Premature babies also have protective anti-viral antibodies

Even premature babies carry anti-viral antibodies transferred from the mother, researchers at Karolinska Institutet in Sweden report in a paper on maternal antibodies in newborns, published in the journal Nature Medicine. The results should change our approach to infection sensitivity in newborns, they say.

Antibodies are transferred from the mother’s blood to the fetus that give the newborn passive defence against infection. Since most of this process takes place during the third trimester of the pregnancy, doctors have regarded very premature babies as being unprotected by such maternal antibodies.

However, now that the total repertoire of maternal anti-viral antibodies has been analyzed in neonates by researchers at Karolinska Institutet and Karolinska University Hospital, another picture is emerging.

“We saw that babies born as early as in week 24 also have maternal antibodies, which surprised us,” says corresponding author Dr Petter Brodin, physician and researcher at the Science for Life Laboratory (SciLifeLab) and the Department of Women’s and Children’s Health, Karolinska Institutet.

The study comprised 78 mother-child pairs. 32 of the babies were very premature (born before week 30) and 46 were full-term. The analysis show that the repertoire of maternal antibodies was the same in both groups.

“I hope that this makes us question some preconceived ideas about the neonate immune system and infection sensitivity so that we can take even better care of newborns,” says Dr Brodin. “Premature babies can be especially sensitive to infection, but that is not because they lack maternal antibodies. We should concentrate more on other possible causes, maybe like their having underdeveloped lung function or weaker skin barriers.”

The study was conducted using a newly developed method for simultaneously analysing the presence of antibodies against all the viruses that can infect humans (with the exception of the Zika virus, which was identified later). The method is developed by US researchers and is based on a so-called bacteriophage display, a technique awarded with the 2018 Nobel Prize in Chemistry.

Briefly, it is based on the ability to make viral particles called bacteriophages display a specific surface protein. In this case, all in all the bacteriophage library displayed over 93,000 different peptides, short-chain proteins, from over 206 species of virus and over 1 000 different strains. The library is mixed with the blood plasma to be tested. Any antibodies in the plasma sample bind with the bacteriophages and can then be detected by the researchers.

The analysis was conducted on samples taken at birth and during the newborns’ first, fourth and twelfth week. The researchers found that the protection offered by the antibodies lasted different durations depending on the virus. This can suggest that their transfer during the fetal stage is regulated rather than random, a possibility the group is now examining further.

The study also shows which parts of the virus proteins that antibodies target, information that is important in the development of vaccines, notes Dr Brodin.

“If all maternal antibodies target a specific part of a virus protein, that is important to know because then it is that part a vaccine should be based on,” he says. “I hope that our results can be used by others to develop better vaccines, such as against the RS virus that causes so much distress for babies every winter.”

Source:

https://ki.se/en/research/protective-antibodies-also-found-in-premature-babies




Researchers define pathway that helps us make antibodies

Our bodies are continuously concocting specific antibodies to thwart invaders like a virus or even pollen, and scientists have new information about how the essential production gets fired up and keeps up.

It’s a key protective mechanism that the scientists want to better understand with the long-term goal of manipulating it to help keep us well, says Dr. Nagendra Singh, immunologist in the Department of Biochemistry and Molecular Biology at the Medical College of Georgia at Augusta University.

“We are trying to design small molecules that can either block or activate this pathway,” says Singh, corresponding author of the study in the journal Nature Communications.

The pathway is called ufmylation, and in this pathway, a polypeptide call Ufm1 is known to target other proteins, connect with them, and change their function. One of those proteins is Ufbp1, and investigators have learned that the Ufbp1 that emerges is key to both immune cells called naïve B cells becoming antibody-producing plasma cells and to plasma cells stepping up production of protective antibodies.

Better understanding how this natural protective mechanism works could ultimately help design better vaccines, the investigators write. In fact, current vaccines help prime B cells to have memories of certain invaders so they can more quickly respond, Singh says.

Selective increases in this Ufbp1 or ufmylation pathway, for example, might one day yield an even more targeted attack on the flu virus.

Conversely, for allergy sufferers, selective intervention might stop the production of antibodies against tree and weed pollens that are already producing itchy, watery eyes and noses this year.

It might enable as well a reduction in antibodies the body inexplicably makes against itself in autoimmune diseases like lupus and arthritis. In fact, the scientists already are looking at making the adjustment in a mouse model of lupus.

They found that Ufbp1 suppresses the enzyme PERK to help B cells differentiate. Proteins have to be properly folded for any cell or body function to happen and PERK is part of the body’s natural “unfolded protein response,” to try to correct problems with improperly folded ones that don’t function correctly and may instead become toxic to cells.

When newly made proteins fold improperly, PERK gets activated which stops new protein production and decreases the misfolded protein pileup. But at this juncture, scientists have learned that Ufbp1 suppresses PERK to ensure ample production of plasma cells. So when there is a deficiency of Ufbp1 in B cells, they found that while B cells survived, the development of plasma cells was impaired.

Inside plasma cells they found that Ufbp1 gets upregulated so the endoplasmic reticulum, basically the manufacturing plant for a cell, can expand and protein folding capacity can expand with it. Conversely, they showed Ufbp1 deficiency in the plasma cells impairs expansion of the endoplasmic reticulum and antibody production.

“We knew that proteins were folded in the endoplasmic reticulum and that an expanded endoplasmic reticulum is the hallmark of secretory cells like plasma cells being made,” Singh says, “but exactly what components were involved, we did not know.”

“What we have found is that the ufmylation pathway is very important in cells that secrete a lot of proteins, like plasma cells,” he says.

“Antibodies work like long-range missiles,” Singh notes, and plasma cells usually shoot them from the bone marrow. B cells also are made in and by the bone marrow, but circulate as well looking for invaders. When they spot one, they must go to the spleen or to a nearby lymph node to become a plasma cell. Plasma cells then move back to the bone marrow and typically take aim from there.

The survival and maintenance of plasma cells is a continuous and delicate balance, which can turn deadly when it goes wrong. Without this balance, plasma cells can grow out of control, become cancerous instead of protective and result in multiple myeloma. One of many next steps for the scientists include looking at whether targeting Ufbp1 holds the key to designing the next generation of multiple myeloma treatments.

