At-line process analytical technology (PAT) for more efficient scale up of biopharmaceutical microfiltration unit operations.

Tangential flow microfiltration (MF) is a cost-effective and robust bioprocess separation technique, but successful full scale implementation is hindered by the empirical, trial-and-error nature of scale-up. We present an integrated approach leveraging at-line process analytical technology (PAT) and mass balance based modeling to de-risk MF scale-up. Chromatography-based PAT was employed to improve the consistency of an MF step that had been a bottleneck in the process used to manufacture a therapeutic protein. A 10-min reverse phase ultra high performance liquid chromatography (RP-UPLC) assay was developed to provide at-line monitoring of protein concentration. The method was successfully validated and method performance was comparable to previously validated methods. The PAT tool revealed areas of divergence from a mass balance-based model, highlighting specific opportunities for process improvement. Adjustment of appropriate process controls led to improved operability and significantly increased yield, providing a successful example of PAT deployment in the downstream purification of a therapeutic protein. The general approach presented here should be broadly applicable to reduce risk during scale-up of filtration processes and should be suitable for feed-forward and feed-back process control.

Chemically modified peptides based on the membrane-proximal external region of the HIV-1 envelope induce high-titer, epitope-specific nonneutralizing antibodies in rabbits.

Broadly neutralizing monoclonal antibodies (bNAbs) 2F5 and 4E10 bind to the membrane proximal external region (MPER) of gp41 and also cross-react with phospholipids. In this study, we investigated if chemical modifications on the MPER adjacent to 2F5 and 4E10 epitopes using mimetics of inflammation-associated posttranslational modifications to induce 2F5- and 4E10-like bNAbs can break tolerance. We synthesized a series of chemically modified peptides spanning the MPER. The serine, threonine, and tyrosine residues in the peptides were modified with sulfate, phosphate, or nitrate moieties and presented in liposomes for rabbit immunizations. All immunizations resulted in high antisera titers directed toward both the modified and unmodified immunogens. Tyrosine modification was observed to significantly suppress antiepitope responses. Sera with strong anti-gp140 titers were purified by affinity chromatography toward the MPER peptide and found to possess a higher affinity toward the MPER than did the bNAbs 2F5 and 4E10. Modest neutralization was observed in the H9 neutralization assay, but neutralization was not observed in the TZM-bl cell or peripheral blood mononuclear cell (PBMC) neutralization assay platforms. Although neutralizing antibodies were not induced by this approach, we conclude that chemical modifications can increase the immune responses to poorly immunogenic antigens, suggesting that chemical modification in an appropriate immunization protocol should be explored further as an HIV-1 vaccine strategy.

Prolyl endopeptidase activity in bronchoalveolar lavage fluid: a novel diagnostic biomarker in a guinea pig model of invasive pulmonary aspergillosis.

Improved diagnostics are needed to detect invasive pulmonary aspergillosis, a life-threatening infection caused by the pathogenic fungus Aspergillus fumigatus. We are investigating secreted fungal proteases as novel biomarkers for the diagnosis of this disease. Although the A. fumigatus genome encodes a multitude of secreted proteases, few have been experimentally characterized. Here, we employed an unbiased combinatorial library of internally quenched fluorogenic probes to detect infection-associated proteolysis in the lungs of guinea pigs experimentally infected with A. fumigatus. Comparative protease activity profiling revealed a prolyl endopeptidase activity that is reproducibly induced during infection but is not observed in healthy animals. This proteolytic activity was found in four independent animal experiments involving two A. fumigatus isolates. We synthesized a small, focused fluorogenic probe library to define the substrate specificity of the prolyl endopeptidase substrate motif and to identify optimal Probe sequences. These efforts resulted in the identification of a panel of six individual substrate-based fluorescent probes capable of detecting infection in guinea pigs with high statistical significance (P<0.005 in most cases). Receiver operating characteristic analyses demonstrated that this fluorogenic assay could detect A. fumigatus infection-associated proteolysis with comparable sensitivity and specificity as existing diagnostic procedures, suggesting that further optimization of the methodology may lead to improved diagnostics options for invasive pulmonary aspergillosis.

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

ß-Carotene Biosynthesis in Probiotic Bacteria.

