PEG修饰对PLL-EGF基因载体靶向结合作用的影响

聚阳离子基因载体系统由于安全性好和便于设计等优点,近年来在基因治疗中的应用发展迅速.在进行基因药物的体内靶向输送时,目前国际上主要通过在基因输送系统中修饰聚乙二醇(PEG)和靶向分子来提高体内输送的稳定性和靶向性.PEG的修饰可能会遮蔽靶向分子的功能呈现,因此建立定量分析方法评价PEG修饰对靶向结合作用的影响非常重要.将连接有表皮生长因子(EGF)的聚赖氨酸(PLL)基因载体作为研究模型,建立BIAcore检测方法,比较PLL-EGF,PEG7000修饰的PLL-EGF,PEG20000修饰的PLL-EGF对表皮生长因子受体(EGFR)的结合和解离速率,评价PEG修饰对PLL-EGF靶向功能呈现的影响.结果表明,PEG7000的修饰降低了EGF和EGFR之间的结合速率,提高了解离速率,整体减弱了靶向分子的靶向结合能力.PEG20000的修饰进一步减弱靶向分子功能的呈现.因此在进行靶向型聚阳离子基因输送系统设计时,考察PEG修饰对靶向结合能力的影响程度非常重要.该研究结果也对其他基因载体系统的设计提供必要的参考.     

Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds

The utility of single-chain Fv proteins as therapeutic agents would be substantially broadened if the circulating lives of these minimal antigen-binding polypeptides were both prolonged and adjustable. Poly(ethylene glycol) (PEG) bioconjugate derivatives of the model single-chain Fv, CC49/218 sFv, were constructed using six different linker chemistries that selectively conjugate either primary amines or carboxylic acid groups. Activated PEG polymers with molecular weights of 2000, 5000, 10 000, 12 000, and 20 000 were included in the sFv bioconjugate evaluation. Additionally, the influence of PEG conjugate geometry in branched PEG strands (U-PEG) and the effect of multimeric PEG-sFv bioconjugates on circulating life and affinity were examined. Although random and extensive PEG polymer conjugations have been achievable in highly active derivatives of the prototypical PEG-enzymes, PEGylation of CC49/218 sFv required stringent adjustment of reaction conditions in order to preserve antigen-binding affinity as measured in either mucin-specific or whole cell immunoassays. Purified bioconjugates with PEG:sFv ratios of 1:1 through 2:1 were identified as promising candidates which exhibit sFv affinity (K(d)) values within 2-fold of the unmodified sFv protein. Interestingly, PEG conjugation to carboxylic acid moieties, using a PEG-hydrazide chemistry, achieved significant activity retention in bioconjugates at a higher PEG:sFv ratio (5:1) than with any of the amine-reactive activated PEG polymers. Prolonged circulating life in mice was demonstrated for each of the PEG conjugates. An increase in PEG polymer length was found to be more effective for serum half-life extension than a corresponding increase in total PEG mass. For example, CC49/218 sFv conjugated to either one strand of PEG-20000, or four strands of PEG-5000, displayed about 20- or 14-fold increased serum half-life, respectively, relative to the unmodified sFv. The demonstrated suitability of established random conjugation chemistries for PEGylation of sFv proteins, in conjunction with innovative site-specific conjugation methods, indicates that production of a panoply of sFv proteins with both engineered affinity and tailored circulating life may now be achievable.     

Comparative Analysis of DNA Nanoparticles and AAVs for Ocular Gene Delivery

Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV), makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs), critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg) than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors.     

