Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

The antibody mining toolbox: An open source tool for the rapid analysis of antibody repertoires

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.     

The effect of the presence of a forage legume on nitrogen and carbon levels in soils under Brachiaria pastures in the Atlantic forest region of the South of Bahia,Brazil

The impact of forest clearance, and its replacement by Brachiaria pastures, on soil carbon reserves has been studied at many sites in the Brazilian Amazonia, but to date there appear to be no reports of similar studies undertaken in the Atlantic forest region of Brazil. In this study performed in the extreme south of Bahia, the changes in C and N content of the soil were evaluated from the time of establishment of grass-only B. humidicola and mixed B. humidicola/Desmodium ovalifolium pastures through 9 years of grazing in comparison with the C and N contents of the adjacent secondary forest. The decline in the content of soil C derived from the forest (C3) vegetation and the accumulation of that derived from the Brachiaria (C4) were followed by determining the ¹³C natural abundance of the soil organic matter (SOM). The pastures were established in 1987, 10 years after deforestation, and it was estimated that until 1994 there was a loss in forest-derived C in the top 30 cm of soil of approximately 20% (9.1 Mg C ha^–1). After the establishment of the pastures, C derived from Brachiaria accumulated steadily such that at the final sampling (1997) it was estimated 13.9 Mg ha^–1 was derived from this source under the grass-only pasture (0–30 cm). Samples taken from all pastures and the forest in 1997 to a depth of 100 cm showed that below 40 cm depth there was no significant contribution of the Brachiaria-derived C and that total C reserves under the grass/legume and the grass-only pastures were slightly higher than under the forest (not significant at P=0.05). The more detailed sampling under the pastures showed that to a depth of 30 cm there was significantly (P<0.05) more C under the mixed pasture than the grass-only pasture. It was estimated that from the time of establishment the apparent rate of C accumulation (0–100 cm depth) under the grass/legume pastures (1.17 Mg ha^–1 yr^–1) was almost double that under the grass-only pastures (0.66 Mg ha^–1 yr^–1). The data indicated that newly incorporated SOM derived from the Brachiaria had a considerably higher C:N ratio than that present under the forest.     

Management swing potential for bioenergy crops

Bioenergy crops are often classified (and subsequently regulated) according to species that have been evaluated as environmentally beneficial or detrimental, but in practice, management decisions rather than species per se can determine the overall environmental impact of a bioenergy production system. Here, we review the greenhouse gas balance and ‘management swing potential’ of seven different bioenergy cropping systems in temperate and tropical regions. Prior land use, harvesting techniques, harvest timing, and fertilization are among the key management considerations that can swing the greenhouse gas balance of bioenergy from positive to negative or the reverse. Although the management swing potential is substantial for many cropping systems, there are some species (e.g., soybean) that have such low bioenergy yield potentials that the environmental impact is unlikely to be reversed by management. High‐yielding bioenergy crops (e.g., corn, sugarcane, Miscanthus, and fast‐growing tree species), however, can be managed for environmental benefits or losses, suggesting that the bioenergy sector would be better informed by incorporating management‐based evaluations into classifications of bioenergy feedstocks.     

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression,but non-specifically enhanced on induction of Tax expression

Background

Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4⁺ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.

Results

Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ_26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.

Conclusions

HTLV-1 gene expression in primary CD4⁺ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.

     

Estimation of biological nitrogen fixation associated withBrachiaria andPaspalum grasses using15N labelled organic matter and fertilizer

Summary Six pasture grasses,Paspalum notatum cv batatais,P. notatum cv pensacola,Brachiaria radicans, B. ruziziensis, B. decumbens andB. humidicola, were grown in concrete cylinders (60 cm diameter) in the field for 31 months. The soil was amended with either a single addition of¹⁵N labelled organic matter or frequent small (2 kg N. ha^–1) additions of¹⁵N enriched (NH₄)₂SO₄. In the labelled fertilizer treatment soil analysis revealed that there was a very drastic change in¹⁵N enrichment in plant-available nitrogen (NO ₃ ^– +NH ₄ ⁺ ) with depth. The different grass cultivars recovered different quantities of applied labelled N, and evidence was obtained to suggest that the roots exploited the soil to different depths thus obtaining different¹⁵N enrichments in soil derived N. This invalidated the application of the isotope dilution technique to estimate the contribution of nitrogen fixation to the grass cultivars in this treatment. In the labelled organic matter treatment the¹⁵N label in the plant-available N declined at a decreasing rate during the experiment until in the last 12 months the decrease was only from 0.274 to 0.222 atom % excess. There was little change in¹⁵N enrichment of available N with depth, hence it was concluded that although the grasses recovered different quantities of labelled N, they all obtained virtually the same¹⁵N enrichment in soil derived N. Data from the final harvests of this treatment indicated thatB. humidicola andB. decumbens obtained 30 and 40% respectively of their nitrogen from N₂ fixation amounting to an input of 30 and 45 kg N.ha^–1 year^–1 respectively.     

Differences in contribution of biological nitrogen fixation to yield performance of common bean cultivars as assessed by the 15N natural abundance technique

Plant and Soil - Identification of variability in biological N2 fixation (BNF) contribution among common bean (Phaseolus vulgaris L.) cultivars under field conditions requires a reliable...