cost formulations of sIgA mAbs as an oral supplement for passive immunization to protect against enteric diseases in the developing world

Characterization of the Physical Stability of a Lyophilized IgG1 mAb After Accelerated Shipping-like Stress

Upon exposure to shaking stress, an IgG1 mAb formulation in both liquid and lyophilized state formed subvisible particles. Since freeze-drying is expected to minimize protein physical instability under these conditions, the extent and nature of aggregate formation in the lyophilized preparation was examined using a variety of particle characterization techniques. The effect of formulation variables such as residual moisture content, reconstitution rate, and reconstitution medium were examined. Upon reconstitution of shake-stressed lyophilized mAb, differences in protein particle size and number were observed by Microflow Digital Imaging (MFI), with the reconstitution medium having the largest impact. Shake-stress had minor effects on the structure of protein within the particles as shown by SDS-PAGE and FTIR analysis. The lyophilized mAb was shake-stressed to different extents and stored for 3 months at different temperatures. Both extent of cake collapse and storage temperature affected the physical stability of the shake-stressed lyophilized mAb upon subsequent storage. These findings demonstrate that physical degradation upon shaking of a lyophilized IgG1 mAb formulation includes not only cake breakage, but also results in an increase in subvisible particles and turbidity upon reconstitution. The shaking-induced cake breakage of the lyophilized IgG1 mAb formulation also resulted in decreased physical stability upon storage

Effect of Formulation Variables on the Stability of a Live, Rotavirus (RV3-BB) Vaccine Candidate using in vitro Gastric Digestion Models to Mimic Oral Delivery

In this work, two different in vitro gastric digestion models were used to evaluate the stability of a live attenuated rotavirus vaccine candidate (RV3-BB) under conditions designed to mimic oral delivery in infants. First, a forced-degradation model was established at low pH to assess the buffering capacity of formulation excipients and to screen for RV3-BB stabilizers. Second, a sequential-addition model was implemented to examine RV3-BB stability under conditions more representative of oral administration to infants. RV3-BB rapidly inactivated at < pH 5.0 (37 °C, 1 h) as measured by an infectivity RT-qPCR assay. Pre-neutralization with varying volumes of infant formula (Enfamil®) or antacid (Mylanta®) conferred partial to full protection of RV3-BB. Excipients with sufficient buffering capacity to minimize acidic pH inactivation of RV3-BB were identified (e.g., succinate, acetate, adipate), however, they concomitantly destabilized RV3-BB in accelerated storage stability studies. Both effects were concentration dependent, thus excipient optimization was required to design candidate RV3-BB formulations which minimize acid-induced viral inactivation during oral delivery while not destabilizing the vaccine during long-term 2–8 °C storage. Finally, a statistical Design -of-Experiments (DOE) study examining RV3-BB stability in the in vitro sequential-addition model identified key formulation parameters likely affecting RV3-BB stability during in vivo oral delivery