Induction of broadly neutralizing antibodies in Germinal Centre simulations.

Vaccines against mutating pathogens such as influenza, HIV, or plasmodium are poorly protective towards new evolving strains. Rare individuals naturally mount broadly neutralizing antibodies covering most strains, but the requirements for their induction are unknown. The antibody response to vaccination has been recapitulated by in silico models that proposed two opposite schemes: A theory of 'frustration' where one epitope at a time leads to optimal antibody breadth through sequential immunizations, that was proven successful for HIV vaccination in primates.

Evaluating the Delivery of Proteins to the Cytosol of Mammalian Cells.

Delivery of proteins to the cytosol of living cells is a promising research tool. Delivery of antibodies in particular bears exciting applications such as in vivo tracking of proteins at endogenous expression levels or interference with cellular processes. In spite of the large number of methods published for protein delivery, successful applications so far are rare.

Antibodies inside of a cell can change its outside: Can intrabodies provide a new therapeutic paradigm?

Challenges posed by complex diseases such as cancer, chronic viral infections, neurodegenerative disorders and many others have forced researchers to think beyond classic small molecule drugs, exploring new therapeutic strategies such as therapy with RNAi, CRISPR/Cas9 or antibody therapies as single or as combination therapies with existing drugs. While classic antibody therapies based on parenteral application can only reach extracellular targets, intracellular application of antibodies could provide specific advantages but is so far little recognized in translational research. Intrabodies allow high specificity and targeting of splice variants or post translational modifications.

Recent Advances with ER Targeted Intrabodies.

ER intrabodies are recombinant antibody fragments produced and retained in the endoplasmatic reticulum (ER) of a cell or an organism with the purpose to induce phenotypes generated by interfering with the intracellular processing or by changing the location of the recognized antigen. The most common application is the generation of functional knockdowns of membrane proteins, which cannot reach their natural location on the cell surface when they are retained in the ER by the intrabody. Phenotypes generated by interfering with the secretion of extracellular or plasma proteins can be analyzed in a similar way.

Specific in vivo knockdown of protein function by intrabodies.

Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e.

The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.

The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER.

Functional knock down of VCAM1 in mice mediated by endoplasmatic reticulum retained intrabodies.

Functional knockdowns mediated by endoplasmatic reticulum-retained antibodies (ER intrabodies) are a promising tool for research because they allow functional interference on the protein level. We demonstrate for the first time that ER intrabodies can induce a knock-down phenotype in mice. Surface VCAM1 was suppressed in bone marrow of heterozygous and homozygous ER intrabody mice (iER-VCAM1 mice).

Delivery of antibodies to the cytosol: debunking the myths.

The use of antibodies to target their antigens in living cells is a powerful analytical tool for cell biology research. Not only can molecules be localized and visualized in living cells, but interference with cellular processes by antibodies may allow functional analysis down to the level of individual post-translational modifications and splice variants, which is not possible with genetic or RNA-based methods. To utilize the vast resource of available antibodies, an efficient system to deliver them into the cytosol from the outside is needed.

Suppression of p75 neurotrophin receptor surface expression with intrabodies influences Bcl-xL mRNA expression and neurite outgrowth in PC12 cells.

Background: Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies.

Targeting antibodies to the cytoplasm.

A growing number of research consortia are now focused on generating antibodies and recombinant antibody fragments that target the human proteome. A particularly valuable application for these binding molecules would be their use inside a living cell, e.g.