Clinical and biomechanical changes following a 4-month toe-out gait modification program for people with medial knee osteoarthritis: a randomized controlled trial.

OBJECTIVE: To compare changes in knee pain, function, and loading following a 4-month progressive walking program with or without toe-out gait modification in people with medial tibiofemoral knee osteoarthritis. DESIGN: Individuals with medial knee osteoarthritis were randomized to a 4-month program to increase walking activity with (toe-out) or without (progressive walking) concomitant toe-out gait modification. The walking program was similar between the two groups, except that the gait modification group was trained to walk with 15° more toe-out. Primary outcomes included: knee joint pain (WOMAC), foot progression angles and knee joint loading during gait (knee adduction moment (KAM)). Secondary outcomes included WOMAC function, timed stair climb, and knee flexion moments during gait. RESULTS: Seventy-nine participants (40 in toe-out group, 39 in progressive walking group) were recruited. Intention-to-treat analysis showed no between-group differences in knee pain, function, or timed stair climb. However, the toe-out group exhibited significantly greater changes in foot progression angle (mean difference = -9.04° (indicating more toe-out), 95% CI: -11.22°, -6.86°; P < 0.001), late stance KAM (mean difference = -0.26 %BW*ht, 95% CI: -0.39 %BW*ht, -0.12 %BW*ht, P < 0.001) and KAM impulse (-0.06 %BW*ht*s, 95% CI: -0.11 %BW*ht*s, -0.01 %BW*ht*s; P = 0.031) compared to the progressive walking group at follow-up. The only between-group difference that remained at a 1-month retention assessment was foot progression angle, with greater changes in the toe-out group (mean difference = -6.78°, 95% CI: -8.82°, -4.75°; P < 0.001). CONCLUSIONS: Though both groups experienced improvements in self-reported pain and function, only the toe-out group experienced biomechanical improvements. TRIALS REGISTRY NUMBER: NCT02019108.

The Silent Sway of Splicing by Synonymous Substitutions.

Alternative splicing diversifies mRNA transcripts in human cells. This sequence-driven process can be influenced greatly by mutations, even those that do not change the protein coding potential of the transcript. Synonymous mutations have been shown to alter gene expression through modulation of splicing, mRNA stability, and translation. Using a synonymous position mutation library in SMN1 exon 7, we show that 23% of synonymous mutations across the exon decrease exon inclusion, suggesting that nucleotide identity across the entire exon has been evolutionarily optimized to support a particular exon inclusion level. Although phylogenetic conservation scores are insufficient to identify synonymous positions important for exon inclusion, an alignment of organisms filtered based on similar exon/intron architecture is highly successful. Although many of the splicing neutral mutations are observed to occur, none of the exon inclusion reducing mutants was found in the filtered alignment. Using the modified phylogenetic comparison as an approach to evaluate the impact on pre-mRNA splicing suggests that up to 45% of synonymous SNPs are likely to alter pre-mRNA splicing. These results demonstrate that coding and pre-mRNA splicing pressures co-evolve and that a modified phylogenetic comparison based on the exon/intron architecture is a useful tool in identifying splice altering SNPs.

Directed DNA shuffling of retrovirus and retrotransposon integrase protein domains.

Chimeric proteins are used to study protein domain functions and to recombine protein domains for novel or optimal functions. We used a library of chimeric integrase proteins to study DNA integration specificity. The library was constructed using a directed shuffling method that we adapted from fusion PCR. This method easily and accurately shuffles multiple DNA gene sequences simultaneously at specific base-pair positions, such as protein domain boundaries. It produced all 27 properly-ordered combinations of the amino-terminal, catalytic core, and carboxyl-terminal domains of the integrase gene from human immunodeficiency virus, prototype foamy virus, and Saccharomyces cerevisiae retrotransposon Ty3. Retrotransposons can display dramatic position-specific integration specificity compared to retroviruses. The yeast retrotransposon Ty3 integrase interacts with RNA polymerase III transcription factors to target integration at the transcription initiation site. In vitro assays of the native and chimeric proteins showed that human immunodeficiency virus integrase was active with heterologous substrates, whereas prototype foamy virus and Ty3 integrases were not. This observation was consistent with a lower substrate specificity for human immunodeficiency virus integrase than for other retrovirus integrases. All eight chimeras containing the Ty3 integrase carboxyl-terminal domain, a candidate targeting domain, failed to target strand transfer in the presence of the targeting protein, suggesting that multiple domains of the Ty3 integrase cooperate in this function.

Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53.

The tumour suppressor p53 is the most frequently mutated gene in human cancer. Reactivation of mutant p53 by small molecules is an exciting potential cancer therapy. Although several compounds restore wild-type function to mutant p53, their binding sites and mechanisms of action are elusive. Here computational methods identify a transiently open binding pocket between loop L1 and sheet S3 of the p53 core domain. Mutation of residue Cys124, located at the centre of the pocket, abolishes p53 reactivation of mutant R175H by PRIMA-1, a known reactivation compound. Ensemble-based virtual screening against this newly revealed pocket selects stictic acid as a potential p53 reactivation compound. In human osteosarcoma cells, stictic acid exhibits dose-dependent reactivation of p21 expression for mutant R175H more strongly than does PRIMA-1. These results indicate the L1/S3 pocket as a target for pharmaceutical reactivation of p53 mutants.

Ensemble-based computational approach discriminates functional activity of p53 cancer and rescue mutants.

The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain ("cancer mutants"). Activity can be restored by second-site suppressor mutations ("rescue mutants"). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC) metric was strongly correlated (r(2) = 0.77) with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i) p53 cancer mutants were more flexible than wild-type protein, (ii) second-site rescue mutations decreased the flexibility of cancer mutants, and (iii) negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.

A vector set for systematic metabolic engineering in Saccharomyces cerevisiae.

A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 µ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering.

All-codon scanning identifies p53 cancer rescue mutations.

In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes. We have used ACS to produce single amino-acid changes in small and large regions of the human tumor suppressor protein p53 to identify single amino-acid substitutions that can restore activity to inactive p53 found in human cancers. Single-tube reactions were used to saturate defined 30-nt regions with all possible codon changes. The same technique was used in 20 parallel reactions to scan the 600-bp fragment encoding the entire p53 core domain. Identification of several novel p53 cancer rescue mutations demonstrated the utility of the ACS approach. ACS is a fast, simple and versatile method, which is useful for protein structure-function analyses and protein design or evolution problems.

Recombinant human collagen and biomimetic variants using a de novo gene optimized for modular assembly.

A collagen-mimetic polymer that can be easily engineered with specific cell-responsive and mechanical properties would be of significant interest for fundamental cell-matrix studies and applications in regenerative medicine. However, oligonucleotide-based synthesis of full-length collagen has been encumbered by the characteristic glycine-X-Y sequence repetition, which promotes mismatched oligonucleotide hybridizations during de novo gene assembly. In this work, we report a novel, modular synthesis strategy that yields full-length human collagen III and specifically defined variants. We used a computational algorithm that applies codon degeneracy to design oligonucleotides that favor correct hybridizations while disrupting incorrect ones for gene synthesis. The resulting recombinant polymers were expressed in Saccharomyces cerevisiae engineered with prolyl-4-hydroxylase. Our modular approach enabled mixing-and-matching domains to fabricate different combinations of collagen variants that contained different secretion signals at the N-terminus and cysteine residues imbedded within the triple-helical domain at precisely defined locations. This work shows the flexibility of our strategy for designing and assembling specifically tailored biomimetic collagen polymers with re-engineered properties.

Mechanism of U insertion RNA editing in trypanosome mitochondria: the bimodal TUTase activity of the core complex.

Expression of the trypanosomal mitochondrial genome requires the insertion and deletion of uridylyl residues at specific sites in pre-mRNAs. RET2 terminal uridylyl transferase is an integral component of the RNA editing core complex (RECC) and is responsible for the guide-RNA-dependent U insertion reaction. By analyzing RNA-interference-based knock-in Trypanosoma brucei cell lines, purified editing complex, and individual protein, we have investigated RET2's association with the RECC. In addition, the U insertion activity exhibited by RET2 as an RECC subunit was compared with characteristics of the monomeric protein. We show that interaction of RET2 with RECC is accomplished via a protein-protein contact between its middle domain and a structural subunit, MP81. The recombinant RET2 catalyzes a faithful editing on gapped (precleaved) double-stranded RNA substrates, and this reaction requires an internal monophosphate group at the 5' end of the mRNA 3' cleavage fragment. However, RET2 processivity is limited to insertion of three Us. Incorporation into the RECC voids the internal phosphate requirement and allows filling of longer gaps similar to those observed in vivo. Remarkably, monomeric and RECC-embedded enzymes display a similar bimodal activity: the distributive insertion of a single uracil is followed by a processive extension limited by the number of guiding nucleotides. Based on the RNA substrate specificity of RET2 and the purine-rich nature of U insertion sites, we propose that the distributive +1 insertion creates a substrate for the processive gap-filling reaction. Upon base-pairing of the +1 extended 5' cleavage fragment with a guiding nucleotide, this substrate is recognized by RET2 in a different mode compared to the product of the initial nucleolytic cleavage. Therefore, RET2 distinguishes base pairs in gapped RNA substrates which may constitute an additional checkpoint contributing to overall fidelity of the editing process.

