Humoral Response PDF

Giada Fregnan/Laura Conte – Lesson 15 – Immunology (prof. Claudia Curcio) – 30/11/21 HUMORAL RESPONSE The antibodies can be involved in direct and indirect activities. Direct activities They are induced as a consequence of the binding of the antibody to the antigen...

Giada Fregnan/Laura Conte – Lesson 15 – Immunology (prof. Claudia Curcio) – 30/11/21 HUMORAL RESPONSE The antibodies can be involved in direct and indirect activities. Direct activities They are induced as a consequence of the binding of the antibody to the antigen, that occurs in the two variable fragments of the arms (Fab regions). Thanks to these direct activities, there can be: 1. Toxin neutralization 2. Virus and microbe inhibition (for instance antibodies are very important in trying to defend ourselves from the Covid infection) 3. Immune complex formation 4. Agglutination (the test used in laboratory to determine the blood group is mediated by the agglutination) 5. Aggregation and internalization of receptors and membrane molecules, to inhibit their effect and block the signaling mediated by these receptors. 1. Toxin neutralization It was first described by Von Behring and Kitasato, who discovered that the sera of the animals immunized against diphtheria or tetanus contained some specific anti-toxic activity mediated by antibodies. For example, the Tetanus toxin, binding the receptor on our neuromuscular cells, can block the contraction of the muscles and can induce paralysis. In the past, both diphtheria and tetanus had a high impact on the health of the population. So, the two scientists proved, for the first time, that the protection against those diseases could be induced injecting people with these anti-sera containing antibodies. The toxin neutralization is used to inhibit the activity of many kinds of toxins (even the ones coming from snakes, for instance) and it is the consequence of the direct binding of the antibody, that is able to interfere with the enzymatic activity of these molecules. The administration of the sera in patients triggers a short protection, because the half-life of the IgM is short, so it confers a transient but immediate immunity. In the meanwhile, the vaccine can induce the development of the memory in these patients, that will produce by themselves the antibodies needed to react against the pathogens. 2. Virus and microbe inhibition The image on the right represents a virus that binds the receptors on the membrane and enters a cell. The virus needs to enter another cell for its survival, in order to use its replicatory machinery and produce new nucleic acid, envelope and capsid proteins. In this way, the progeny viruses can be produced and they can spread, infecting other cells of the body. The antibodies can bind the surface of the virus, blocking its binding with the target cells and avoiding the production of new virions. This phenomenon is called adsorption and it is achieved when molecules on the viral envelope interact with molecules on the target cells. 1 Giada Fregnan/Laura Conte – Lesson 15 – Immunology (prof. Claudia Curcio) – 30/11/21 This is the reason why, especially for COVID-19, the production of antibodies induced by vaccines is so important: the antibodies block the entrance of the virus into our cells. The following table represents the two types of receptors used by SARS-CoV2 to enter other cells: ACE2 (in grey) and TMPRSS2 (in orange). These receptors are present in many organs, and this is why this virus has a high potential to infect our cells. Using antibodies, the binding of the virus to the receptor can be blocked, avoiding the internalization and the replication of the virus. 3. Immunocomplex formation Immunocomplexes are nets formed by the antigen and the immunoglobulin. They are normally formed and then phagocytosed. If produced in large amount, they can activate the complement and trigger an important damage in our cells. The immunoglobulins can bind the antigens recognizing the epitopes and form these nets. The IgM are the best immunoglobulin sub-class to form immunocomplexes, especially when there are low titers of antibodies, because they have 5-10 binding sites. Not all immunocomplexes are similar, their difference depends on the concentrations of both the antigens and the antibodies. In case of high concentrations of antibodies or antigens, the immunocomplexes will be smaller, while the same amount of antigen and immunoglobulin will correspond to larger immune complexes. 2  Giada Fregnan/Laura Conte – Lesson 15 – Immunology (prof. Claudia Curcio) – 30/11/21 4. Agglutination It is the formation of an immune complex with the antibody and a large cell with the antigen, for example the red blood cells. Red blood cell IgM Agglutination is routinely used in laboratories to determine the blood groups. On the left, there’s an image of the reaction. In the four circles there are the three different anti-sera (anti-A, anti-B and anti- D, which is 0) and the control. Adding in each circle a sample of the serum with the undefined blood group, the technician can determine the blood type by visualizing the agglutination. In this case reported, the reaction occurs in the first sample, so the red blood cells of the serum presented antigen A on their surfaces. This method can also be used to identify specific microbes or antigens. 5. Aggregation and internalization of membrane molecules and receptors mediated by antibodies It allows the inhibition of several processes, such as proliferation. The receptor, once it is bound by the antibody, is internalized and degraded. In this way, no more receptors will be present on the cell membrane. The aggregation and internalization can occur also for membrane self-molecules, causing pathologies such as Myasthenia gravis. In normal conditions, the acetylcholine (ACh) released by stimulated neurons in the neuromuscular junction binds the ACh receptors on muscle cells. On the contrary, in Myasthenia gravis, auto- antibodies can bind the ACh receptors, causing 3 Giada Fregnan/Laura Conte – Lesson 15 – Immunology (prof. Claudia Curcio) – 30/11/21 their internalization and degradation. Therefore, on the surface of the muscle, there won’t be any receptor and the muscle will become less responsive to acetylcholine. This mechanism is also used in cancer therapy. For instance, ERBB-2 receptor, also called HER-2/neu receptor, is involved in the development and progression of some types of breast cancer (its ligand is