There also are individuals born with some key ufmylation pathway components missing and, while not a lot is known about the impact, it can result in a disease that damages your brain called encephalopathy as well as blood disorders, Singh says. Protein misfolding is also a known factor in conditions like Parkinson’s and Alzheimer’s.

Singh notes that misfolding likely happens in all of us, but at low, harmless levels.

Source:

http://www.augusta.edu/mcg/




EAF4 module determines physicochemical, biophysical attributes of monoclonal antibodies

Postnova Analytics Inc. reports how its Electrical Asymmetrical Flow Field Flow Fractionation (EAF4) and Size Exclusion Chromatography (SEC) modules have been used to determine the physicochemical and biophysical attributes of monoclonal antibodies.

EAF4 technology uniquely combines the principle of Electrical and Asymmetrical Field Flow Fractionation in just one system. In a Postnova Analytics EAF2000 system – Electrical and Cross Flow Fields are applied simultaneously across the FFF channel enabling separation by size and simultaneous measurement of particle charge based on electrophoretic mobility. Combining these two powerful separation techniques in a single platform opens the door to characterizing complex proteins, antibodies and viruses as well as charged nanoparticles, colloids, or polymers that have proven intractable to other techniques.

A new poster describes how the EAF4 module enabled simultaneous measurement of size and surface charge characteristics (electrophoretic mobility) of antibodies and proteins. While no aggregates were detected by SEC, the EAF4 system showed antibody aggregates represented 10% of the total mass injected. The researchers concluded that the FFF open channel design may allow for better recovery of injected mass than SEC, which is particularly important when seeking to quantify aggregates present in small amounts.

In the described work, a reference monoclonal antibody (RM 8671 mAb), from the U.S. National Institute of Standards and Technology (NIST), was used to compare separation, aggregation quantification, and recovery parameters for an EAF4-UV-MALS versus SEC-UV-MALS techniques. The NIST mAb provides a representative test molecule for development of novel technology for therapeutic protein characterization.




Research offers new hope for seasonal allergy sufferers

One in three people is plagued by an allergy, triggered by foodstuffs, fungi, house dust mites or on a seasonal basis due to pollen. The latter group is the largest: around 800 million people worldwide suffer from some form of allergy to pollen, with the usual symptoms such as a runny nose, cough and severe breathing problems. One in five Austrians is allergic to pollen. MedUni Vienna researchers have now set themselves the task of immunizing camels with pollen allergens to obtain heavy single-chain antibodies for the passive treatment of pollen allergies.

Why camels? “Camels have a rare capacity for producing antibodies consisting of only one chain,” explains Sabine Flicker, head of the Antibody Working Group at MedUni Vienna’s Institute of Pathophysiologyand Allergy Research: “The isolated single-chain antibodies are tested for their efficacy in preventing specific immunoglobulin E antibodies (IgE) from binding to allergens, thereby suppressing the triggering of a pollen allergy.”

An allergic reaction normally involves allergens coming into contact with the IgE antibodies produced on sensitization. These “arm” specific cells, primarily the mast cells. When the allergens are incorporated a second time, they bind to cell-bound IgE antibodies, thus activating the mast cells. These then release messenger substances that are responsible for allergic inflammation and other symptoms – the allergy is “born”.

Stop sign for the allergy

In a new joint project approved by the FWF (Austrian Science Fund) and RFBR (Russian Foundation for Basic Research), MedUni Vienna researchers led by Sabine Flicker will be working with Sergei Tillib from the Russian Academy of Sciences. The project will involve injecting allergens into camels to immunize them. A high-performance technology, nanobody technology, is used to obtain allergen-specific single-chain antibodies from the blood of the immunized camels. This is the first time that this advanced technique is being used to produce allergen-specific antibodies. It is hoped that the process can be used to generate a large number of specific single-chain antibodies. Says Flicker: “Nanobody technology therefore represents a significant improvement over the methods previously used for obtaining recombinant monoclonal antibodies.”

“We are able to manufacture the single-chain antibodies as recombinant proteins in the laboratory and test them for their protective potential. Those single-chain antibodies that prevent IgE from binding to allergens act as a stop sign to the allergy, as it were,” explains the MedUni Vienna researcher.

According to the scientists, these new findings could lead to a local treatment (nasal spray, eyedrops) to combat seasonal pollen allergies in eight to ten years time.

Source:

https://www.meduniwien.ac.at/web/en/about-us/news/detailsite/2019/news-im-maerz-2019/new-hope-for-seasonal-allergy-sufferers/

Posted in: Medical Research News | Medical Condition News

Tags: Allergen, Allergy, Antibodies, Antibody, Blood, Cell, Cough, Immunoglobulin, Inflammation, Laboratory, Nasal Spray, Research




Reduced antibody adaptability may make the elderly more vulnerable to influenza

The influenza vaccine may be less effective in the elderly because their B cells are less capable of producing antibodies that can adapt to protect against new viral strains, researchers report February 19 in the journal Cell Host & Microbe. With age, B cells and the antibodies they secrete acquire fewer mutations that would provide flexible protection against the ever-changing flu virus.

“The major implication is that when a newly circulating influenza virus infects elderly individuals, they don’t have quite the right tool to fight it because their antibodies are not as protective,” says senior study author Patrick Wilson of the University of Chicago. “Our findings could be used by the vaccine community to make better vaccines and improve protection of the elderly population.”

The detrimental effect of aging on the immune system is thought to be a major cause of illness and death in elderly adults by increasing susceptibility to bacterial, fungal, and viral infections. The great majority of influenza deaths occur within populations older than 65 years, and aged individuals have a significantly reduced antibody response to influenza vaccination. As a result, influenza is a leading cause of death in the elderly, and the vaccine protects only a fraction of this population.

To understand the underlying mechanisms, Wilson and his team compared how B cells and antibodies from elderly and younger adults respond to vaccination with different flu strains. While B cells from younger subjects showed a continued recent accumulation of mutations, the elderly appeared to have an essentially fixed B-cell repertoire, lacking recent adaptations that would allow the evolution of B cells to divergent influenza virus strains.

Moreover, antibodies from the elderly are less potent and less capable of protecting against the flu virus. The antibodies of elderly subjects target only conserved proteins and structures of the influenza virus, with fewer mutations that would enable effective responses against evolving viral strains. By contrast, antibodies from younger individuals are better able to recognize recently mutated molecules on the flu virus.