A 'random steady-state' model for the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase enzyme complexes.

The multienzyme complexes, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, involved in the central metabolism of Escherichia coli consist of multiple copies of three different enzymes, E1, E2 and E3, that cooperate to channel substrate intermediates between their active sites. The E2 components form the core of the complex, while a mixture of E1 and E3 components binds to the core. We present a random steady-state model to describe catalysis by such multienzyme complexes. At a fast time scale, the model describes the enzyme catalytic mechanisms of substrate channeling at a steady state, by polynomially approximating the analytic solution of a biochemical master equation. At a slower time scale, the structural organization of the different enzymes in the complex and their random binding/unbinding to the core is modeled using methods from equilibrium statistical mechanics. Biologically, the model describes the optimization of catalytic activity by substrate sharing over the entire enzyme complex. The resulting enzymatic models illustrate the random steady state (RSS) for modeling multienzyme complexes in metabolic pathways.

Predicting positive p53 cancer rescue regions using Most Informative Positive (MIP) active learning.

Many protein engineering problems involve finding mutations that produce proteins with a particular function. Computational active learning is an attractive approach to discover desired biological activities. Traditional active learning techniques have been optimized to iteratively improve classifier accuracy, not to quickly discover biologically significant results. We report here a novel active learning technique, Most Informative Positive (MIP), which is tailored to biological problems because it seeks novel and informative positive results. MIP active learning differs from traditional active learning methods in two ways: (1) it preferentially seeks Positive (functionally active) examples; and (2) it may be effectively extended to select gene regions suitable for high throughput combinatorial mutagenesis. We applied MIP to discover mutations in the tumor suppressor protein p53 that reactivate mutated p53 found in human cancers. This is an important biomedical goal because p53 mutants have been implicated in half of all human cancers, and restoring active p53 in tumors leads to tumor regression. MIP found Positive (cancer rescue) p53 mutants in silico using 33% fewer experiments than traditional non-MIP active learning, with only a minor decrease in classifier accuracy. Applying MIP to in vivo experimentation yielded immediate Positive results. Ten different p53 mutations found in human cancers were paired in silico with all possible single amino acid rescue mutations, from which MIP was used to select a Positive Region predicted to be enriched for p53 cancer rescue mutants. In vivo assays showed that the predicted Positive Region: (1) had significantly more (p<0.01) new strong cancer rescue mutants than control regions (Negative, and non-MIP active learning); (2) had slightly more new strong cancer rescue mutants than an Expert region selected for purely biological considerations; and (3) rescued for the first time the previously unrescuable p53 cancer mutant P152L.

Computationally Optimised DNA Assembly of synthetic genes.

Gene synthesis is hampered by two obstacles: improper assembly of oligonucleotides; oligonucleotide defects incurred during chemical synthesis. To overcome the first problem, we describe the employment of a Computationally Optimised DNA Assembly (CODA) algorithm that uses the degeneracy of the genetic code to design overlapping oligonucleotides with thermodynamic properties for self-assembly into a single, linear, DNA product. To address the second problem, we describe a hierarchical assembly strategy that reduces the incorporation of defective oligonucleotides into full-length gene constructs. The CODA algorithm and these biological methods enable fast, simple and reliable assemblies of sequence-correct full-length genes.

TY3 GAG3 protein forms ordered particles in Escherichia coli.

The yeast retrovirus-like element Ty3 GAG3 gene encodes a Gag3 polyprotein analogous to retroviral Gag. Gag3 lacks matrix, but contains capsid, spacer, and nucleocapsid domains. Expression of a Ty3 Gag3 or capsid domain optimized for expression in Escherichia coli was sufficient for Ty3 particle assembly. Virus-like ordered particles assembled from Gag3 were similar in size to immature particles from yeast and contained nucleic acid. However, particles assembled from the CA domain were variable in size and displayed much less organization than native particles. These results indicate that assembly can be driven through interactions among capsid subunits in the particle, but that the nucleocapsid domain, likely in association with RNA, confers order upon this process.