Susceptibility to deadly diarrheal diseases is partly due to widespread pediatric vitamin A deficiency. To increase vitamin A coverage in malnourished children, we propose to engineer a probiotic bacterium that will produce ß-carotene in the intestine, which will be metabolized to vitamin A. Such a therapy has the potential to broadly stimulate mucosal immunity and simultaneously reduce the incidence and duration of diarrheal disease. To that end, a ß-carotene-producing variant of the probiotic Escherichia coli strain Nissle 1917 (EcN-BETA) was generated. Notably, the strain produces ß-carotene under anaerobic conditions, reflective of the gut environment. EcN-BETA also retains ß-carotene production capability after lyophilization, suggesting that it may be amenable to dry formulation. Moreover, EcN-BETA activates murine dendritic cells in vitro, suggesting that the presence of ß-carotene may not diminish the immunostimulatory capacity of EcN. Finally, we present a framework through which further improvements may enable approaches such as the one described in this report to yield innovative life-saving therapies for the developing world.

Rational design of membrane proximal external region lipopeptides containing chemical modifications for HIV-1 vaccination.

The inability to generate broadly neutralizing antibody (bnAb) responses to the membrane proximal external region (MPER) of HIV-1 gp41 using current vaccine strategies has hampered efforts to prevent the spread of HIV. To address this challenge, we investigated a novel hypothesis to help improve the anti-MPER antibody response. Guided by structural insights and the unique lipid reactivity of anti-MPER bnAbs, we considered whether amino acid side chain modifications that emulate hydrophilic phospholipid head groups could contribute to the generation of 2F5-like or 4E10-like neutralizing anti-MPER antibodies. To test this hypothesis, we generated a series of chemically modified MPER immunogens through derivatization of amino acid side chains with phosphate or nitrate groups. We evaluated the binding affinity of the chemically modified peptides to their cognate monoclonal antibodies, 2F5 and 4E10, using surface plasmon resonance. The modifications had little effect on binding to the antibodies and did not influence epitope secondary structure when presented in liposomes. We selected five of the chemically modified sequences to immunize rabbits and found that an immunogen containing both the 2F5 and 4E10 epitopes and a phosphorylated threonine at T676 elicited the highest anti-peptide IgG titers, although the high antipeptide titers did not confer higher neutralizing activity. These data indicate that side chain modifications adjacent to known neutralizing antibody epitopes are capable of eliciting antibody responses to the MPER but that these chemically modified gp41 epitopes do not induce neutralizing antibodies.

Proteolytic fingerprinting of complex biological samples using combinatorial libraries of fluorogenic probes.

Proteases have garnered interest as candidate biomarkers and therapeutic targets for many human diseases. A key challenge is the identification and characterization of disease-relevant proteases in the complex milieu of biological fluids such as serum, plasma, and bronchoalveolar lavage, in which a multitude of hydrolases act in concert. This unit describes a protocol to map the global proteolytic substrate specificities of complex biological samples using a concise combinatorial library of internally quenched fluorogenic peptide probes (IQFPs). This substrate profiling approach provides a global and quantitative comparison of protease specificities between different biological samples. Such a comparative analysis can lead to the identification of disease-specific 'fingerprints' of proteolytic activities with potential utility in diagnosis and therapy.

Comparative analysis of the substrate preferences of two post-proline cleaving endopeptidases, prolyl oligopeptidase and fibroblast activation protein α.

Post-proline cleaving peptidases are promising therapeutic targets for neurodegenerative diseases, psychiatric conditions, metabolic disorders, and many cancers. Prolyl oligopeptidase (POP; E.C. 3.4.21.26) and fibroblast activation protein α (FAP; E.C. 3.4.24.B28) are two post-proline cleaving endopeptidases with very similar substrate specificities. Both enzymes are implicated in numerous human diseases, but their study is impeded by the lack of specific substrate probes. We interrogated a combinatorial library of proteolytic substrates and identified novel and selective substrates of POP and FAP. These new sequences will be useful as probes for fundamental biochemical study, scaffolds for inhibitor design, and triggers for controlled drug delivery.

Design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens.