The findings suggest that antibodies from aged individuals arise from cross-reactive memory B cells generated early in life, with reduced adaptation to recent influenza virus strains. For example, 47% of antibodies generated from the elderly individuals bound to six or more strains of the flu virus compared to only 12% for the young adults. In addition, antibodies from the elderly individuals had higher affinity to historical strains that were circulating during their childhood and lower affinity to more contemporary strains.

Despite these observations, vaccination remains the best way to protect elderly individuals from influenza virus infection. “We are not saying that people shouldn’t be vaccinated or that the current vaccines are useless for elderly individuals,” says first author Carole Henry of the University of Chicago.

Currently, the researchers are working to understand the underlying biological basis for their observations. From a clinical standpoint, the findings suggest that vaccines driving protective mutations in B cells should be a priority to improve influenza immunity in the elderly. “More recent vaccines developed especially for the elderly population are now on the market and could help induce more protective antibodies,” Wilson says. “The next step will be to evaluate antibody adaptability in elderly individuals immunized with these vaccines.”

Source:

http://www.cellpress.com/




Influenza vaccine may be less effective in elderly patients, finds study

The influenza vaccine may be less effective in the elderly because their B cells are less capable of producing antibodies that can adapt to protect against new viral strains, researchers report February 19 in the journal Cell Host & Microbe. With age, B cells and the antibodies they secrete acquire fewer mutations that would provide flexible protection against the ever-changing flu virus.

“The major implication is that when a newly circulating influenza virus infects elderly individuals, they don’t have quite the right tool to fight it because their antibodies are not as protective,” says senior study author Patrick Wilson of the University of Chicago. “Our findings could be used by the vaccine community to make better vaccines and improve protection of the elderly population.”

The detrimental effect of aging on the immune system is thought to be a major cause of illness and death in elderly adults by increasing susceptibility to bacterial, fungal, and viral infections. The great majority of influenza deaths occur within populations older than 65 years, and aged individuals have a significantly reduced antibody response to influenza vaccination. As a result, influenza is a leading cause of death in the elderly, and the vaccine protects only a fraction of this population.

To understand the underlying mechanisms, Wilson and his team compared how B cells and antibodies from elderly and younger adults respond to vaccination with different flu strains. While B cells from younger subjects showed a continued recent accumulation of mutations, the elderly appeared to have an essentially fixed B-cell repertoire, lacking recent adaptations that would allow the evolution of B cells to divergent influenza virus strains.

Moreover, antibodies from the elderly are less potent and less capable of protecting against the flu virus. The antibodies of elderly subjects target only conserved proteins and structures of the influenza virus, with fewer mutations that would enable effective responses against evolving viral strains. By contrast, antibodies from younger individuals are better able to recognize recently mutated molecules on the flu virus.

The findings suggest that antibodies from aged individuals arise from cross-reactive memory B cells generated early in life, with reduced adaptation to recent influenza virus strains. For example, 47% of antibodies generated from the elderly individuals bound to six or more strains of the flu virus compared to only 12% for the young adults. In addition, antibodies from the elderly individuals had higher affinity to historical strains that were circulating during their childhood and lower affinity to more contemporary strains.

Despite these observations, vaccination remains the best way to protect elderly individuals from influenza virus infection. “We are not saying that people shouldn’t be vaccinated or that the current vaccines are useless for elderly individuals,” says first author Carole Henry of the University of Chicago.

Currently, the researchers are working to understand the underlying biological basis for their observations. From a clinical standpoint, the findings suggest that vaccines driving protective mutations in B cells should be a priority to improve influenza immunity in the elderly. “More recent vaccines developed especially for the elderly population are now on the market and could help induce more protective antibodies,” Wilson says. “The next step will be to evaluate antibody adaptability in elderly individuals immunized with these vaccines.”




Type 1 diabetes: Developing an early warning system

Type 1 diabetes starts out as a sneak attack. For unknown reasons, the immune system makes antibodies against insulin and other proteins in the insulin-producing islet cells in the pancreas, targeting these healthy cells for destruction. Clinical trials have suggested that, if the attack is stopped early with immune-modulating therapies, full-blown type 1 diabetes might be prevented.

But there’s been a big hurdle in getting this early-intervention strategy to work: You need to figure out who has the bad-actor antibodies before too much damage occurs.

“Identifying these patients as early as possible is a really high priority for the field,” said Brian Feldman, MD, PhD, an endocrinologist who has been studying the problem. Existing tests are expensive, require a lot of technical expertise to run, and don’t do a good job of finding the earliest antibodies the immune system produces.

Now, Feldman’s team at Stanford and the University of California-San Francisco has published a proof-of-concept paper in PLOS ONE showing that they have a new method to find the bad-actor antibodies much earlier than traditional testing. (Feldman was at Stanford until recently, when he moved to UCSF to accept an endowed professorship position.)

The work expands on the team’s prior success at building an inexpensive nanotech-based microchip for diagnosing type 1 diabetes. In the new study, Feldman and his colleagues adjusted the microchip to detect immunoglobulin M (IgM) antibodies against insulin. IgM antibodies are the first to appear in the autoimmune response that precedes full-blown type 1 diabetes. The body later switches to making a different class of antibodies, IgG, which are detected by traditional tests.

The researchers used the microchip on blood samples collected as part of TrialNet, a large study following people who have close relatives with type 1 diabetes. They tested blood samples collected every six months for several years from six individuals who started out without type 1 diabetes, but went on to develop the disease. As a control, the researchers also used the microchip to test blood samples from eight healthy volunteers.

In three of the six people who developed type 1 diabetes, the new test found anti-insulin antibodies much earlier than traditional tests: For two subjects, the new method found the bad antibodies a year earlier, and in one person, the bad antibodies were detected four years earlier than via the traditional test. The microchip did not register any false positives on blood from healthy volunteers.

“The biggest surprise was how far ahead we could detect these antibodies,” Feldman said. Before the study, the team had hoped that, at best, they might find bad-actor antibodies a few months in advance.

If the result holds up in larger trials, the extra warning time could make a big difference for the success of immune-modulating preventive strategies for diabetes. “The earlier you apply these next-generation therapies, the better they work,” Feldman said.

