[Lecture Notes] Fundamentals of Biotechnology - L6: Immune technology

Immune technology

In the human body foreign macromolecules trigger an immune response. Invading microorganisms have proteins on their surfaces called antigens. The immune molecules which recognise them are called antibodies.

B cell antibodies are secreted into the lymph and the B cells generate billions of different antibody structures to diversify the the amount of foreign macromolecules they can detect. Some of the B cell antibodies which are bound to the cell surface are called B cell receptors. Some of these B cell receptors will inevitably recognise a foreign body - the B cell with the receptors that bind the foreign body will begin to divide rapidly, thus amplifying the amount of receptive antibody. This is the immune response. During rapid division, slight mutation of the antibody occurs in order to better bind the antigen on the invader. The immune system also has a memory after the infection has been wiped out so that if re-infected, some of the matching antibodies remain in the body which rapidly speed up the next immune response should this invader be seen again. This is the principle of vaccination which injects inactivated or destroyed virus or pathogen particles into the body to trigger an immune response without the corresponding infection. The body then keeps a record of the vaccination.

Antigen: any molecule foreign to the organism which raises an immune response. These are typically proteins of bacteria or viruses. A protein which raises a strong immune response is highly antigenic, such as lipoproteins. DNA and polysaccarides can be antigenic.

Humoral immunity: immunoglobulin mediated immunity (antibodies in the blood plasma).

Cell mediated immunity: T lymphocyte mediated immunity, which are T helper and T cytotoxic cells which bind protein fragments.

Epitope: The region of the foreign protein which is recognised by the antibody thus triggering the immune response. Large proteins may have many epitopes binding to many different antibodies.

T cells recognise antigens of other cells’ surfaces, such as virus infected cells. The recognition proteins on the surface are acalled class I and class II major histocompatibility complexes (MHCs or human leukocyte antigens (HLAs)). The genes that code for these proteins are unique for each individual.

The huge possible number of antibodies cannot be achieved through having a separate gene for each one, this would require enourmous genomes. Gene shuffling is used in a process called VDJ recombination to generate exponential variety in the antibodies. This process occurs in the bone marrow during the development of the B-cell and is done by RAN1 and RAG2 which nick the NDA, then NHEJ enzymes reconnect the pieces of DNA imperfectly causing inversions and deletions. A few thousand gene pieces can make billions of antibodies.

Antibody structure

The most common type of antibody is an immunoglobulin G (IgG) and has a gamma heavy chain. 75% of blood serum antibodies have this heavy chain and are IgGs. IgG can move across the placenta barrier during pregnancy. IgG is made of two light chains which contain the variable reagion and two heavy chains. THey have a Y shape and each half of the antibody and light chain/heavy chain complex is bound using a disulphide bridge. The kappa and lambda gene loci code for the light chains. The light and heavy chains ahve 1 to 4 constant regions and one constant region each. The variable regions are what bind the antigen and are called the paratope. So the paratope binds the epitope.

Chromosome 2 has the kappa locus which codes for light chains. 75 V chains exist, 5 J and 1 C segment. 30 V chains are functional and you can combine these chains so that each light chain is coded for by 1 V, J and C segment each. Further modification during the translation phase and post-translational modification gives rise to more variety in the light chain. The heavy chain is similarly coded for but the locus is on chromosome 14 and there are 39 V, 27 D and 6 J chains, and again alternate splicing, insertions or deletions are what gives rise to higher variety

Antibodies are made of three pieces, two of which are the same. The Fab fragments are the two tips of the Y shape of the antibody and the Fc fragment is the lower section made of the heavy chains. Fab means: Fragment antibody binding, and Fc means: Fragment crystallisable.

Clinical applications of antibodies

If an organism is injected with an antigen it will produce different antibodies because different antibodies can bind to the different epitopes on the antigen. This micture is a polyclonal antibody mixture because many differnt B cell clones will produce somewhat different antibodies in response to the immune trigger.

Antibodies which are actually clinically useful come from 1 B cell line and so will be called monoclonal antibodies because the mixture will contain only that 1 antibody which the B cells produce. B cells have a limited lifespan and so can be fused with myeloma cells (which are B cell cancer derivatives). They will express the antibody but are an immortal cell line. The myeloma cell used does not make its own antibodies. The resulting immortal B cell line is called a hybridoma. This is done by injecting a mouse with an antigen and collecting its B cells which are then fused with the myeloma cancer cells which are then cultered to make lots of antibody.

