Expanding the Malaria Antibody Toolkit: Development and Characterisation of RH5, CyRPA, and CSP Recombinant Human Monoclonal Antibodies.

Malaria, an infection caused by apicomplexan parasites of the genus , continues to exact a significant toll on public health with over 200 million cases world-wide, and annual deaths in excess of 600,000. Considerable progress has been made to reduce malaria burden in endemic countries in the last two decades. However, parasite and mosquito resistance to frontline chemotherapies and insecticides, respectively, highlights the continuing need for the development of safe and effective vaccines.

Bovine colostrum-derived antibodies against SARS-CoV-2 show great potential to serve as prophylactic agents.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to impose a serious burden on health systems globally. Despite worldwide vaccination, social distancing and wearing masks, the spread of the virus is ongoing. One of the mechanisms by which neutralizing antibodies (NAbs) block virus entry into cells encompasses interaction inhibition between the cell surface receptor angiotensin-converting enzyme 2 (ACE2) and the spike (S) protein of SARS-CoV-2.

Polymer-grafted chromatography media for the purification of enveloped virus-like particles, exemplified with HIV-1 gag VLP.

Polymer-grafted chromatography media, especially ion exchangers, are high performance materials for protein purification. However, due to the pore size limitation, conventional chromatography beads are usually not considered for the downstream processing of large biomolecules such as virus-like particles (VLPs). Contrariwise, since the outer surface of the chromatography beads provides satisfactory binding capacity for VLPs and impurities of smaller size can bind inside of the beads, conventional porous beads should be considered for VLP capture and purification.

Separation of virus-like particles and extracellular vesicles by flow-through and heparin affinity chromatography.

Separation of enveloped virus-like particles from other extracellular vesicles is a challenging separation problem due to the similarity of these bionanoparticles. Without simple and scalable methods for purification and analytics, it is difficult to gain deeper insight into their biological function. A two-step chromatographic purification method was developed.

Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysis.

The rapid quantification of enveloped virus-like particles (VLPs) requires orthogonal methods to obtain reliable results. Three methods-nanoparticle tracking analysis (NTA), size-exclusion HPLC (SE-HPLC) with UV detection, and detection with multi-angle light scattering (MALS)-for quantification of enveloped VLPs have been compared, and the lower and upper limits of detection and quantification have been evaluated. NTA directly counts the enveloped VLPs, and a particle number is obtained with a lower limit of detection (LLOD) of 1.

Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith.

Purification of HIV-1 gag virus-like particles and separation of other extracellular particles.

Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes.

Phenylboronic acid as a multi-modal ligand for the capture of monoclonal antibodies: development and optimization of a washing step.

In this work, phenylboronic acid (PBA) was thoroughly investigated as a synthetic ligand for the purification of an immunoglobulin G (IgG) from a clarified cell supernatant from Chinese Hamster Ovary (CHO) cell cultures. In particular, the study was focused on the development of a washing step and in the optimization of the elution step using a serum containing supernatant. From the different conditions tested, best recoveries - 99% - and purifications - protein purity of 81% and a purification factor of 16 out of a maximum of 20 - were achieved using 100mM d-sorbitol in 10mM Tris-HCl as washing buffer and 0.

New generation of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids.

Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160nm. Such nanoparticles have a negative surface charge (-11 to -18mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing.

Homologous recombination is facilitated in starving populations of Pseudomonas putida by phenol stress and affected by chromosomal location of the recombination target.

Homologous recombination (HR) has a major impact in bacterial evolution. Most of the knowledge about the mechanisms and control of HR in bacteria has been obtained in fast growing bacteria. However, in their natural environment bacteria frequently meet adverse conditions which restrict the growth of cells.

Molecular characterization of Rif(r) mutations in Pseudomonas aeruginosa and Pseudomonas putida.

The rpoB gene encoding for beta subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rif(r)) phenotype of bacteria. Here we have characterized rpoB/Rif(r) system in Pseudomonas aeruginosa and Pseudomonas putida as a test system for studying mutational processes. We found that in addition to the appearance of large colonies which were clearly visible on Rif selective plates already after 24h of plating, small colonies grew up on these plates for 48 h.

Elevated mutation frequency in surviving populations of carbon-starved rpoS-deficient Pseudomonas putida is caused by reduced expression of superoxide dismutase and catalase.

RpoS is a bacterial sigma factor of RNA polymerase which is involved in the expression of a large number of genes to facilitate survival under starvation conditions and other stresses. The results of our study demonstrate that the frequency of emergence of base substitution mutants is significantly increased in long-term-starved populations of rpoS-deficient Pseudomonas putida cells. The increasing effect of the lack of RpoS on the mutation frequency became apparent in both a plasmid-based test system measuring Phe(+) reversion and a chromosomal rpoB system detecting rifampin-resistant mutants.

