The origin of large molecules in primordial autocatalytic reaction networks

Large molecules such as proteins and nucleic acids are crucial for life, yet their primordial origin remains a major puzzle. The production of large molecules, as we know it today, requires good catalysts, and the only good catalysts we know that can accomplish this task consist of large molecules. Thus the origin of large molecules is a chicken and egg problem in chemistry. Here we present a mechanism, based on autocatalytic sets (ACSs), that is a possible solution to this problem. We discuss a mathematical model describing the population dynamics of molecules in a stylized but prebiotically plausible chemistry. Large molecules can be produced in this chemistry by the coalescing of smaller ones, with the smallest molecules, the `food set', being buffered. Some of the reactions can be catalyzed by molecules within the chemistry with varying catalytic strengths. Normally the concentrations of large molecules in such a scenario are very small, diminishing exponentially with their size. ACSs, if present in the catalytic network, can focus the resources of the system into a sparse set of molecules. ACSs can produce a bistability in the population dynamics and, in particular, steady states wherein the ACS molecules dominate the population. However to reach these steady states from initial conditions that contain only the food set typically requires very large catalytic strengths, growing exponentially with the size of the catalyst molecule. We present a solution to this problem by studying `nested ACSs', a structure in which a small ACS is connected to a larger one and reinforces it. We show that when the network contains a cascade of nested ACSs with the catalytic strengths of molecules increasing gradually with their size (e.g., as a power law), a sparse subset of molecules including some very large molecules can come to dominate the system.Comment: 49 pages, 17 figures including supporting informatio

Generation, Characterization and Epitope Mapping of Two Neutralizing and Protective Human Recombinant Antibodies against Influenza A H5N1 Viruses

The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development.We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model.Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines

Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries

Phage display of combinatorial antibody libraries is a very efficient method for selecting recombinant antibodies against a wide range of molecules. It has been applied very successfully for the generation of therapeutic antibodies for more than a decade. To increase robustness and reproducibility of the selection procedure, we developed a semi-automated selection method for the generation of recombinant antibodies from phage display libraries. In this procedure, the selection targets are specifically immobilised to magnetic particles which can then by automatically handled by a magnetic particle processor. At present up to 96 samples can be handled simultaneously. Applying the processor allows standardisation of panning parameters such as washing conditions, incubation times, or to perform parallel selections on same targets under different buffer conditions. Additionally, the whole protocol has been streamlined to carry out bead loading, phage selection, phage amplification between selection rounds and magnetic particle ELISA for confirmation of binding activity in microtiter plate formats. Until now, this method has been successfully applied to select antibody fragments against different types of target, such as peptides, recombinant or homologous proteins, or chemical compounds

Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis)

<p>Abstract</p> <p>Background</p> <p>The cotton boll weevil (<it>Anthonomus grandis</it>) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. <it>In vitro </it>directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of <it>Bacillus thuringiensis</it>.</p> <p>Results</p> <p>Bioassays carried out with <it>A. grandis </it>larvae revealed that the LC₅₀of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability.</p> <p>Conclusions</p> <p>The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control <it>A. grandis</it>.</p

The 4C5 Cell-Impermeable Anti-HSP90 Antibody with Anti-Cancer Activity, Is Composed of a Single Light Chain Dimer

MAb 4C5 is a cell impermeable, anti-HSP90 murine monoclonal antibody, originally produced using hybridoma technology. We have previously shown that mAb 4C5 specifically recognizes both the α- and to a lesser extent the β-isoform of HSP90. Additionally, in vitro and in vivo studies revealed that by selectively inhibiting the function of cell-surface HSP90, mAb 4C5 significantly impairs cancer cell invasion and metastasis. Here we describe the reconstitution of mAb 4C5 into a mouse-human chimera. More importantly we report that mAb 4C5 and consequently its chimeric counterpart are completely devoid of heavy chain and consist only of a functional kappa light chain dimer. The chimeric antibody is shown to retain the original antibody's specificity and functional properties. Thus it is capable of inhibiting the function of surface HSP90, leading to reduced cancer cell invasion in vitro. Finally, we present in vivo evidence showing that the chimeric 4C5 significantly inhibits the metastatic deposit formation of MDA-MB-453 cells into the lungs of SCID mice. These data suggest that a chimeric kappa light chain antibody could be potentially used as an anti-cancer agent, thereby introducing a novel type of antibody fragment, with reduced possible adverse immunogenic effects, into cancer therapeutics

Selection of Diethylstilbestrol-Specific Single-Chain Antibodies from a Non-Immunized Mouse Ribosome Display Library