Repeated Delivery of Adeno-Associated Virus Vectors to the Rabbit Airway

Efficient local expression from recombinant adeno-associated virus (rAAV)-cystic fibrosis (CF) transmembrane conductance regulator (CFTR) vectors has been observed in the airways of rabbits and monkeys for up to 6 months following a single bronchoscopic delivery. However, it is likely that repeated administrations of rAAV vectors will be necessary for sustained correction of the CF defect in the airways. The current study was designed to test the feasibility of repeated airway delivery of rAAV vectors in the rabbit lung. After two doses of rAAV-CFTR to the airways, rabbits generated high titers of serum anti-AAV neutralizing antibodies. Rabbits then received a third dose of a rAAV vector containing the green fluorescent protein (GFP) reporter gene packaged in either AAV serotype 2 (AAV2) or serotype 3 (AAV3) capsids. Each dose consisted of 1 ml containing 5 x 10(9) DNase-resistant particles of rAAV vector, having no detectable replication-competent AAV or adenovirus. Three weeks later, GFP expression was observed in airway epithelial cells despite high anti-AAV neutralizing titers at the time of delivery. There was no significant difference in the efficiency of DNA transfer or expression between the rAAV3 and rAAV2 groups. No significant inflammatory responses to either repeated airway exposure to rAAV2-CFTR vectors or to GFP expression were observed. These experiments demonstrate that serum anti-AAV neutralizing antibody titers do not predict airway neutralization in vivo and that repeated airway delivery rAAV allows for safe and effective gene transfer.     

Mapping a neutralizing epitope onto the capsid of adeno-associated virus serotype 8

Adeno-associated viruses (AAVs) are small single-stranded DNA viruses that can package and deliver nongenomic DNA for therapeutic gene delivery. AAV8, a liver-tropic vector, has shown great promise for the treatment of hemophilia A and B. However, as with other AAV vectors, host anti-capsid immune responses are a deterrent to therapeutic success. To characterize the antigenic structure of this vector, cryo-electron microscopy and image reconstruction (cryo-reconstruction) combined with molecular genetics, biochemistry, and in vivo approaches were used to define an antigenic epitope on the AAV8 capsid surface for a neutralizing monoclonal antibody, ADK8. Docking of the crystal structures of AAV8 and a generic Fab into the cryo-reconstruction for the AAV8-ADK8 complex identified a footprint on the prominent protrusions that flank the 3-fold axes of the icosahedrally symmetric capsid. Mutagenesis and cell-binding studies, along with in vitro and in vivo transduction assays, showed that the major ADK8 epitope is formed by an AAV variable region, VRVIII (amino acids 586 to 591 [AAV8 VP1 numbering]), which lies on the surface of the protrusions facing the 3-fold axis. This region plays a role in AAV2 and AAV8 cellular transduction. Coincidently, cell binding and trafficking assays indicate that ADK8 affects a postentry step required for successful virus trafficking to the nucleus, suggesting a probable mechanism of neutralization. This structure-directed strategy for characterizing the antigenic regions of AAVs can thus generate useful information to help re-engineer vectors that escape host neutralization and are hence more efficacious.     

In vitro resolution of covalently joined AAV chromosome ends

We have developed an assay for a key step in the replication of adeno-associated virus (AAV) DNA. We demonstrate the covalently joined ends of linear AAV DNA can be resolved in vitro to the open duplex configuration. Only extracts prepared from human cells that have been infected with both adenovirus and AAV are capable of carrying out the reaction. The reaction is initiated by a site-specific and strand-specific endonucleolytic cut at a terminal resolution site near the end of the AAV terminal palindrome. During resolution the orientation of the terminal palindrome is inverted, and the 3' viral strand is extended by DNA synthesis. The size of the newly synthesized 3' strand is nearly identical to that found in viral particles. These observations provide direct biochemical evidence for an essential step in the model for AAV DNA replication.     

High temperature biocatalysis by chemically modified cytochrome C

Chemically modified cytochrome c with poly(ethylene glycol) (PEG) showed activity at temperatures higher than 100 degrees C and to be highly thermostable. The molecular size of PEG moieties and the coupling site affected the thermal stabilization. An optimal PEG/protein mass ratio of 2.8 was found, producing a fully thermostable biocatalyst at 80 degrees C. Site-directed mutagenesis on yeast cytochrome c showed an increased thermostabilization when lysine 79 residue, localized at the edge of the active site, was replaced by a nonreactive residue. Tertiary, secondary, and active-site structures were analyzed by fluorescence, CD, and UV/visible spectroscopies. Besides its disordered structure, the pegylated protein showed a lower unfolding rate at the active-site than the unmodified ones. A shell-like structure seems to protect the heme environment, in which PEG is coiled on the protein surface with a primary shield of rigid water molecules solvating the hydrophilic region of bound-PEG, and the PEG hydrophobic regions interacting with the hydrophobic clusters on protein surface.     