Liposomes (phospholipid bilayer vesicles) are versatile and robust delivery systems for induction of antibody and T lymphocyte responses to associated subunit antigens. In the last 15 years, liposome vaccine technology has matured and now several vaccines containing liposome-based adjuvants have been approved for human use or have reached late stages of clinical evaluation. Given the intensifying interest in liposome-based vaccines, it is important to understand precisely how liposomes interact with the immune system and stimulate immunity. It has become clear that the physicochemical properties of liposomal vaccines - method of antigen attachment, lipid composition, bilayer fluidity, particle charge, and other properties - exert dramatic effects on the resulting immune response. Here, we present a comprehensive review of the physicochemical properties of liposomal vaccines and how they influence immune responses. A discussion of novel and emerging immunomodulators that are suitable for inclusion in liposomal vaccines is also presented. Through a comprehensive analysis of the body of liposomal vaccine literature, we enumerate a series of principles that can guide the rational design of liposomal vaccines to elicit immune responses of a desired magnitude and quality. We also identify major unanswered questions in the field, pointing the direction for future study.

Robust substrate profiling method reveals striking differences in specificities of serum and lung fluid proteases.

Proteases are candidate biomarkers and therapeutic targets for many diseases. Sensitive and robust techniques are needed to quantify proteolytic activities within the complex biological milieu. We hypothesized that a combinatorial protease substrate library could be used effectively to identify similarities and differences between serum and bronchoalveolar lavage fluid (BALF), two body fluids that are clinically important for developing targeted therapies and diagnostics. We used a concise library of fluorogenic probes to map the protease substrate specificities of serum and BALF from guinea pigs. Differences in the proteolytic fingerprints of the two fluids were striking: serum proteases cleaved substrates containing cationic residues and proline, whereas BALF proteases cleaved substrates containing aliphatic and aromatic residues. Notably, cleavage of proline-containing substrates dominated all other protease activities in both human and guinea pig serum. This substrate profiling approach provides a foundation for quantitative comparisons of protease specificities between complex biological samples.

Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr.

BACKGROUND: The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers. METHODOLOGY AND PRINCIPAL FINDINGS: As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures. CONCLUSIONS: This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.

The virulence of the opportunistic fungal pathogen Aspergillus fumigatus requires cooperation between the endoplasmic reticulum-associated degradation pathway (ERAD) and the unfolded protein response (UPR).

The filamentous fungal pathogen Aspergillus fumigatus secretes hydrolytic enzymes to acquire nutrients from host tissues. The production of these enzymes exerts stress on the endoplasmic reticulum (ER), which is alleviated by two stress responses: the unfolded protein response (UPR), which adjusts the protein folding capacity of the ER, and ER-associated degradation (ERAD), which disposes of proteins that fail to fold correctly. In this study, we examined the contribution of these integrated pathways to the growth and virulence of A. fumigatus, focusing on the ERAD protein DerA and the master regulator of the UPR, HacA. A ΔderA mutant grew normally and showed no increase in sensitivity to ER stress. However, expression of the UPR target gene bipA was constitutively elevated in this strain, suggesting that the UPR was compensating for the absence of DerA function. To test this, the UPR was disrupted by deleting the hacA gene. The combined loss of derA and hacA caused a more severe reduction in hyphal growth, antifungal drug resistance and protease secretion than the loss of either gene alone, suggesting that DerA and HacA cooperate to support these functions.  Moreover, the ΔderA/ΔhacA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasted the wild type virulence of ΔderA and the reduced virulence of the ΔhacA mutant. Taken together, these data demonstrate that DerA cooperates with the UPR to support the expression of virulence-related attributes of A. fumigatus.

Antibody response to polyhistidine-tagged peptide and protein antigens attached to liposomes via lipid-linked nitrilotriacetic acid in mice.