Ty3 nucleocapsid controls localization of particle assembly.

Expression of the budding yeast retrotransposon Ty3 results in production of viruslike particles (VLPs) and retrotransposition. The Ty3 major structural protein, Gag3, similar to retrovirus Gag, is processed into capsid, spacer, and nucleocapsid (NC) during VLP maturation. The 57-amino-acid Ty3 NC protein has 17 basic amino acids and contains one copy of the CX(2)CX(4)HX(4)C zinc-binding motif found in retrovirus NC proteins. Ty3 RNA, protein, and VLPs accumulate in clusters associated with RNA processing bodies (P bodies). This study investigated the role of the NC domain in Ty3-P body clustering and VLP assembly. Fifteen Ty3 NC Ala substitution and deletion mutants were examined using transposition, immunoblot, RNA protection, cDNA synthesis, and multimerization assays. Localization of Ty3 proteins and VLPs was characterized microscopically. Substitutions of each of the conserved residues of the zinc-binding motif resulted in the loss of Ty3 RNA packaging. Substitution of the first two of four conserved residues in this motif caused the loss of Ty3 RNA and protein clustering with P bodies and disrupted particle formation. NC was shown to be a mediator of formation of Ty3 RNA foci and association of Ty3 RNA and protein with P bodies. Mutations that disrupted these NC functions resulted in various degrees of Gag3 nuclear localization and a spectrum of different particle states. Our findings are consistent with the model that Ty3 assembly is associated with P-body components. We hypothesize that the NC domain acts as a molecular switch to control Gag3 conformational states that affect both assembly and localization.

Amino Acid Metabolism and Fluxes.

By the mid1960s, the pioneering work of Umbarger and Gerhart and Pardee had shown us that carbon flow through a biosynthetic pathway was controlled by allosteric inhibition of the first enzyme of the pathway by its end product; and, studies of the lac operon by Jacob and Monod had established that genes were controlled by an operator-repressor mechanism. During the intervening forty-plus years, knowledge and technologies have continued to explode in unanticipated ways. Today, we understand in great detail the molecular mechanisms of the many levels of metabolic and genetic regulation that control carbon flow through the amino acid biosynthetic pathways. Traditional experimental approaches are not sufficient for the integration and reconstruction of complex biological systems using data mostly generated by high-throughput experiments. Only with computational methods and adequate modeling tools will we be able to reconstruct and query these large and complicated systems. Due to complicated enzyme reaction mechanisms and the frequent lack of rate constant measurements needed for solving differential equations, most investigators have turned their attention to the development of abstract, top-down modeling tools. For example, Palsson and colleagues have used metabolic flux balance analysis (FBA) methods to simulate steady-state metabolite flux through E. coli pathways representing hundreds of enzyme steps. Recently, Yang et al. have developed a bottom-up, enzyme mechanism modeling language, kMech (kinetic mechanism), for the mathematical simulation of metabolic pathways.

Optimizing scaleup yield for protein production: Computationally Optimized DNA Assembly (CODA) and Translation Engineering.

Translation Engineering combined with synthetic biology (gene synthesis) techniques makes it possible to deliberately alter the presumed translation kinetics of genes without altering the amino acid sequence. Here, we describe proprietary technologies that design and assemble synthetic genes for high expression and enhanced protein production, and offers new insights and methodologies for affecting protein structure and function. We have patented Translation Engineering technologies to manage the complexity of gene design to account for codon pair usage, translational pausing signals, RNA secondary structure and user-defined sequences such as restriction sites. Failure to optimize for codon pair-encoded translation pauses often results in the relatively common occurrence of a slowly translated codon pair that slows the rate of protein elongation and decreases total protein production. Translation Engineering technology improves heterologous expression by tuning the gene sequence for translation in any well-characterized host, including cell-free expression techniques characterized by "broken"Escherichia coli systems used in kits for today's molecular tools market. In addition, we have patented a novel gene assembly method (Computationally Optimized DNA Assembly; CODA) that uses the degeneracy of the genetic code to design oligonucleotides with thermodynamic properties for self-assembly into a single, linear DNA product. Fast translational kinetics and robust protein expression are optimized in synthetic "Hot Rod" genes that are guaranteed to express in E. coli at high levels. These genes are optimized for codon usage and other properties known to aid protein expression, and importantly, they are engineered to be devoid of mRNA secondary structures that might impede transcription, and over-represented codon pairs that might impede translation. Hot Rod genes allow translating ribosomes and E. coli RNA polymerases to maintain coupled translation and transcription at maximal rates. As a result, the nascent mRNA is produced at a high level and is sequestered in polysomes where it is protected from degradation, even further enhancing protein production. In this review we demonstrate that codon context can profoundly influence translation kinetics, and that over-represented codon pairs are often present at protein domain boundaries and appear to control independent protein folding in several popular expression systems. Finally, we consider that over-represented codon pairs (pause sites) may be essential to solving problems of protein expression, solubility, folding and activity encountered when genes are introduced into heterologous expression systems, where the specific set of codon pairs controlling ribosome pausing are different. Thus, Translation Engineering combined with synthetic biology (gene synthesis) techniques may allow us to manipulate the translation kinetics of genes to restore or enhance function in a variety of traditional and novel expression systems.