Single amino acid changes can influence titer, heparin binding, and tissue tropism in different adeno-associated virus serotypes

Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors.     

Adeno-associated virus type 12 (AAV12): a novel AAV serotype with sialic acid- and heparan sulfate proteoglycan-independent transduction activity

Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy. Recent isolations of novel AAV serotypes have led to significant advances by broadening the tropism and increasing the efficiency of gene transfer to the desired target cell. However, a major concern that remains is the strong preexisting immune responses to several vectors. In this paper, we describe the isolation and characterization of AAV12, an AAV serotype with unique biological and immunological properties. In contrast to those of all other reported AAVs, AAV12 cell attachment and transduction do not require cell surface sialic acids or heparan sulfate proteoglycans. Furthermore, rAAV12 is resistant to neutralization by circulating antibodies from human serum. The feasibility of rAAV12 as a vector was demonstrated in a mouse model in which muscle and salivary glands were transduced. These characteristics make rAAV12 an interesting candidate for gene transfer applications.     

Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis

ABSTRACT

Site-specific antibody-drug conjugates (ADCs) are designed to overcome the heterogeneity observed with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. Despite significantly enhanced homogeneity, however, the production of site-specific ADCs yields some process-related species heterogeneity, including stereoisomers, unconjugated antibody, underconjugated species, and overconjugated species. An elevated level of size variants, such as heavy chain-light chain species (half ADC), heavy chain-heavy chain-light chain species, and light chain species, is also observed with the final site-specific ADC product. To understand the root cause of heterogeneity generated during the ADC conjugation process, we designed time-course studies for each conjugation step, including reduction, oxidation, conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample analysis. On the basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidation as a result of alternative disulfide bond arrangements. During oxidation, two hinge cysteines formed an intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in the unconjugated antibody. Time-course data also showed that the elevated level of size variants, especially heavy chain-heavy chain-light chain species and light chain species, resulted from the quenching step, where the quenching reagent engaged in a disulfide bond exchange reaction with the ADC and broke the disulfide bonds connecting the heavy chain and light chain. Underconjugated and overconjugated species arose from the equilibrium established during the conjugation reaction.     

Effect of oriented or random PEGylation on bioactivity of a factor VIII inhibitor blocking peptide

Peptides as therapeutic substances are efficient agents in the treatment of several diseases. However, they often have to be chemically modified in order to be suited as therapeutic agent. Conjugation to large carrier molecules is often required. A critical step is the identification of available sites for chemical reaction, without influencing bioactivity. Peptide 238/S1 with the sequence NH(2)-PYWKWQYKYD-COOH previously selected from a combinatorial decapeptide library, has the ability to block inhibitory antibodies against blood clotting factor VIII (FVIII) and therefore, it constitutes a lead for developing a drug to treat patients suffering from development of such antibodies. The aims of this study were (i) to identify sites of the peptide, which are suited for modification without losing bioactivity and (ii) to find out the influence of molecular size of polyethylene glycol (PEG) for bioactivity of the peptide. The contribution of each amino acid residue to biological functionality was investigated by mutational analysis. This method confirmed that the N-terminus is crucial for activity, whereas both lysine residues could be exchanged by other L-amino acids. Using mutational analysis it was possible to identify peptides with higher reactivity compared to the wild type 238/S1. PEGylation experiments demonstrated that conjugation of the peptide to PEG 20,000 resulted in a loss of reactivity, while PEG 5,000 could maintain the bioactivity when conjugated in a site directed manner. The peptide lost its neutralization properties when PEG was coupled to the N-terminus, again indicating that this part of the peptide is important for functionality.     

Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle

BACKGROUND: The overlapping approach was developed recently to expand the adeno-associated viral (AAV) packaging capacity. In this approach, a gene is split into two partially overlapping fragments and separately packaged into an upstream and a downstream vector, respectively. Transgene expression is achieved in co-infected cells after homologous recombination. Despite the promising proof-of-principle results in the lung, the efficiency has been very disappointing in skeletal muscle. Here we examined two potential rate-limiting factors including AAV serotype and the transgene sequence. METHODS: To study serotype effect, we delivered AAV-2, -5 and -6 overlapping vectors (5 x 10(8) vg particles of the upstream and the downstream vectors, respectively) and 5 x 10(8) vg particles of the intact gene vector to the tibialis anterior muscles of 7-week-old C57Bl/6 mice, respectively. To determine the effect of transgene sequence, we compared LacZ and alkaline phosphatase (AP) overlapping vectors. Transduction efficiency was quantified 6 weeks later by scoring the percentage of transgene-positive myofibers. RESULTS: AAV-2 overlapping vectors barely resulted in detectable transduction. Transduction efficiency was significantly improved in AAV-5 and AAV-6. The highest level was achieved in AAV-6 that reached 42% and 96% of that of the intact gene vector for the LacZ gene and the AP gene, respectively. Surprisingly, AAV-6 overlapping vector resulted in higher transduction than did AAV-2 and AAV-5 intact gene vectors. CONCLUSIONS: Our findings suggest that AAV serotype and the transgene sequence play critical roles in the overlapping approach. AAV-6 holds great promise for overlapping vector-mediated muscle gene therapy.     

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

Icosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production.     

Therapeutic anti-methamphetamine antibody fragment-nanoparticle conjugates: synthesis and in vitro characterization

Treatments specific to the medical problems caused by methamphetamine (METH) abuse are greatly needed. Toward this goal, we are developing new multivalent anti-METH antibody fragment-nanoparticle conjugates with customizable pharmacokinetic properties. We have designed a novel anti-METH single chain antibody fragment with an engineered terminal cysteine (scFv6H4Cys). Generation 3 (G3) polyamidoamine dendrimer nanoparticles were chosen for conjugation due to their monodisperse properties and multiple amine functional groups. ScFv6H4Cys was conjugated to G3 dendrimers via a heterobifunctional PEG cross-linker that is reactive to a free amine on one end and a thiol group on the other. PEG modified dendrimers were synthesized by reacting the PEG cross-linker with dendrimers in a stoichiometric ratio of 11:1, which were further reacted with 3-fold molar excess of anti-METH scFv6H4Cys. This reaction resulted in a heterogeneous mix of G3-PEG-scFv6H4Cys conjugates (dendribodies) with three to six scFv6H4Cys conjugated to each dendrimer. The dendribodies were separated from the unreacted PEG modified dendrimers and scFv6H4Cys using affinity chromatography. A detailed in vitro characterization of the PEG modified dendrimers and the dendribodies was performed to determine size, purity, and METH binding function. The dendribodies were found to have affinity for METH identical to that of the unconjugated scFv6H4Cys in saturation binding assays, whereas the PEG modified dendrimers had no affinity for METH. These data suggest that an anti-METH scFv can be successfully conjugated to a PEG modified dendrimer nanoparticle with no adverse effects on METH binding properties. This study is a critical step toward preclinical characterization and development of a novel nanomedicine for the treatment of METH abuse.     

Immune responses to AAV in a phase I study for Canavan disease

BACKGROUND: Canavan disease is a rare leukodystrophy with no current treatment. rAAV-ASPA has been developed for gene delivery to the central nervous system (CNS) for Canavan disease. This study represents the first use of a viral vector in an attempt to ameliorate a neurodegenerative disorder. METHODS: Subjects received intracranial infusions via six cranial burr holes. Adeno-associated virus, serotype 2 (AAV2), mediated intraparenchymal delivery of the human aspartoacylase cDNA at a maximum dose of 1 x 10(12) vector genomes per subject. The immune response and safety profiles were monitored in the follow-up of ten subjects. RESULTS: Following rAAV2 administration, we found no evidence of AAV2 neutralizing antibody titers in serum for the majority of subjects tested (7/10). In a subset (3/10) of subjects, low to moderately high levels of AAV2 neutralizing antibody with respect to baseline were detected. In all subjects, there were minimal systemic signs of inflammation or immune stimulation. In subjects with catheter access to the brain lateral ventricle, cerebrospinal fluid was examined and there was a complete absence of neutralizing antibody titers with no overt signs of brain inflammation. CONCLUSIONS: rAAV2 vector administration to the human CNS appears well tolerated. The low levels of immune response to AAV2 detected in 3/10 subjects in this study suggest at this dose and with intraparenchymal administration this approach is relatively safe. Long-term monitoring of subjects and expansion to phase II/III will be necessary in order to make definitive statements on safety and efficacy.     