The researchers have lots of ideas for what to do next. Although the current study only looked for antibodies against insulin, three other types of antibodies also presage type 1 diabetes; the microchip technology could easily be adapted to look for all four at once. The researchers would also like to greatly expand the number of people studied, and try the microchip method for people who don’t have relatives with type 1 diabetes, since most individuals who develop the disease don’t have an affected family member.

A big advantage of the microchip is its low-cost, easy-to-use design, Feldman said. “We’re excited about that because we have an interest in making state-of-the-art diagnostics much more available, both in our own country and globally,” he said.

Photo by Norbert von der Groeben




NIH researchers discover that anti-flu antibodies can inhibit two different viral proteins

Researchers from the National Institutes of Health have discovered that antibodies that may form the basis of a universal flu vaccine inhibit a second viral protein in addition to the one that they bind. The study, to be published January 25 in the Journal of Experimental Medicine, reveals that antibodies that recognize the viral surface protein hemagglutinin can also inhibit the viral neuraminidase, and that this enhances antibody neutralization of the virus and the activation of innate immune cells with anti-viral activity.

Hemagglutinin and neuraminidase are yin-yang proteins present on the surface of the influenza virus. The former mediates virion attachment and fusion with host cell membranes, while the latter is an enzyme that releases budding progeny virions from the cell surface that remain attached via the hemagglutinin binding.

Hemagglutinin consists of a head domain that contains the receptor binding site that attaches to host cell membranes and a stem domain that connects the head to the virion membrane. Current flu vaccines induce antibodies that recognize the hemagglutinin head and inhibit its ability to mediate viral entry. But the hemagglutinin head undergoes rapid mutation to escape existing antibodies. This generates vaccine-resistant strains of the influenza virus each year, necessitating the yearly mad dash to create a matched vaccine.

The hemagglutinin stem domain, in contrast, is far more resistant to mutations, providing a target for universal flu vaccines, as has been shown by dozens of studies in animal models.

“Hemagglutinin stem-specific antibodies are perhaps the most promising approach for improving the duration and effectiveness of influenza vaccination,” write the authors of the study, which was led by Jonathan W. Yewdell, a senior investigator at the National Institute of Allergy and Infectious Diseases, National Institutes of Health. “It is therefore critical to better understand how anti-stem antibodies provide protection from the virus.”

Stem-binding antibodies can block viral entry into host cells by inhibiting hemagglutinin cell fusion activity, but as Yewdell’s lab reports, they also inhibit the release of newly replicated virions by blocking neuraminidase molecules in close proximity to hemagglutinin on the virion.

Experiments in mice confirmed that the ability of anti-stem antibodies to inhibit neuraminidase enabled animals to better survive a severe influenza infection. Yewdell and colleagues think that this effect may be largely due to the role that neuraminidase normally plays in preventing the activation of innate immune cells with anti-viral activity. In support of this idea, the researchers found that the FDA-approved neuraminidase inhibitor oseltamivir (Tamiflu) further boosted the ability of anti-stem antibodies to activate immune cells exposed to influenza virus.

“The ability of neuraminidase inhibitors to enhance… immune cell activation [by anti-stem antibodies] bound to viruses or infected cells suggests the possible clinical synergy between neuraminidase inhibitors and [anti-stem antibodies] in humans,” the authors write. In addition, this new understanding of how anti-stem antibodies exert their protective effects should aid the design of universal flu vaccines targeting the hemagglutinin stem domain.




Cell Culture Clarification Expressing Recombinant Antibodies

An interview with Tina Stoschek and Marcus Gerlach, discussing maximizing monoclonal antibody generation using lab-scale clarification of mammalian cell cultures expressing recombinant antibodies.

Monoclonal antibodies are increasingly important as targeted therapeutics. Please give an overview of this industry and the challenges faced when producing these antibodies at scale.

The production of monoclonal antibodies in varying scale, from small batches in academia and industry up to bulk manufacturing in biopharmaceutical industry is an elaborate process. The recombinant antibodies are expressed in mammalian suspension cells and the cultivation process is more complex compared to bacterial expression systems (e.g. lower cell growth, sensitive cell physiology, complex nutrition).

3d rendered medically accurate illustration of antibodies

Shutterstock | Sebastian Kaulitzki

This complexity leads to increased time requirements and higher cost of goods. Process design to a fast, cost-saving and stable biomanufacturing process without loss of product quality and product titer is one of the major challenges.

What work are you doing at Tubulis to improve antibody research and maximize monoclonal antibody generation?

Tubulis is a spin-off project located at the LMU Munich, focusing on the development of next-generation Antibody Drug Conjugates (ADCs). In our research facilities, each individual process step is seen as an elementary factor and is continuously optimized to achieve the best product quality and quantity.

In order to fulfil this requirement, the use of state-of-the-art technologies is essential. Our department is equipped with the infrastructure for efficient upstream process development (USP), downstream process development (DSP) and we established bioanalytic methods for product characterization.

We can boost the manufacturing process at any time by means of scale-up and high-throughput approaches. In addition, we can produce high titers in a short period of time. Despite highly standardized workflows, the on-going optimization of the process is always present – like the implementation of the straight forward cell culture harvest using Sartoclear Dynamics Lab V Kits.

What is Tub-tag® conjugation? Why is it important to be able to conjugate a defined drug-to-antibody ratio?

The Tub-tag® technology enables the development of highly stable and homogeneous ADCs. It is a recombinant, chemoenzymatic conjugation technology enabling site-specific antibody functionalization. In general, the conjugation of highly-potent cytotoxic molecules to a target-specific antibody allows tumor-targeted drug delivery in cancer treatment.

Antibodies binding to cancer cell surface receptors

Shutterstock | Alpha Tauri 3D Graphics

The antibody-mediated drug delivery approach decreases the minimum effective dose and potentially reduces the side effects in comparison to conventional chemotherapy. During the last years it has been recognized that site-specific and homogeneous conjugation to antibody-products has the potential to improve the therapeutic index of the resulting antibody-drug-conjugate (ADC).

The drug-to-antibody ratio (DAR) of an ADC can impact the therapeutic efficacy, as unconjugated antibodies reduce potency, while a high DAR can affect pharmacokinetics and cause toxicity. This creates the requirement to control and determine a defined payload distribution of ADCs. With Tub-tag®, Tubulis has developed a next-generation ADC platform technology that addresses current and pressing challenges in ADC conjugation.