Monoclonal antibodies need to be humanised as to not raise an immune response in us humans. Since the heavy chain is functionally redundant in the recognition of an epitope, it can be replaced with a human version (this is the C region). This lowers the immune response in humans but is still not perfect. This is called a hybrid or chimeric antibody. Not the entire V region is involved in epitope detection and so the bits that aren’t can be humanised as well. It was found that only 3 sections of the V chain are responsible for binding antigens and changing the structure around these areas has no effect on the antibody, therefore this section of the V chain can be humanised as well. These detection regions are hypervariable and called complementary determining regions (CDRs). There are three CDRs on the light and 3 on the heavy chain. Splicing together the coding regions for human antibodies and the CDRs yields a fully humanised mAb (fig 6.10). Many of these humanised mAbs are being given as clinical treatment such as herceptin and humira which is a TNFalpha antibody.

Antibody engineering

Antibodies are of interest due to the extremely high binding specificity, and that they can be attached to other molecules. The genes coding for the variable chains are cloned into bacteria separately so that antibody fragments are expressed. These ar Fab, Fv and single chain Fvs (scFv). Fv fragments can be linked together using peptide chains wheras Fabs are linked by disulphide bridges. Other molecules such as tags or therapeutic drugs can be attached to the scFv fragments which can allow for targeted drug delivery.

Camel antibodies are very different, they have only the heavy chains with “nanobodies” (Nb) on them which contain the variable epitope binding regions. Nanobodies are very small and easier to work with, much smaller than human antibody fragments. They are stable, have high heat resistance and have a high binding affinity. Nanobodies can recognise concave and convex structures meaning that they are able to bind to enzyme active sites deep within a protein that normally only a small molecule would be able to access. They can also pass through the kidney and the blood brain barrier due to their size. This means they may be used as biosensors or humanised for treatment.

Diabodies are scFv fragmetns which have been joined together by polypeptide linkers. Each scFv has a different paratope and therefore can be used to bring two different molecules together, such as an anti-cancer drug to a cancer cell.

ELISA

An ELISA is used to detect protein in a sample and to give an indication of its concentration. An antibody specific for the target protein is made and the protein is treated as an antigen. The antigen is then marked with an enzyme that produces colour from a substrate if the antibody binds the protein. The target protein is immobilised on a microwell wall and the antibody and colour generating enzyme are added. Then the well is washed so that only bound antibody and enzyme are left. Then the colour changing substrate is added and measuring the colour change signal can be used to to detect if the protein bound the antibody and the amount. This can be done using a second antibody after the wash step, the second antibody recognises the first antibody and the second one itself has the detection system bound to it. So if the first antibody is present because it bound to the protein then the second antibody will bind to it and the not be washed away and the detection system will generate a signal. These are called secondary antibodies because they will recognise any antibody from a specifit organism and so does not require any engineering.

ELISA kits can be easy to make for diagnostics and quickly screen for diseases even before they become symptomatic. Pregnancy kits are an ELISA which is an assay for hCG which is produced in the placenta. It passes out through the urine when pregnant. The test is a piece of paper which acts like a column, wicking urine across it. Anti-hCG antibodies are attached to part of the paper and as urine passes over it, hCG binds to the antibody and this complex continues its journey down the paper. This complex reaches part of the test which has a secondary antibody which is impregnated into the paper in the shape of a plus. This has a colour detection system and when the anti-hGC/hCG complex binds to it it will turn a different colour. Then the urine keeps passing and reaches a control window which has another secondary antibody which recognises only anti-hCG and so binds regardless of whether the woman is pregmant (there is always excess anti-hCG). This is just a test for whether the pregnancy test is working (fig 6.17).

Imaging with antibodies

Immunocytochemistry: visualising antigens in cultured cells. Immunohistochemistry: visualising antigens in tissue sections. There are many ways of preparing cells for visualisations such as with formaldehyde, acetone or methanol, or by freezing and taking a 2d slice of the organism, or by embedding in wax. Regardless of the preservation technique, cells are made permeable to the antigen next. The primary antibody is added and enters the cells and binds to the antigen of interest, the secondary antibody is then added and allowed to bind to the primary antibody. The secondary antibody is carrying the detection mechanism so that the primary antibody/secondary antibody/antigen complex can be visualised. These detection systems can be enzymatic or by exploiting fluorescence. Alkaline phosphatase is common.

Fluorescence with antibodies can be exploited to distinguish one cell type from another by finding fluorescent antibodies that bind to surface antigens.

FACS is fluorescence activated cell sorting. The cells do not need to be prepared like previously discussed. One can separate HT and KT cells from other blood cells based on whether or not they have CD4 or CD8 antigens presented on their surface. Different fluorescent markers are added to two different monoclonal antibodies which either bind to CD4 or CD8 which will be used to differentiate the cells. Single cell drops of liquid are passed through a machine and a fluorescence detector measures the colour of the fluorescence and an electric field guides the drop into the correct container based on the measurement. If no signal is detected then the droplet goes into a third container. Flow cytometry is a similar method but instead of sorting the cells they are just discarded after measurement.