Low modulus biomimetic microgel particles with high loading of hemoglobin

We synthesized extremely deformable red blood cell-like microgel particles and loaded them with bovine hemoglobin (Hb) to potentiate oxygen transport. With similar shape and size as red blood cells (RBCs), the particles were fabricated using the PRINT (particle replication in nonwetting templates) technique. Low cross-linking of the hydrogel resulted in very low mesh density for these particles, allowing passive diffusion of hemoglobin throughout the particles. Hb was secured in the particles through covalent conjugation of the lysine groups of Hb to carboxyl groups in the particles via EDC/NHS coupling. Confocal microscopy of particles bound to fluorescent dye-labeled Hb confirmed the uniform distribution of Hb throughout the particle interior, as opposed to the surface conjugation only. High loading ratios, up to 5 times the amount of Hb to polymer by weight, were obtained without a significant effect on particle stability and shape, though particle diameter decreased slightly with Hb conjugation. Analysis of the protein by circular dichroism (CD) spectroscopy showed that the secondary structure of Hb was unperturbed by conjugation to the particles. Methemoglobin in the particles could be maintained at a low level and the loaded Hb could still bind oxygen, as studied by UV-vis spectroscopy. Hb-loaded particles with moderate loading ratios demonstrated excellent deformability in microfluidic devices, easily deforming to pass through restricted pores half as wide as the diameter of the particles. The suspension of concentrated particles with a Hb concentration of 5.2 g/dL showed comparable viscosity to that of mouse blood, and the particles remained intact even after being sheared at a constant high rate (1000 1/s) for 10 min. Armed with the ability to control size, shape, deformability, and loading of Hb into RBC mimics, we will discuss the implications for artificial blood.     

Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity. To optimize PEGylation, scFvs have been recombinantly produced in a vector that adds an unpaired cysteine (c) near the scFv carboxy terminus (scFv-c), thus providing a specific site for thiol conjugation. To evaluate the general applicability of this unpaired cysteine for PEGylation of scFv-c, conjugation efficiency was determined for four different scFvs and several PEG molecules having thiol reactive groups. The effect of the PEG molecular format on scFv-c PEG malignant cell binding was also addressed. ScFvs produced as scFv-c and purified by anti E-TAG affinity chromatography were conjugated using PEG molecules with maleimide (Mal) or o-pyridyl disulfide (OPSS). Conjugations were performed at pH 7.0, with 2 molar excess TCEP/scFv and PEG-(Mal) or PEG-OPSS, using 5:1 (PEG/scFv). PEG-Mal conjugation efficiency was also evaluated with 1:5 (PEG/scFv). PEGylation efficiency was determined for each reaction by quantitation of the products on SDS-PAGE. ScFv-c conjugation with unifunctional maleimide PEGs resulted in PEG conjugates incorporating 30-80% of the scFv-c, but usually above 50%. Efficiency of scFv-c conjugation to both functional groups of the bifunctional PEG-(Mal)2 varied between the PEG and scFv-c molecules studied. A maximum of 45% of scFv-c protein was conjugated as PEG- (scFv-c)2 using the smallest PEG-(Mal)2 (2 kDa). No significant increase in scFv-c conjugation was observed by the use of greater than a 5 molar excess of PEG/scFv-c. Under the same conjugation conditions, PEG as OPSS yielded less than 10% PEG-scFv-c. PEG-(scFv)2 conjugates had increased binding in ELISA using malignant cell membranes, when compared with unmodified scFv-c. PEGylated-scFv binding was comparable with unmodified scFv-c. In summary, scFv-c can be PEGylated in a site-specific manner using uni- or bivalent PEG-Mal, either linear or branched. ScFv-c was most efficiently conjugated to smaller PEG-Mal molecules, with the smallest, 2 kDa PEG-Mal, usually PEGylating 60-90% of the scFv-c. ScFv-c conjugation to form PEG-(scFv-c)2 reached greatest efficiency at 45%, and its purified form demonstrated greater binding than the corresponding scFv-c.     