Please give an overview of the new lab-scale method for fast filtration from Sartorius. How has this affected your work?

Sartoclear Dynamics Lab V Kits enables a one-step process for cell culture harvesting at lab-scale. The direct addition of diatomaceous earth (DE) to the cell supernatant makes the standard centrifugation step redundant. The harvested supernatant can be directly filtered with a bottle top filter and a vacuum pump.news

During filtration, DE forms a porous filter cake which helps to clarify cells and cell particles and prevents blocking of the filter. The implementation of Sartoclear Dynamics Lab V Kits, harvesting has become a fast and reliable process step in antibody manufacturing.

How does the Sartoclear Dynamics Lab V kit compare with the previous gold standard? What are the advantages and limitations?

The gold standard for cell culture harvesting includes a clarification step via centrifugation to remove cells and larger debris. Especially for higher lab-scale volumes (~ 2000 mL) limited centrifuge space leads to multiple runs and increases the time for clarification.

In a second step, the remaining supernatant is filtrated to remove smaller cell debris. Besides slow flow rates in this step, the main disadvantage is filter clogging what leads to a considerable loss of product and a higher consumption of filter membranes.

Using Sartoclear Dynamic V Lab Kits clarification and filtration is achieved within one step, circumventing the centrifugation step and related issues like centrifuge capacity and availability. Preparation and filtration time are reduced due to a direct addition of DE to cell culture broth and filtration under reduced pressure.

Why is it important to remove cell debris and small particles from the supernatant? How does this affect the end result?

The recovery of the expressed recombinant antibody is a multi-step process, requiring prior-clarification of the cell culture broth to obtain a particle free solution for subsequent protein A affinity chromatography. But beside the removal of submicron particles to avoid high particle load on the protein A column, it has to be considered that a shear-less removal of cells also minimizes the formation of smaller cell debris and furthermore the release of intracellular enzymes or DNA from damaged cells.

A harsh centrifugation step can destruct the cell membrane, leading to the contact of intracellular molecules to the product. In the end, this can lead to altered glycosylation pattern and tertiary structure and functionality changes of the recombinant antibody.

Is there a difference in protein yield or mAb aggregation with the Sartoclear Dynamics Lab V method versus previous methods?

The analysis of DE clarified samples via size exclusion chromatography showed a similar content of high molecular weight species and heavy chain fragment compared to the sample which was harvested with a centrifugation step.

Furthermore, the total IgG titer was determined with analytical protein A affinity chromatography and compared to the conventional clarification method via centrifugation. A higher total IgG titer was determined for the samples clarified with the Sartoclear Dynamics® Lab V Kit.

What limitations are there for the Sartoclear kit?

At the moment, we also use the Sartoclear Dynamics® Lab V Kits for the clarification of lab-scale bioreactor batches (~2000 mL). In this case the harvest of high-density mammalian cell culture samples with Sartoclear Dynamics® Lab V Kits is limited by 1000 mL per Kit. A blockage of the filter membrane was not observed for cell densities of 1*107 c/ml.

What’s next for your research and Tubulis?

Tubulis is currently in the final phase of the preclinical validation of the Tub-tag® technology for the development of next-generation ADCs. Based upon our platform technologies we are working towards developing first proprietary ADCs. In addition, we are interested to partner our technology with interested Biotechnology and Pharma companies.

Where can readers find more information?

About Tubulis: www.tubulis.com

Sartoclear Dynamics: https://www.sartorius.com/en/products/lab-filtration-purification/harvesting-devices

About Tina Stoschek

B.Sc. and M.Sc. of Biology from the Ludwig-Maximilian University Munich (LMU). After her university degree she worked at Roche Pharma dealing with cell culture harvest in industrial scale. Since 2017 she is working for Tubulis®, a spin-off project at the LMU Munich where she is focusing on cell culture cultivation and antibodies.

About Marcus Gerlach

Marcus Gerlach completed his undergraduate studies in Biochemistry at Bielefeld University. After an Internship at the Institute for Molecular Bioscience in Brisbane he completed his doctoral studies at Bielefeld University in the group of Norbert Sewald, focusing on the chemo-enzymatic derivatization of proteins and peptides. Since 2017 he has been working at Tubulis®, developing and characterizing site-specific antibody conjugates for research and cancer treatment.




Roadmap reveals shortcut to recreate key HIV antibody for vaccines

HIV
Credit: CC0 Public Domain

HIV evades the body’s immune defenses through a multitude of mutations, and antibodies produced by the host’s immune system to fight HIV also follow convoluted evolutionary pathways that have been challenging to track.

This complexity has made it difficult for researchers to develop a preventive HIV vaccine that elicits effective antibodies similar to those that evolve in some people living with HIV. This is a task akin to retracing a traveler’s exact journey knowing only the destination, with few clues to the myriad possible origins and routes.

Now, a team led by Duke Human Vaccine Institute researchers, publishing online Dec. 11 in the journal Immunity, reported that they have filled in a portion of the roadmap toward effective neutralization of HIV, identifying the steps that a critical HIV antibody takes to develop and maintain its ability to neutralize the virus.

In their study, lead author Mattia Bonsignori, M.D., and colleagues focused on a particular class of broadly neutralizing antibodies known as VRC01, which targets a conserved region of the HIV envelope called the CD4 binding site. This antibody lineage has long been considered a critical component of a protective vaccine-induced immune response because of its ability to neutralize a vast majority of HIV variants, despite their diversity.

“These broadly neutralizing antibodies undergo a long and convoluted maturation process,” said Bonsignori, a member of the Duke Human Vaccine Institute. “There has been extensive study of them in the field, but until now, we have not been able to start at the unmutated ancestors—the origin—because it’s been so difficult to retrace the sequence of the many mutations, deletions and changes.”

The researchers extrapolated the un-mutated common ancestor of the VRC01 lineage and reconstructed the maturation pathways that resulted in the broadest antibodies and those that would be detrimental to HIV transmission.

Using that roadmap, the researchers then found that it’s possible for this class of antibodies to get to broad neutralization using a strategic detour along their developmental pathway. This detour is essentially a shortcut around what had been a major impediment that blocked previous attempts to induce the antibodies’ neutralizing properties.