Particulate delivery systems enhance antibody responses to subunit antigens. However, covalent attachment of protein antigens can disrupt protein structure and mask critical epitopes, altering the antibody response to the antigen. In this report, we evaluate noncovalent metal chelation via nitrilotriacetic acid (NTA) as a nondestructive method to attach peptide and protein antigens to liposomes. Two model antigens, ovalbumin (OVA) and a peptide derived from the membrane-proximal region of HIV-1 gp41 (N-MPR), were polyhistidinylated and attached to liposomes via monovalent NTA (mono-NTA; K(D) [equilibrium dissociation constant], ∼10 µM), trivalent NTA (tris-NTA; K(D), ∼1 nM), or a covalent linkage. Attachment of N-MPR, but not OVA, to liposomes via an NTA lipid elicited stronger antibody responses in BALB/c mice than a formulation in which unassociated antigen was simply admixed with control liposomes lacking NTA. However, the tris-NTA linkage did not increase antibody responses to either N-MPR or OVA compared to the level for the mono-NTA linkage, despite the greater liposomal association of the antigen. For both antigens, covalently attaching them to a lipid elicited significantly stronger antibody responses than NTA-anchored antigens (OVA titer, 3.4 × 10(6) versus 1.4 × 10(6) to 1.6 × 10(6) [P < 0.001]; N-MPR titer, 4.4 × 10(4) versus 5.5 × 10(2) to 7.6 × 10(2) [P < 0.003]). The data indicate that NTA linkages may increase antibody titers to weak antigens such as N-MPR, but NTA-mediated attachment remains inferior to covalent conjugation. Moreover, enhancements in antigen-liposome affinity do not result in increased antibody titers. Thus, additional improvements of NTA-mediated conjugation technology are necessary to achieve an effective, nondestructive method for increasing the humoral response to antigens in particulate vaccines.

Tris-nitrilotriacetic acids of subnanomolar affinity toward hexahistidine tagged molecules.

Nitrilotriacetic acid (NTA) has moderate affinity (10 µM) for hexahistidine (His6) and is widely used in the purification of His6-tagged proteins. The affinity can be increased significantly (10 nM) through multivalency such as using a tris-NTA. We show that the binding affinity of tris-NTA is dependent on the flexibility and length of the spacer between the mono-NTA and the scaffold: the shorter the spacer, the higher the affinity. A series of biotinylated tris-NTA having different spacers were synthesized and used to prepare tris-NTA sensor chips for surface plasmon resonance measurement of binding affinity. Subnanomolar affinity can be achieved with a short spacer. The new high-affinity tris-NTA enables the formation of stable complexes with hexahistidine containing molecules and provides a convenient method to noncovalently attach proteins to various surfaces.

All-trans retinoic acid potentiates the antibody response in mice to a lipopeptide antigen adjuvanted with liposomal lipid A.

Retinoic acid (RA), the bioactive metabolite of retinol, is essential for robust humoral immunity in animals and humans. Recent interest in RA as a vaccine adjuvant has been encouraged by reports showing cooperative enhancement of antibody responses to tetanus toxin in rodents by all-trans RA (ATRA) and a Toll-like receptor-3 agonist. We hypothesized that RA would augment the antibody response to a co-delivered lipopeptide immunogen derived from the membrane proximal region (MPR) of HIV-1 gp41. The MPR is weakly immunogenic and could benefit from potent new humoral adjuvants. When co-formulated in liposomes and administered to BALB/C mice, ATRA alone did not elicit serum anti-peptide antibodies to an MPR-derived lipopeptide. However, addition of ATRA, but not 13-cis RA, to a liposomal formulation containing the Toll-like receptor-4 agonist monophosphoryl lipid A resulted in a fourfold enhancement of serum anti-peptide IgG titers as compared with a formulation containing lipid A alone (P=0.00039). The difference did not arise from biophysical changes in the liposome formulation, including vesicle size, vesicle charge and liposome association of antigen. Thus, ATRA warrants further study as a vaccine adjuvant.

Role of lipid structure in the humoral immune response in mice to covalent lipid-peptides from the membrane proximal region of HIV-1 gp41.