Vaccination

After infection, some B cells called memory cells remain in the body. If these B cells encounter the same pathogen they divide very rapidly and the immune response is not delayed trying to find a suitable antibody. The response can be so fast that no symptoms are even shown. Vaccination uses this immune memory by taking derivatives of infectious agents which can raise an immune response but not cause the disease. Most vaccines are made by taking the disease and heating it to kill it or denaturing it chemically. Sometimes this cannot be done due to cost or the danger of working with these pathogens.

Attenuated vaccines are those where a live pathogen is injected but has been engineered so that it does not cause any harm. Toxins or protein expression is turned on so no disease symptoms are shown. This keeps the antigen markers but removes risk of disease. A similar strain of the organism can be used as well, one which is non pathogenic but similar enough to raise an immune response to that disease. Attenuated viruses are more difficult to make as attenuated viruses can return to their infectious form due to the mutation rate.

Subunit vaccines are only effective against a subset of the molecules associated with a certain disease. They are made using recobinant DNA technology. Part of a protein that raises an immune response is made, this protein was likely identified on the surface of a pathogen. The gene for that protein is then expressed in a CHO cell and is tested. The success of a subunit vaccine depends on the protein folding correctly. Small sections of the protein which raise the immune response can be created and added to a carrier molecule to stimulate an immune response as well (figure 6.23).

Multivalent vaccines such as the MMR vaccine have multiple epitopes from different pathogens in them so that only one injection needs to be given. The different protein fragments tend to be from related organisms however.

Vector vaccines are created by engineering a bacterium or virus to express an antigen which is typically expressed by a disease causing pathogen. Homologous recombination is used to achieve this and this has the dual function of making the vaccine recipient resistant to both the organism injected but the disease causing organism as well. A virus called the vaccinia virus is typically used. Genes are inserted into its genome using homologous recombination (when DNA segments are exchanged between two other segments).

Many infectious agents have had their genomes sequenced, and this can be used to find new antigens for vaccines. All the genes from these infectious genomes are expressed in an expression library and the proteins ar eisolated. These protein mixtures are screened for an immune response, typically in mice, and when a group of proteins raises such a response, that pool is further sub-divided. This is repeated until a pure isolate of a protein is fuond that raises the immune response. These proteins cna then be used in subunit vaccines or multivariate vaccines(fig 6.26).

When a bacterium invades a host, the metabolism of that bacteria must change in order to adapt to its new living conditions. Part of this will involve expressing new proteins intracellularly which are different to proteins expressed on its surface. Identifying these expressed proteins which only come about post-infection can be useful in the creation of new vaccines. How are the genes which are switched on post-infection detected? When a phagocyte engulfs an invading bacterium, the bacterium will find itself in a low pH environment, the cytoplasm of the phagocyte. fluorescent protein genes or other tags can be fused to genes which scientists suspect are switched on under intracellular conditions. These fusion genes are inserted into the pathogen and the pathogen is allowed to infect a host. The amount of signal detection corresponds to the amount that the gene of interest is expressed.

This can be done on a larger scale testing for all genes. FACS sorting can be used for this. A gene library for GFP-fused genes of the host can be created and expressed. Each pathogen will have the GFP gene fused to a unique gene and these pathogens can be put under a simulated intracellular environment, such as a low pH. Some pathogens will express proteins (which will fluoresce) because they were tagged, and some will not express any fluorescent proteins implying that those proteins are not related to infection. FACS can then sort these cells and sequencing can identify which genes are involved with invasion which we did not previously know about.

DNA Vaccines

DNA vaccines work on the principle of injecting DNA which codes for a certain antigen instead of injecting the antigen itself. This might be a plasmid carrying the gene for the antigen along with a strong promoter. This removes the need to purify antigens. The DNA vaccine would be injected into the patient, most likely into muscle tissue, and the cells would express the gene over a period of a few weeks allowing the antigen to build up to concentrations where an immune response can be triggered - this immune response would be localised to the place of injection which mitigates side effects elsewhere in the body. DNA can be stored dry at room temperature eliminating the need for a full cold chain like in traditional vaccines. A positively charged microbead is used to bind the DNA’s negative backbone for delivery. This allows for a slow release of DNA which raises a better immune respnse than an immediate large dose.

References: my notes are made from, and follow the structure of my course textbook which is Biotechnology 2nd edition by David P. Clark, which can be found for purchase here.