“That was where everyone was stuck—we knew that if we could figure out how to engage the ancestor antibodies, we would be on our way,” Bonsignori said. “But we always hit this roadblock, where a particular sugar on HIV envelope blocked the development of the antibody during the early stages of antibody maturation, and everything got stuck.”

Bonsignori said the solution is to bypass that sugar, slipping around this impediment rather than trying to blast through it.

“What we found by reconstructing the different pathways was that you can get to the end using an easier route,” Bonsignori said. “Now we can use this information to design immunogens that will properly engage the immune system to circumvent this roadblock.”


Researchers map pathways to protective antibodies for an HIV vaccine



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Minimizing Antibody Size to Maximize Research Potential

An interview with Dr. Dave Fancy from Jackson ImmunoResearch at SfN 2018, discussing the use of secondary antibodies in neurobiology and the future of nanobodies.

How are secondary antibodies used in neurobiology?

Neuroscientists have been using secondary antibodies for years. They are generally used to provide or amplify the signal from a primary antibody bound to a specific target. For example, if a researcher wants to find out the location of a particular protein within a cell, they can run an IHC or immunofluorescence experiment. This involves first applying a primary antibody, followed by a labeled secondary antibody that can be visualized using an imaging technique such as fluorescence microscopy.

Immunofluorescent image using antibodies to visualize tumor cells - photo by Carl DupontCarl Dupont | Shutterstock

What makes secondary antibodies so attractive to researchers is that they allow researchers to use a primary antibody that’s unlabeled or carry out multi-colored labeling, for example, to simultaneously observe a whole host of different proteins. This means researchers can look at multiple proteins in a neuron, in a cell, in a synapse and more; with each color corresponding to a different protein. It also gives researchers the flexibility to choose colors that give the best contrast.

Finally, secondary antibodies give researchers the flexibility to detect proteins and compounds in the presence of other antibodies without cross-reactions. Developing secondary antibodies that are minimally cross-reactive is something we are passionate about at Jackson.  

You recently launched Alpaca Secondary Antibodies. Why did you feel it was important to add another antibody type to your extensive collection?

Firstly, alpaca antibodies are another species to the antibodies researchers are used to working with, such as mouse and rabbit. These antibodies, therefore, provide another avenue for multi-labeling experiments and add a whole extra repertoire to the research toolbox.

Also, researchers can take alpaca secondary antibodies and further manipulate them down to the nanobody component (the protein-binding VHH component) which makes them small.

This is particularly useful for super-resolution imaging, where researchers want to see fine spots and a traditional mouse, human or rabbit antibodies have several rotational degrees of freedom. This creates a very large circle of fluorescence in the final image, which makes the image difficult to analyze. Using a nanobody, in theory, allows researchers to decrease the size of this circle and increase imaging resolution to 1 or 2 nanometers of scale.

Our goal is to break our alpaca secondary antibody into smaller and smaller pieces so they can be used for techniques such as CLARITY. In this process, brain samples are made transparent by the removal of lipids, and antibodies are added to visualize specific proteins. However, the process is slow as the antibodies take a long time to re-enter the tissues.

Working with a nanobody or VHH domain, for example, makes this process faster. These components can penetrate hydrogels and tissues much faster than normal antibodies. This development will significantly cut down the amount of time it takes to introduce a secondary antibody to a sample.

Overall, our Alpaca range adds a whole extra layer of specificity that we can take advantage of, while also adding flexibility with an extended tool kit. Over the next few years, we hope to exploit more advantages relating to using the VHH domain, and we’re looking forward to the alpaca secondary antibody

What advantages do nanobodies provide over conventional mouse and rabbit antibodies?

There are many advantages to nanobodies. The first is their small size, which allows them to penetrate in vitro systems very quickly and pass through the blood-brain-barrier.

They’re also very stable and easy to engineer. Researchers can use just two PCR primers, and produce their own library of nanobodies. Compare this to working with a normal antibody, which requires pairing up the heavy and light chains – this process is not efficient and very difficult to get right.

Antibody labelled cells - photo taken by Carl DupontCarl Dupont | Shutterstock

Can camelid nanobodies be used in the same experiment as human and mouse antibodies without cross-reactivity? Why is this useful for researchers?

If a researcher makes a nanobody to target a particular protein, more often than not, they are going to see a lot of other proteins. At Jackson, we work hard to reduce the cross-reactivity of our antibodies so that researchers can use them alongside other proteins or other antibodies.

Our process is simple; we create the antibody, purify the nanobody, extract the VHH domain, then check for cross-reactivity. If the antibody doesn’t meet our standards, it goes back into the system to be cleaned up. This process can take weeks or even months, but it’s an integral part of producing a high-quality antibody.

Secondary antibodies are a lot tougher to make than primary antibodies because they have to be highly specific for researchers to be able to use them in multi-labeling experiments, and the same will be true for nanobodies.

The length of the binding site in nanobodies is smaller – instead of having a large paratope, nanobodies have a very small one, which is likely to make them more cross-reactive. That said, we developed IgG2 and IgG3 antibodies that showed some cross-reactivity with human antibodies but were able to remove this successfully.

What does the next year look like for Jackson ImmunoResearch?

The next year looks really good for us. There have been many novel techniques developed over the past few years, and we’ve been building antibodies that work with these. Techniques such as CLARITY or super-resolution microscopy are demanding smaller and smaller antibodies, and we are meeting that demand. Fab fragments are another antibody fragment that we have developed and work well with these techniques. I think they could become popular.

Globally, antibodies are huge, especially for pathology, so our main range of products are doing well. The development of CAR-T cell and immunology-based tools have brought this field to life in recent years – it’s the perfect time to be an immunologist. We think the future’s bright in the nanobody and antibody world.

About Dr. Dave Fancy

Headshot image of Dr. Dave Fancy Dave Fancy is the COO of Jackson ImmunoResearch, a position he has held for 5 years. Prior to his role at Jackson, Dave worked as a senior scientist at SDIX, where he focused on the development of polyclonal and monoclonal antibodies for immunoassays.

Dave received a Ph.D. in Organic Chemistry from the University of Texas at Austin in 1997. He then became a postdoctoral research fellow in structural biology at the The University of Texas Southwestern Medical Center, and after two years became an Assistant Professor.

In his current role, Dave leads the development and commercialization of antibody-based products and services for researchers working in the life sciences.  