Experimental design to optimize an <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> type b conjugate vaccine made with hydrazide-derivatized tetanus toxoid

The introduction of type b Haemophilus influenzae conjugate vaccines into routine vaccination schedules has significantly reduced the burden of this disease; however, widespread use in developing countries is constrained by vaccine costs, and there is a need for a simple and high-yielding manufacturing process. The vaccine is composed of purified capsular polysaccharide conjugated to an immunogenic carrier protein. To improve the yield and rate of the reductive amination conjugation reaction used to make this vaccine, some of the carboxyl groups of the carrier protein, tetanus toxoid, were modified to hydrazides, which are more reactive than the ε -amine of lysine. Other reaction parameters, including the ratio of the reactants, the size of the polysaccharide, the temperature and the salt concentration, were also investigated. Experimental design was used to minimize the number of experiments required to optimize all these parameters to obtain conjugate in high yield with target characteristics. It was found that increasing the reactant ratio and decreasing the size of the polysaccharide increased the polysaccharide:protein mass ratio in the product. Temperature and salt concentration did not improve this ratio. These results are consistent with a diffusion controlled rate limiting step in the conjugation reaction. Excessive modification of tetanus toxoid with hydrazide was correlated with reduced yield and lower free polysaccharide. This was attributed to a greater tendency for precipitation, possibly due to changes in the isoelectric point. Experimental design and multiple regression helped identify key parameters to control and thereby optimize this conjugation reaction.     

Cloning of infectious adeno-associated virus genomes in bacterial plasmids

We describe the construction of two Escherichia coli hybrid plasmids, each of which contains the entire 4.7-kb DNA genome of the human parvovirus, adeno-associated virus (AAV) type 2. Because the AAV genome was inserted into the plasmid DNA using BglII linkers the entire virus genome can be recovered by in vitro cleavage of the purified recombinant plasmid. Transfection of these recombinant DNAs into an adenovirus-transformed human cell line in the presence of helper adenovirus resulted in efficient rescue and replication of the AAV genome and production of fully infectious virus particles. These AAV-plasmid recombinant DNA molecules should be useful both for site-specific mutagenesis of the viral genome and to study the potential of AAV as a eukaryotic vector.     

Complement activation and cell uptake responses toward polymer-functionalized protein nanocapsules

Self-assembling protein nanocapsules can be engineered for various bionanotechnology applications. Using the dodecahedral scaffold of the E2 subunit from pyruvate dehydrogenase, we introduced non-native surface cysteines for site-directed functionalization. The modified nanoparticle's structural, assembly, and thermostability properties were comparable to the wild-type scaffold (E2-WT), and after conjugation of poly(ethylene glycol) (PEG) to these cysteines, the nanoparticle remained intact and stable up to 79.7 ± 1.8 °C. PEGylation of particles reduced uptake by human monocyte-derived macrophages and MDA-MB-231 breast cancer cells, with decreased uptake as PEG chain length is increased. In vitro C4-depletion and C5a-production assays yielded 97.6 ± 10.8% serum C4 remaining and 40.1 ± 6.0 ng/mL C5a for E2-WT, demonstrating that complement activation is weak for non-PEGylated E2 nanoparticles. Conjugation of PEG to these particles moderately increased complement response to give 79.7 ± 6.0% C4 remaining and 87.6 ± 10.1 ng/mL C5a. Our results demonstrate that PEGylation of the E2 protein nanocapsules can modulate cellular uptake and induce low levels of complement activation, likely via the classical/lectin pathways.