Dual role of NER in mutagenesis in Pseudomonas putida.

Nucleotide excision repair (NER) is one of the most important repair systems which counteracts different forms of DNA damage either induced by various chemicals or irradiation. At the same time, less is known about the functions of NER in repair of DNA that is not exposed to exogenous DNA-damaging agents. We have investigated the role of NER in mutagenesis in Pseudomonas putida.

Oxidative DNA damage defense systems in avoidance of stationary-phase mutagenesis in Pseudomonas putida.

Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNA-protecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida.

Study of factors which negatively affect expression of the phenol degradation operon pheBA in Pseudomonas putida.

Transcription of the plasmid-borne phenol catabolic operon pheBA in Pseudomonas putida is activated by the LysR-family regulator CatR in the presence of the effector molecule cis,cis-muconate (CCM), which is an intermediate of the phenol degradation pathway. In addition to the positive control of the operon, several factors negatively affect transcription initiation from the pheBA promoter. First, the activation of the pheBA operon depends on the extracellular concentration of phenol.

Study of involvement of ImuB and DnaE2 in stationary-phase mutagenesis in Pseudomonas putida.

Several bacterial species carry in their genomes a so-called "mutagenesis" gene cluster encoding ImuB which is similar to Y-family DNA polymerases, and DnaE2 related to the catalytic subunit DnaE of Pol III. Y-family DNA polymerases are known to be involved in stationary-phase mutagenesis and DnaE2 homologues characterized so far have expressed a mutator phenotype. In this study, we raised a question about the involvement of ImuB and DnaE2 in stationary-phase mutagenesis.

Involvement of DNA mismatch repair in stationary-phase mutagenesis during prolonged starvation of Pseudomonas putida.

One of the popular ideas is that decline in methyl-directed mismatch repair (MMR) in carbon-starved bacteria might facilitate occurrence of stationary-phase mutations. We compared the frequency of accumulation of stationary-phase mutations in carbon-starved Pseudomonas putida wild-type and MMR-defective strains and found that knockout of MMR system increased significantly emergence of base substitutions in starving P. putida.

A DNA polymerase V homologue encoded by TOL plasmid pWW0 confers evolutionary fitness on Pseudomonas putida under conditions of environmental stress.

Plasmids in conjunction with other mobile elements such as transposons are major players in the genetic adaptation of bacteria in response to changes in environment. Here we show that a large catabolic TOL plasmid, pWW0, from Pseudomonas putida carries genes (rulAB genes) encoding an error-prone DNA polymerase Pol V homologue which increase the survival of bacteria under conditions of accumulation of DNA damage. A study of population dynamics in stationary phase revealed that the presence of pWW0-derived rulAB genes in the bacterial genome allows the expression of a strong growth advantage in stationary phase (GASP) phenotype of P.

Involvement of error-prone DNA polymerase IV in stationary-phase mutagenesis in Pseudomonas putida.

In this work we studied involvement of DNA polymerase IV (Pol IV) (encoded by the dinB gene) in stationary-phase mutagenesis in Pseudomonas putida. For this purpose we constructed a novel set of assay systems that allowed detection of different types of mutations (e.g.

Different spectra of stationary-phase mutations in early-arising versus late-arising mutants of Pseudomonas putida: involvement of the DNA repair enzyme MutY and the stationary-phase sigma factor RpoS.

Stationary-phase mutations occur in populations of stressed, nongrowing, and slowly growing cells and allow mutant bacteria to overcome growth barriers. Mutational processes in starving cells are different from those occurring in growing bacteria. Here, we present evidence that changes in mutational processes also take place during starvation of bacteria.

Growth medium composition-determined regulatory mechanisms are superimposed on CatR-mediated transcription from the pheBA and catBCA promoters in Pseudomonas putida.

Expression of the phenol degradation pathway in Pseudomonas putida strain PaW85 requires coordinated transcription of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and phenol monooxygenase, respectively, and the chromosomally encoded catechol degradation catBCA operon. Transcriptional activation from the pheBA and catBCA promoters is regulated by CatR and the catechol degradation pathway intermediate cis,cis-muconate. Here it is shown that physiological control mechanisms are superimposed on this regulatory system.

Critical nucleotides in the interaction of CatR with the pheBA promoter: conservation of the CatR-mediated regulation mechanisms between the pheBA and catBCA operons.

The promoter of the plasmid-borne pheBA genes encoding enzymes for phenol degradation resembles the catBCA promoter and is activated by CatR, the regulator of the chromosomally encoded catechol-degradative catBCA genes in Pseudomonas putida. In this study, site-directed mutagenesis of the pheBA promoter region was performed. The interrupted inverted repeat sequence of the CatR recognition binding site (RBS) of the pheBA promoter is highly homologous to that of the catBCA promoter.