Ty3 capsid mutations reveal early and late functions of the amino-terminal domain.

The Ty3 retrotransposon assembles into 50-nm virus-like particles that occur in large intracellular clusters in the case of wild-type (wt) Ty3. Within these particles, maturation of the Gag3 and Gag3-Pol3 polyproteins by Ty3 protease produces the structural proteins capsid (CA), spacer, and nucleocapsid. Secondary and tertiary structure predictions showed that, like retroviral CA, Ty3 CA contains a large amount of helical structure arranged in amino-terminal and carboxyl-terminal bundles. Twenty-six mutants in which alanines were substituted for native residues were used to study CA subdomain functions. Transposition was measured, and particle morphogenesis and localization were characterized by analysis of protein processing, cDNA production, genomic RNA protection, and sedimentation and by fluorescence and electron microscopy. These measures defined five groups of mutants. Proteins from each group could be sedimented in a large complex. Mutations in the amino-terminal domain reduced the formation of fluorescent Ty3 protein foci. In at least one major homology region mutant, Ty3 protein concentrated in foci but no wt clusters of particles were observed. One mutation in the carboxyl-terminal domain shifted assembly from spherical particles to long filaments. Two mutants formed foci separate from P bodies, the proposed sites of assembly, and formed defective particles. P-body association was therefore found to be not necessary for assembly but correlated with the production of functional particles. One mutation in the amino terminus blocked transposition after cDNA synthesis. Our data suggest that Ty3 proteins are concentrated first, assembly associated with P bodies occurs, and particle morphogenesis concludes with a post-reverse transcription, CA-dependent step. Particle formation was generally resistant to localized substitutions, possibly indicating that multiple domains are involved.

DNA deformation energy as an indirect recognition mechanism in protein-DNA interactions.

Proteins that bind to specific locations in genomic DNA control many basic cellular functions. Proteins detect their binding sites using both direct and indirect recognition mechanisms. Deformation energy, which models the energy required to bend DNA from its native shape to its shape when bound to a protein, has been shown to be an indirect recognition mechanism for one particular protein, Integration Host Factor (IHF). This work extends the analysis of deformation to two other DNA-binding proteins, CRP and SRF, and two endonucleases, I-CreI and I-PpoI. Known binding sites for all five proteins showed statistically significant differences in mean deformation energy as compared to random sequences. Binding sites for the three DNA-binding proteins and one of the endonucleases had mean deformation energies lower than random sequences. Binding sites for I-PpoI had mean deformation energy higher than random sequences. Classifiers that were trained using the deformation energy at each base pair step showed good cross-validated accuracy when classifying unseen sequences as binders or nonbinders. These results support DNA deformation energy as an indirect recognition mechanism across a wider range of DNA-binding proteins. Deformation energy may also have a predictive capacity for the underlying catalytic mechanism of DNA-binding enzymes.

Pressure dissociation of integration host factor-DNA complexes reveals flexibility-dependent structural variation at the protein-DNA interface.

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236-39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379-401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF-DNA interfaces while the resulting energetic compensation maintains the same net binding energy.

GRA1 protein vaccine confers better immune response compared to codon-optimized GRA1 DNA vaccine.

The present study evaluates immunogenicity and protection potency of a codon-optimized GRA1 DNA vaccine, wild type GRA1 DNA vaccine and an adjuvanted recombinant GRA1 protein vaccine candidate in BALB/c mice against lethal toxoplasmosis. Of the three GRA1 vaccines tested, the recombinant GRA1 protein vaccine results reveal significant increase in immune response and prolonged survival against acute toxoplasmosis compared to DNA vaccinations. Immune response and protection conferred by codon-optimized GRA1 DNA vaccine was slightly better than wild type GRA1 DNA vaccine.