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.     

Quantification of the virus-host interaction in human T lymphotropic virus I infection

Background

Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.

Results

In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.

Conclusion

These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.     

Bacterial expression and characterization of nine polypeptides encoded by segments of the envelope gene of human immunodeficiency virus

Nine envelope (Env) polypeptides, encoding different regions of HIV gp120 and gp41 Env proteins, and accounting for approx. 96% of the entire Env precursor glycoprotein complex (gp160) were expressed in Escherichia coli at levels ranging from approx. 2 to 20% of total cellular protein. The recombinant polypeptides were produced either as hybrid products fused to the cII gene fragment of the lambda vector or in an unfused form without interfering cII products. Partially purified protein fractions of each polypeptide were characterized serologically by Western-blot analysis against a panel of well characterized human immunodeficiency virus (HIV)-positive human reference sera. Most of the Env polypeptides were highly immunoreactive with anti-gp120/gp41 antibodies present in the sera of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related diseases, but the patterns of reactivity were different. These results demonstrate that some of the antigenic determinants residing on the viral gp160 complex are retained on the surfaces of the recombinant Env polypeptides, and suggest that these sites are differentially immunogenic. These results are therefore interpreted in the context of an ongoing process towards using bacterially expressed HIV Env polypeptides to help define biological and structural epitopes to aid in the development of more sensitive diagnostic and therapeutic reagents in the fight against AIDS.     

Assessing Low-Intensity Relationships in Complex Networks

Many large network data sets are noisy and contain links representing low-intensity relationships that are difficult to differentiate from random interactions. This is especially relevant for high-throughput data from systems biology, large-scale ecological data, but also for Web 2.0 data on human interactions. In these networks with missing and spurious links, it is possible to refine the data based on the principle of structural similarity, which assesses the shared neighborhood of two nodes. By using similarity measures to globally rank all possible links and choosing the top-ranked pairs, true links can be validated, missing links inferred, and spurious observations removed. While many similarity measures have been proposed to this end, there is no general consensus on which one to use. In this article, we first contribute a set of benchmarks for complex networks from three different settings (e-commerce, systems biology, and social networks) and thus enable a quantitative performance analysis of classic node similarity measures. Based on this, we then propose a new methodology for link assessment called z* that assesses the statistical significance of the number of their common neighbors by comparison with the expected value in a suitably chosen random graph model and which is a consistently top-performing algorithm for all benchmarks. In addition to a global ranking of links, we also use this method to identify the most similar neighbors of each single node in a local ranking, thereby showing the versatility of the method in two distinct scenarios and augmenting its applicability. Finally, we perform an exploratory analysis on an oceanographic plankton data set and find that the distribution of microbes follows similar biogeographic rules as those of macroorganisms, a result that rejects the global dispersal hypothesis for microbes.     

The Semiglades: The Collision of Restoration,Social Values,and the Ecosystem Concept

Defining success targets in restoration and how social values affect them are two commonly discussed issues in restoration today. We believe that how success is commonly defined—with vague terms such as “healthy ecosystem” or cited as a return to a previous, historic state—needs to be reevaluated. With the increasing number of novel ecosystems, there is an increasing conflict between the ecosystem concept, social values, and restoration. This arises from the fact that ecosystems are defined by the values of the scientists describing them, necessarily constraining the ecosystem to a generally static concept. It is not directly the concept, but how it is perceived through our filter of social values that represses the creativity and innovation needed in restoration today. Within restoration, we feel that the ecosystem concept does a disservice by ignoring the increasing number of novel systems, and that hinders real progress in a time when hesitation can be costly. To best illustrate this, we offer the example of restoration of the Florida Everglades and how it has become a novel system in pattern and process. We suggest renaming the Everglades “The Semiglades” in hopes of opening a dialog to expose social/ecosystem biases and include novel landscapes in management and planning.