The membrane proximal region (MPR) of HIV-1 gp41 is a desirable target for development of a vaccine that elicits neutralizing antibodies since the patient-derived monoclonal antibodies, 2F5 and 4E10, bind to the MPR and neutralize primary HIV isolates. The 2F5 and 4E10 antibodies cross-react with lipids and structural studies suggest that MPR immunogens may be presented in a membrane environment. We hypothesized that covalent attachment of lipid anchors would enhance the humoral immune response to MPR-derived peptides presented in liposomal bilayers. In a comparison of eight lipids conjugated to an extended 2F5 epitope peptide, a sterol, cholesterol hemisuccinate (CHEMS), was found to promote the strongest anti-peptide IgG titers (6.4 x 10(4)) in sera of BALB/C mice. Two lipid anchors, palmitic acid and phosphatidylcholine, failed to elicit a detectable serum anti-peptide IgG response. Association with the liposomal vehicle contributed to the ability of a lipopeptide to elicit anti-peptide antibodies, but no other single factor, such as position of the lipid anchor, peptide helical content, lipopeptide partition coefficient, or presence of phosphate on the anchor clearly determined lipopeptide potency. Conjugation to CHEMS also rendered a 4E10 epitope peptide immunogenic (5.6 x 10(2) IgG titer in serum). Finally, attachment of CHEMS to a peptide spanning both the 2F5 and 4E10 epitopes elicited serum IgG antibodies that bound to each of the individual epitopes as well as to recombinant gp140. Further research into the mechanism of how structure influences the immune response to the MPR may lead to immunogens that could be useful in prime-boost regimens for focusing the immune response in an HIV vaccine.

A multiplexed immunoassay for detection of antibodies against avian influenza virus.

Avian influenza (AI) is a highly contagious disease in poultry and outbreaks can have dramatic economic and health implications. For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies against AI virus (AIV) proteins. In this study, we report the development of a multiplexed fluorescence microsphere immunoassay (FMIA) for detection of antibodies against AIV proteins in poultry. Recombinant nucleoprotein (NP), matrix protein (M1), and non-structural protein 1 (NS1) were expressed using a baculovirus expression system, purified and covalently coupled to fluorescent xMAP microspheres. Using these reagents, a triplex bead assay was developed for the Luminex platform. The assay displayed minimal cross reactivity when screened against a panel of reference sera raised against common avian viruses. For detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commercially available ELISA. The assay was also employed to investigate the early kinetics of antibody response in chickens infected with AIV. Our results suggest that NP should be the protein of choice when detecting AI infections in commercial chickens, as the immune response was higher and persisted longer than that of M1 and NS1 proteins. This report provides a framework from which a more robust assay could be developed to profile exposure to many AIV subtypes in a single test.

Facile synthesis of multivalent nitrilotriacetic acid (NTA) and NTA conjugates for analytical and drug delivery applications.

High-affinity nitrilotriacetic acids (NTA) have great potential in the molecular manipulation of His-tagged proteins. We have developed a facile method to synthesize multivalent NTA and its conjugates. Starting with appropriately protected lysine, we synthesized the mono-NTA synthons functionalized with either an amino group or a carboxylic group. We then obtained tri-NTA through the condensation of the amino NTA and the carboxylic NTA. Using amino tri-NTA as the key intermediate, we synthesized a series of tri-NTA conjugates with a variety of functional units including biotin, dialkyl, fluorescein, and a hydroxybenzimidate moiety. The biotin-tri-NTA was employed to convert a Biacore streptavidin chip into a high-affinity tri-NTA chip. The equilibrium dissociation constants of tri-NTA/His-tagged protein complexes measured by surface plasmon resonance are in the 20 nM range. Histidine(6)-tagged yeast cytosine deaminase (His6-yCD) was incorporated onto the liposome surface by the lipid-tri-NTA conjugate without any activity loss. Fluorescein-tri-NTA formed a stable 1:1 complex with His6-yCD without significant fluorescence quenching. Specific tri-NTA derivatives for the radiolabeling and coupling of two His-tagged proteins to each other are described. Thus, we have added to the toolbox a number of high-affinity tri-NTA adaptors for the manipulation of His-tagged molecules.

Creating multiple time-shared laser traps with simultaneous displacement detection using digital signal processing hardware.

We present a design for implementing multiple laser traps for single-molecule studies through time-sharing using commercially available digital signal processing hardware in a computer running a standard multitasking operating system. This design enables four to six independent laser traps with a visitation frequency of 10,000s(-1)trap(-1) and a timing jitter of +/-0.5 micros to be created. The design also achieves nanometer-resolution detection of displacement in all of the traps simultaneously via back focal-plane interferometry and only a single quadrant photodiode detector. Practical design considerations and limitations together with the use of fiberlasers in laser traps are discussed. Using this device, the mechanokinetics of multiple molecular motors or adhesion proteins may be measured simultaneously. We present the example biological application of two kinesin-coated beads in separate traps moving on different portions of a microtubule.