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Discovery of A Classical Lymphatic System in the Brain Will Revolutionize Neuro-Immunology

Until now, it was thought that the brain lacked the type of classical lymphatic drainage found in the rest of the body. Even though it was known that the brain was monitored by immune cells it wasn’t understood how they got there and moved around.

Researchers at the University of Virginia Medical School have just discovered a functioning classical lymphatic system that serves the brain. In order to make this discovery a special technique for preparing and mounting brain tissue for microscopic examination was developed. Without this technique the presence of the brain’s lymphatic vessels would have been missed again. According to the researchers, the lymphatic structures in the brain are well-hidden which explains why they have remained undiscovered for so long.

The current work has been done primarily in mice, but similar structures in human brain tissues have already been identified. These findings will revolutionize our understanding of the connection between the central nervous system and the immune system, and has tremendous implication for the study of neuro-immunological diseases such as Alzheimer’s Disease, and Multiple Sclerosis.

References

Loveau A, Smirnov I, Keyes TJ, Eccles JD, Rouhani SJ, Peske JD, Derecki NC, Castle D, Mandell JW, Lee KS, Harris TH, and Kipnis J. 2015. Nature. Jun 1. doi: 10.1038/nature14432. [Epub ahead of print] PMID: 26030524

University of Virginia Health System. “Missing link found between brain, immune system; major disease implications.” ScienceDaily. www.sciencedaily.com/releases/2015/06/150601122445.htm (accessed June 5, 2015).

Recently two healthcare workers infected with Ebola Virus (EBOV) were treated with a “mystery serum” which turned out to be a cocktail of monoclonal antibodies (Mabs) to EBOV, similar to receiving anti-venom for a snake bite. [see Mystery Serum Links below] What is unique about these Mabs is that they were produced in a plant, specifically, Nicotiana benthamiana, a relative of the tobacco plant. Antibodies produced in plants have been nicknamed “plantibodies”.

Why Make Proteins In Plants?

The ability to make mammalian, viral, and other proteins in plants has been around for about 25 years now. Early methods of expressing foreign proteins in plants were highly inefficient, but new methods have solved many of the early issues (ref). and produce high yields. Yields of 300mg/kg leaf fresh weight and 1-2 mg/g fresh weight (up to 10% of total soluble protein) have been reported (4,5). Some of the advantages of plant-based expression systems are (5):

• Relatively inexpensive,  requiring no special culture equipment, bioreactors, media, components, or sterile environment.
• Fast: can grow high yields of the desired protein within a few days to a weekThe material retains biological activity and immunogenicity.
• Multi-vector systems consisting of replication-defective constructs can be used to minimize the chance of spurious replication.
• Unique storage possibilities in that proteins can be stored in the form of seeds or in freeze-dried leaves.

In addition to EBOV antigens, proteins of Foot & Mouth Disease Virus, HIV-1, malaria, rotovirus, Influenza virus, HPV, Yersinia pestis, Dengue Virus, and HbsAg, and monoclonal antibodies all have been produced in plants (5).

How Do They Do It?

There are two key genetic components to the technology for expressing exogenous proteins in plants. The first is a plant expression vector derived from a plant virus. The two most popular types of vectors are based on Tobacco Mosaic Virus (TMV) or Potato Virus X (PVX) (1-7, Wikipedia).

The second is the bacterium Agrobacterium tumefaciens (Agrobacterium), that infects certain plants and causes tumors. Agrobacterium cultures are used as the gene transfer and delivery system to induce protein expression in the plants.

Here’s how it works:

1. The gene of interest is cloned into a plant expression vector plasmid.
2. The plasmid is introduced into Agrobacterium by transformation/conjugation.
3. The plant is infected with Agrobacterium by a process called agroinfiltration.
4. Agrobacterium then transfers the vector and gene of interest into the plant where it is expressed at high copy number, producing large quantities of the protein of interest.

SIDE BAR: Transient Expression by Agroinfiltration  (Wikipedia)

Agroinfiltration is a technique used to induce transient expression of genes in plants. A suspension of Agrobacterium tumefaciens is mechanically infused into the leaves of plants, where it transfers the desired gene to plant cells. The benefits of agroinfiltration compared to other plant transformation methods are speed and convenience. Once inside the leaf the Agrobacterium transfers the gene of interest in high copy numbers into the plant cells. The gene is then transiently expressed.

Plantibodies: Plant-Generated Monoclonal Antibodies

Monoclonal antibodies (Mab), such as the anti-EBOV antibodies, can also be produced in plants by agroinfiltration. Expressing complete antibody molecules is trickier because you need to express two different polypeptide chains and get them to assemble into a molecule consisting of two heavy chains (HC) and two light chains (LC), and retain antigen-binding specificity and capability (6,7).

Step 1: Generate monoclonal antibodies by immunizing mice and making hybridomas producing the Mab by the standard methods for making Mabs.

Step 2: “Humanize” the antibody by swapping gene and protein sequences common to mouse antibodies with those found in human antibodies, to prevent or decrease the chance of the recipient producing an immune reaction against the MAbs.

Step 3: Clone the Mab genes into plant expression vectors such as TMV- or PVX-based vectors; infect plants and let them grow (for about a week). Seems to work best if the HC and LC are expressed from non-competing vector systems such as HC in a TMV-based vector and LC in a PVX-based vector.

Step 4: Harvest the plant material, extract and purify the MAbs.

Conclusion

Although the production of proteins and antibodies in plants has not received much attention to date, the successful use of the anti-EBOV cocktail to treat people infected by EBOV will most likely propel this technology into the limelight in the near future.