Single chain variable fragments (scFvs) against diethylstilbestrol (DES) were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM) complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3) for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of one clone (30-1). The measured KD was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies

Bottom-Up Assembly of Hydrogels from Bacteriophage and Au Nanoparticles: The Effect of Cis- and Trans-Acting Factors

Hydrogels have become a promising research focus because of their potential for biomedical application. Here we explore the long-range, electrostatic interactions by following the effect of trans-acting (pH) and cis-acting factors (peptide mutation) on the formation of Au-phage hydrogels. These bioinorganic hydrogels can be generated from the bottom-up assembly of Au nanoparticles (Au NP) with either native or mutant bacteriophage (phage) through electrostatic interaction of the phage pVIII major capsid proteins (pVIII). The cis-acting factor consists of a peptide extension displayed on the pVIII that mutates the phage. Our results show that pH can dictate the direct-assembly and stability of Au-phage hydrogels in spite of the differences between the native and the mutant pVIII. The first step in characterizing the interactions of Au NP with phage was to generate a molecular model that identified the charge distribution and structure of the native and mutant pVIII. This model indicated that the mutant peptide extension carried a higher positive charge relative to the native pVIII at all pHs. Next, by monitoring the Au-phage interaction by means of optical microscopy, elastic light scattering, fractal dimension analysis as well as Uv-vis and surface plasmon resonance spectroscopy, we show that the positive charge of the mutant peptide extension favors the opposite charge affinity between the phage and Au NP as the pH is decreased. These results show the versatility of this assembly method, where the stability of these hydrogels can be achieved by either adjusting the pH or by changing the composition of the phage pVIII without the need of phage display libraries

Identification of Peptide Mimotopes of Trypanosoma brucei gambiense Variant Surface Glycoproteins

The control of human African trypanosomiasis or sleeping sickness, a deadly disease in sub-Saharan Africa, mainly depends on a correct diagnosis and treatment. The aim of our study was to identify mimotopic peptides (mimotopes) that may replace the native proteins in antibody detection tests for sleeping sickness and hereby improve the diagnostic sensitivity and specificity. We selected peptide expressing phages from the PhD.-12 and PhD.-C7C phage display libraries with mouse monoclonal antibodies specific to variant surface glycoprotein (VSG) LiTat 1.3 or LiTat 1.5 of Trypanosoma brucei gambiense. The peptide coding genes of the selected phages were sequenced and the corresponding peptides were synthesised. Several of the synthetic peptides were confirmed as mimotopes for VSG LiTat 1.3 or LiTat 1.5 since they were able to inhibit the binding of their homologous monoclonal to the corresponding VSG. These peptides were biotinylated and their diagnostic potential was assessed with human sera. We successfully demonstrated that human sleeping sickness sera recognise some of the mimotopes of VSG LiTat 1.3 and LiTat 1.5, indicating the diagnostic potential of such peptides

Anti-HIV-1 Response Elicited in Rabbits by Anti-Idiotype Monoclonal Antibodies Mimicking the CD4-Binding Site

Antibodies against conserved epitopes on HIV-1 envelope glycoproteins (Env), such as the gp120 CD4-binding site (CD4bs), could contribute to protection against HIV-1. Env-based immunogens inducing such a response could be a major component of future anti-HIV-1 strategies. In this proof-of-concept study we describe the generation of two anti-idiotype (AI) murine antibodies mimicking the CD4bs epitope. Sera were collected from long-term non-progressor patients to obtain CD4bs-directed IgG, through sequential purification steps. The purified IgG were then used as Fab fragments to immunize mice for hybridoma generation. Two hybridomas (P1 and P2), reacting only against the CD4bs-directed IgG, were identified and characterized. The P1 and P2 antibodies were shown to recognize the idiotype of the broadly neutralizing anti-CD4bs human mAb b12. Both P1 and P2 Fabs were able to induce a strong anti-gp120 response in rabbits. Moreover, the rabbits' sera were shown to neutralize two sensitive tier 1 strains of HIV-1 in an Env-pseudotype neutralization assay. In particular, 3/5 rabbits in the P1 group and 1/5 in the P2 group showed greater than 80% neutralizing activity against the HXB2 pseudovirus. Two rabbits also neutralized the pseudovirus HIV-MN. Overall, these data describe the first anti-idiotypic vaccine approach performed to generate antibodies to the CD4bs of the HIV-1 gp120. Although future studies will be necessary to improve strength and breadth of the elicited neutralizing response, this proof-of-concept study documents that immunogens designed on the idiotype of broadly neutralizing Abs are feasible and could help in the design of future anti-HIV strategies