DNA origami can accurately measure how antibodies interact with several antigens

Using DNA origami – DNA-based design of precise nanostructures – scientists at Karolinska Institutet in collaboration with researchers at University of Oslo, have been able to demonstrate the most accurate distance between densely packed antigens in order to get the strongest bond to antibodies in the immune system. The study, which is published in the journal Nature Nanotechnology, may be of significance to the development of vaccines and immunotherapy used in cancer.

Vaccines work by training the immune system with harmless mixtures of antigens (foreign substances that trigger a reaction in the immune system), from a virus, for example. When the body is then exposed to the virus, the immune system recognizes the antigens that the virus carries and is able to effectively prevent an infection.

Today, many new vaccines make use of something called “particle display”, which means that the antigens are introduced into the body/presented to the immune system in the form of particles with lots of antigens densely packed on the surface. Particle display of antigens works in some cases better as a vaccine than simply providing free antigens and one example is the HPV vaccine, which protects against cervical cancer.

Antibodies, or immunoglobulins, perhaps the most important part of the body’s defence against infection, bind antigens very effectively. The antibodies have a Y-shaped structure where each “arm” can bind an antigen. In this way, each antibody molecule can usually bind two antigen molecules.

Were able to accurately measure distances for best binding

In the current study, the researchers examined how closely and how far apart from each other the antigens can be packed without significantly affecting the ability of an antibody to bind both molecules simultaneously.

“We have for the first time been able to accurately measure the distances between antigens that result in the best simultaneous binding of both arms of different antibodies. Distances of approximately 16 nanometres provide the strongest bond”, says Björn Högberg, docent at the Department of Medical Biochemistry and Biophysics, Karolinska Institutet, who led the study.

The study also shows that immunoglobulin M (IgM), the first antibody involved in an infection, is significantly larger reach, that is the ability to bind two antigens, than previously thought. IgM also has a significantly greater reach than the IgG antibodies produced at a later stage of an infection.

DNA-origami a relatively new technique

The technology the scientists used is based on a relatively new technique known as DNA origami, which has been in use since 2006, that allows precise nanostructures to be designed using DNA. However, it is only in recent years that scientists have learned to use this technique in biological research. The application used in the study is newly developed.

“By putting antigens on these DNA origami structures, we can manufacture surfaces with precise distances between the antigens and then measure how different types of antibodies bind to them. Now we can measure exactly how antibodies interact with several antigens in a manner that was previously impossible”, says Björn Högberg.

The results can be used to better understand the immune response, for example why B lymphocytes, a type of white blood cell, are so effectively activated by particle display vaccines, and to design better antibodies for immunotherapy when treating cancer.

Important insight for tailor-made treatment

The research has been conducted in close collaboration with the Laboratory of adaptive immunity and homeostasis led by Jan Terje Anderson, at the University of Oslo and Oslo University Hospital.

“We study the relationship between the structure and function of antibodies. Such insight is important when we design the next generation of vaccines and antibodies for tailor-made treatment of serious diseases. We have long been looking for new methods that can help us get detailed insight into how different antibodies bind to the antigens. The collaboration with Björn Högberg has opened completely new doors,” says Jan Terje Andersen.

Source:

https://ki.se/en/news/dna-origami-a-precise-measuring-tool-for-optimal-antibody-effectiveness




Bio-Rad publishes new paper on generation, characterization of complex-specific antibodies

Published in the Peer-Reviewed Journal mAbs, the Paper Introduces Complex-Specific Antibodies for Pharmacokinetic Analysis of Biotherapeutics

Bio-Rad Laboratories, Inc., a global leader of life science research and clinical diagnostic products, has published new findings on the generation and characterization of drug-target complex-specific antibodies for pharmacokinetic (PK) analysis of biotherapeutics.

The paper published in the journal mAbs discusses antibodies that recognize the drug only when bound to its target. Called Type 3 antibodies, these antibodies differ from other anti-idiotypic antibodies that specifically detect free antibody drug by binding the paratope of the drug (Type 1) or total drug by binding outside the paratope of the drug (Type 2). PK assays form part of the totality of evidence required for approval of an original biologic or biosimilar drug, and anti-idiotypic antibodies are critical reagents used in these types of ligand binding assays (LBAs). By describing the generation and characterization of Type 3 specificities for the development of LBAs, the authors demonstrate the advantages of these antibodies as tools for drug quantification.

The paper describes the successful generation of Type 3 antibodies directed against several approved antibody drugs using Bio-Rad’s innovative custom antibody generation service, based on the Human Combinatorial Antibody Library (HuCAL®) technology and Cys-Display®, a modified phage display method. This recombinant production ensures a consistent and secure batch-to-batch supply, which is important for assay reproducibility.

Using Type 3 antibodies, Bio-Rad’s team demonstrate increased sensitivity and specificity across several assay formats, and quantify monovalent antibody fragments such as ranibizumab, which is difficult to achieve with commonly used LBAs like bridging assays. Additionally, the team introduce a derivative of the Type 3 specificity, termed Type 4, providing an alternative when the drug target is not easily available or costly to produce, or when selection of Type 3 is not possible.

Drug development relies on ligand binding assays, and the robustness, accuracy and reproducibility of these assays depends on the quality of critical reagents used. This paper is important in characterizing drug-target complex-specific reagents as useful tools in those assays, demonstrating several advantageous properties, to ultimately improve the accuracy of conclusions and accelerate the drug development process.”

Stefan Harth, R&D Team Leader, Custom Antibodies, Bio-Rad, and lead author on the paper

The reagents discussed in the paper represent a valuable addition to the ligand binding assay toolbox, and the reagents offer bioanalysts options for more sophisticated PK assay design to support biotherapeutic development. The original Type 3 and Type 4 reagents enable simple and robust assays that support the development of simplified rapid tests for therapeutic drug monitoring.”​

Amanda Turner, Bio-Rad Product Manager, Life Science Group




Researchers generate six antibodies to diagnose and treat Zika virus

Researchers have generated six Zika virus antibodies that could be used to test for and possibly treat a mosquito-borne disease that has infected more than 1.5 million people worldwide.

The antibodies “may have the dual utility as diagnostics capable of recognizing Zika virus subtypes and may be further developed to treat Zika virus infection,” corresponding author Ravi Durvasula, MD, and colleagues report in a study published in the journal PLOS ONE.