Mystery Serum Links

http://www.the-scientist.com/?articles.view/articleNo/40700/title/Serum-to-Stop-Ebola-/

http://www.forbes.com/sites/davidkroll/2014/08/05/ebola-secret-serum-small-biopharma-the-army-and-big-tobacco/

http://arstechnica.com/science/2014/08/bio-high-tech-treatment-for-ebola-may-have-saved-two-us-citizens/

References—Want To Read More?

1. Gelvin SB. 2003. Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev. 67(1):16-37. ⊂⊃
2. Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y. 2004. In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci U S A. 101(18): 6852-6857. ⊂⊃
3. Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y. 2005. Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol. 23(6): 718-723.  ⊂⊃
4. Giritch A, Marillonnet S, Engler C, van Eldik G, Botterman J, Klimyuk V, and Gleba Y. 2006. Rapid high-yield expression of full-size IgG antibodies in plants coinfected with non-competing viral vectors. Proc Natl Acad Sci U S A. 103(40): 14701–14706.  ⊂⊃
5. Hefferon KL. 2012. Plant virus expression vectors set the stage as production platforms for biopharmaceutical proteins. Virology 433: 1–6.  ⊂⊃
6. Olinger GG, Pettitt J, Kim D, Working C, Bohorov O, Bratcher B, Hiatt E, Hume SD, Ashley K. Johnson AK, Morton J, Pauly M, Whaley KJ, Lear CM, Biggins JE, Scully C, Hensley L, and Zeitlin L. 2012. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proc Natl Acad Sci U S A. 109(44): 18030-18035.  ⊂⊃
7. Pettitt J, Zeitlin L, Kim DH, Working C, Johnson JC, Bohorov O, Bratcher B, Hiatt E, Hume SD, Johnson AK, Morton J, Pauly MH, Whaley KJ, Ingram MF, Zovanyi A, Heinrich M, Piper A, Zelko J, and Olinger GG. 2013. Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail. Sci. Transl. Med. 5(199):199ra113, pp.1-6.  ⊂⊃

Wikipedia Sources

http://en.wikipedia.org/wiki/Agroinfiltration

http://en.wikipedia.org/wiki/Agrobacterium_tumefaciens

http://en.wikipedia.org/wiki/Transfer_DNA

Companies Involved In Plantibody Research

USAMRIID

http://www.mappbio.com/

http://www.leafbio.com/

http://www.kbpllc.com/AboutUs.aspx

Icon Genetics GmbH, Weinbergweg 22, 06120 Halle, Germany; and Bayer BioScience N.V., Technologiepark 38, B-9052 Gent, Belgium
http://www.icongenetics.com/html/home.htm

 

Instead of hiding from your immune system Ebola Virus (EBOV) infects cells of your immune system first. A number of other viruses also do this, including Measles Virus (MV), and of course HIV. With EBOV, lymphocytes are NOT infected, but the infection eventually leads to their depletion.

Once the virus infects your immune cells and begins to replicate, it evolves to have an affinity for other cell types, especially liver cells. Many different cell surface proteins, found on many different types of cells, can act as receptors for the virus making it possible to infect many other cell types.

The incubation period for EBOV infection can be anywhere from 2-21 days

Pathogenic Mechanisms

EBOV has two main, and complementary, pathogenic mechanisms that make it so deadly:

1. It turns on the inflammatory response full-blast. This results in increased vascular permeability, hemorrhage, shock, and ultimately, death.
2. It turns off the activation of virus-specific immune responses so you get little-to-no anti-viral immunity to control the replication of the virus inside your body.

Ebola also has a liking for liver cells and causes liver failure. And guess what?! Most of the blood clotting factors are made in the liver. Bad news.

The infection process goes something like this:

1. EBOV infects Dendritic Cells (DCs), monocytes, and macrophages (MO), and also activates neutrophils. These are all cells belonging to your early or “innate” immune system, which is your front-line defense against foreign invaders.
2. These events cause the release of massive amounts of pro-inflammatory cytokines such as TNFa, which increase vascular permeability, fluid leakage, and shock. This effect is known as “cytokine storm”. Tissue Factor (TF) is also produced, which disrupts normal blood clotting and contributes to hemorrhaging.
3. Virus replication escalates in the infected cells
4. In the infected cells, the production of interferon-gamma (IFNg) is turned off. Downstream interactions of DCs and MO with lymphocytes (T cells and B cells) to initiate an anti-viral immune response, which are dependent on IFNg, are disrupted.
5. The maturation of DCs into functional antigen-presenting cells is inhibited, which, along with the decrease in IFNg, blocks activation of T cells and B cells preventing development of virus-specific immunity.
6. Virus replication is rampant in infected cells resulting in rapid spread internally, high viral loads, and nothing to keep the virus in check.
7. The virus spreads to other cell types, in particular liver cells, eventually leading to liver failure, increasing the potential for hemorrhaging.
8. Once symptoms appear, death typically occurs within 7-14 days. If the patient survives, recovery can be a very long and difficult process.

What About A Vaccine?

A wide variety of vaccine candidates and approaches have been tested or are currently being tested. These include killed viruses, live attenuated viruses, subunit vaccines, DNA vaccines, recombinant vaccines using other viruses as vectors. The approaches to create an Ebola vaccine are the same as those taken to produce a vaccine for other organisms, and the difficulties and pitfalls are also the same.  The primary issue is finding the balance between potency/efficacy and safety, especially in the case of live or recombinant vaccines. It is also clear that a durable and potent vaccine needs to generate both cellular (T cell-mediated) and humoral (antibody-mediated) immunity. Furthermore, variation and changes in the viral envelope protein (GP) make it difficult to neutralize with antibodies from either natural immunity or a vaccine. Work remains in progress.

Reference

Mansour Mohamadzadeh M, Chen L, and Schmaljohn AL. 2007. How Ebola and Marburg viruses battle the immune system. Nature Reviews Immunology 7: 556-567.