Dr. Durvasula is professor and chair of the department of medicine of Loyola Medicine and Loyola University Chicago Stritch School of Medicine. First author is Adinarayana Kunamneni, PhD, a research assistant professor in Loyola’s department of medicine.

Zika is spread mainly by mosquitos. Most infected people experience no symptoms or mild symptoms such as a rash, mild fever and red eyes. But infection during pregnancy can cause miscarriages, stillbirths and severe birth defects such as microcephaly.

Zika virus is a textbook example of an emerging disease that appears quickly, often in remote areas with little or no public health infrastructure. There is no effective vaccine or drug to treat the disease.

“The recent Zika virus outbreak is a health crisis with global repercussions,” Drs. Durvasula, Kunamneni and colleagues write in the PLOS ONE study. “Rapid spread of the disease within the epidemic regions, coupled with migration of infected persons, has underscored the need for rapid, robust and inexpensive diagnostic tools and therapeutics.”

Antibodies could be key to diagnosing and treating Zika virus. An antibody is a Y-shaped protein made by the immune system. When a virus, bacterium or other pathogen invades the body, antibodies bind to antigens associated with the bug, marking it for the immune system to destroy.

Using a technology called ribosome display, researchers generated six synthetic antibodies that bind to the Zika virus. The antibodies, which are inexpensive to produce, could be used in a simple filter paper test to detect the Zika virus in the field. (If the filter paper turns color, the Zika virus is present.)

Because the Zika virus is evolving, it’s useful to have six different antibodies. In the event the virus mutates, it’s likely at least one of the antibodies still would match the virus and thus could still be used in diagnosis and treatment.

An antibody-based test for the Zika virus likely would be cheap and fast, and thus could easily be used to monitor mosquito populations for Zika. If the virus is present in an area, officials could respond by stepping up mosquito-abatement efforts. They also could educate the public – especially women who are pregnant or could become pregnant – on how to avoid mosquito bites by applying mosquito repellent, wearing long pants and long-sleeve shirts, eliminating standing water, etc.

The antibodies are “neutralizing,” meaning that when they bind to the Zika virus, they prevent the virus from infecting cells. This effectively renders the virus harmless. The neutralizing property potentially could lead to the development of a drug that an at-risk woman could take to prevent the virus from infecting her fetus.

It will take further research to validate the antibodies’ potential for diagnosing and treating Zika virus, researchers said.​

Source:

https://www.loyolamedicine.org/




A Stable and Specific way to Target Cells in the Nervous System

An interview with Eugenia Kuteeva conducted at SfN 2018, by Alina Shrourou, BSc.

How can Neuroscience markers be used to advance our understanding of the brain?

Neuroscience markers are monoclonal antibodies, developed for identification of the main anatomical and histochemical cell types in the central nervous system. They can therefore be used as markers of the structural and chemical neuroanatomy. These antibodies can also be employed in multiplexing studies, to look, for example, at the expression of your protein of interest in a particular cell type in the nervous system.

Please outline the Neuroscience Marker Panel provided by Atlas Antibodies and which neural lineage and signalling markers they can target.

Our Neuroscience marker panel antibodies are a part of our catalogue of monoclonal antibodies. They target the main cell types in the nervous system, including neurons, astrocytes and oligodendrocytes. We will soon be adding also some microglial markers, which are currently under development. And already now we provide markers for the main neurotransmitter systems, including GABA, glutamate, acetylcholine, dopamine and serotonin systems.

How do Atlas Antibodies develop and validate these markers?

It is important to mention that we have chosen the targets for development of the Neuroscience markers antibodies, based on scientific knowledge on most relevant proteins for identification of different cell types in the brain.

We have an established protocol for development of our monoclonal antibodies, which starts with a careful selection of the most appropriate antigen for immunization. Following immunization, a number of ELISA positive clones are tested in functional applications in order to select the optimal ones for establishing the hybridomas, from which the purified monoclonal antibodies are finally produced. We test the purified monoclonal antibodies thoroughly in all relevant applications before approving and releasing them as products. In addition, all PrecisA Monoclonals are supported with isotype and, when relevant, epitope data.

We put a great effort into the proper validation of our antibodies by following the directions of the International Working Group for Antibody Validation (IWGAV), which recommends the five pillars of validation – genetic, orthogonal, independent antibody, expression of tagged proteins and immunocapture mass spectrometry. We apply these guidelines when validating our antibodies, which we do in the application specific manner.

Furthermore, the majority of PrecisA Monoclonals within the Neuroscience marker panel are tested and approved for use in both human, mouse and rat tissues, which can be beneficial for e.g. translational studies.

What are the other areas of research you provide the antibodies for?

Alongside our Neuroscience marker panel monoclonals, we also have monoclonal antibodies targeting early development of the nervous system, cortical layers markers, as well as organelle, SOX proteins, stem cells and EMT markers to name a few.

On our website, we provide information about the applications and species, which antibodies have been approved for. For the neuroscience marker panel in particular, we have tested and approved them for immunohistochemistry use in human, mouse, and rat tissues.

What can Atlas Antibodies provide to the neuroscience research community by being at Neuroscience 2018?

We put a great effort into developing the most specific and selective antibodies that meet the researchers needs. We take a great care in ensuring that the antibodies are functioning properly for our customers in all recommended applications.

We are here to meet with and talk to scientists in the neuroscience community. We always look forward to events like SfN to be able to discuss our products with our customers in person and to help them to meet their research challenges by providing technical and scientific advice.

 

Researchers often come to us to discuss which antibodies can be useful for their studies. For example, whether we have antibodies against their specific targets of interest, or which antibodies can be used for identifying specific cell types, groups of cells or particular structures in the nervous system. They may also have questions on experimental protocols. We are present here with our many years of neuroscience expertise to try to meet up and help scientist with their questions.

About Eugenia Kuteeva

Eugenia Kuteeva is a Principle Scientist working with development and validation of novel monoclonal antibodies (PrecisA Monoclonals) at Atlas Antibodies since 2012. Dr. Kuteeva holds a PhD degree in Neuroscience from the Department of Neuroscience at Karolinska Institute. She has a broad experience in life sciences from both academia and industry. Dr. Kuteeva has received several prestigious grants and has published more than 20 peer reviewed scientific papers, reviews and book chapters.