not known yet). In order to be activated, it needs the crosslinking of different receptors, that are usually overexpressed in cancer. Thanks to this interaction, the signal transduction triggers the proliferation of tumoral cells and the deregulation of the apoptosis. Moreover, these signals stimulate the migration of cancer cells, causing the development of metastases. Crosslinking 4  Giada Fregnan/Laura Conte – Lesson 15 – Immunology (prof. Claudia Curcio) – 30/11/21 In this case the effect of the antibodies (such as Herceptin, a monoclonal antibody, or Trastuzumab) is very important, because they can bind the receptors and mediate their capping, endocytosis and degradation. In this way, the receptors won’t bind each other and the signal necessary to activate the proliferation pathway will be inhibited. Indirect activities They are mainly mediated by the constant fragment (Fc domain) of the immunoglobulins. As discussed in the previous lesson, some receptors can bind the Fc portions of the antibodies to mediate their different biological functions. In particular, Fc receptors are constituted by an alpha chain, that binds the Fc region of the antibody, and other chains (β, γ and ζ), similar to the ones present in the CD3 of the TCR, responsible for the transduction of the signals. There are many kinds of Fc receptors (FcR): - High-affinity receptors: such as CD64, made up of an α-chain and two γ-chains, containing two ITAM sequences, involved in the activation of the signals. - Low-affinity receptors: such as CD32, consisting of one α-chain with an ITAM or an ITIM sequence, and CD16 (on NK- cells), whose structure can be similar to CD64 (with two chains devoted to the signaling transduction thanks to the ITAM motif) or it can just consist of one chain unable to transduce any signal (because of the absence of the ITAM motif). - pH dependent receptors: FcRn (Neonatal Fc receptor), important for the transport of the IgG from the maternal to the fetal circulation 5  Giada Fregnan/Laura Conte – Lesson 15 – Immunology (prof. Claudia Curcio) – 30/11/21 This scheme sums up the two main functions addressed by Fc receptors (transport and the activation of the immune response): They facilitate the transcytosis of IgA The indirect activities of the antibodies are: 1. Immune phagocytosis (opsonization) 2. Cytotoxic activity by granulocytes, macrophages and NK cells 3. Induction of mast cell and granulocyte degranulation 4. Activation of the complement 1. Immune phagocytosis If the antigen is completely covered by antibodies, the Fc receptors on the surface of the macrophage can bind the Fc fragments of the antibodies and elicit a more efficient phagocytosis, that is called opsonization. This mechanism allows an easier internalization of the antibodies. 6  Giada Fregnan/Laura Conte – Lesson 15 – Immunology (prof. Claudia Curcio) – 30/11/21 2. Antibody dependent cellular cytotoxicity (ADCC) The cytotoxic activity is mainly mediated by NK cells (thanks to the CD16) and macrophages. The picture shows a pathogen covered by antibodies and an immune cell presenting the FcR (CD16), that bind the Fc portion of the immunoglobulins. Thanks to the recognition of the antibodies by the FcR, all the granules of the immune cell are recruited near the pathogen. In this way, the activation of the killing program can occur and the secretion of these granules, containing perforin and granzyme, will be specifically directed to the pathogen. Therefore, the presence of the antibodies on the surface of the pathogen can drive the lytic activity of the killer cells. 3. Mast cell and granulocyte degranulation Basophils and mast cells present high-affinity FcR on their membranes. These receptors, also called FcRε, can bind the IgE. This interaction triggers a survival signal for this subtype of this type of immunoglobulins, that normally have a very short half-life (2/3 days). Therefore, the binding of the antigen to the IgE and its crosslink with the FcRε on basophils or mast cells lead to the degranulation and the release of several vasoactive mediators. These vasoactive mediators can be divided into: - Immediate phase reaction: the degranulation causes the release of histamine, proteoglycans, proteases and TNF - Late phase reaction (after 6-8 hours): there is the occurrence of the secretion of lipid mediators and the transcription of cytokines and chemokines, that mediate the inflammatory and immunoregulatory signals. 7  Giada Fregnan/Laura Conte – Lesson 15 – Immunology (prof. Claudia Curcio) – 30/11/21 This mechanism is very important because it causes the allergic reaction and it represents a protection against parasites, especially helminths (nematodes). In fact, this reaction can trigger the induction of diarrhea or sickness, which are a way to eliminate parasites from the body. 4. Antibody feedback It is activated according to the amount of IgG. Once the antigen is fought by the immune system, the antibody production has to be switched off when it’s no longer needed, because it is expensive for the body. One possibility is mediated by the receptor FcγIIB: the increased IgG production parallels the ability of IgG to bind the antigens and forming immune complexes. These latter, then, bind the FcɣRIIB and trigger a feedback that blocks the excessive production of antibodies. Therefore, on the B cells there are both BCR and FcγRIIB. Here, the presence of the antigen leads to the crosslink of the BCR, that will trigger the formation of a structure similar to a bridge, constituted by immunoglobulins, BCR and antigens. Once the recognition of the antigen has occurred, the BCR will start to activate the chains (α and β), responsible for the signal transduction. Hence the chains will be phosphorylated to trigger the cellular activation and antibody production. As the infection progresses, immunoglobulins and antigens will interact forming the immunocomplexes. 8 Giada Fregnan/Laura Conte – Lesson 15 – Immunology (prof. Claudia Curcio) – 30/11/21 When the infection decreases these immune complexes can be bound by FcγRIIB. In this way, the phosphatase removes the phosphate groups form the phosphorylated inositols, therefore removing the substrate to the PLC. As a consequence, the Calcium pathway will be inhibited. Therefore, the B cells are no longer activated. 9 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 MONOCLONAL ANTIBODIES (mAb) Monoclonal antibodies represent one of the most important discoveries in biotechnology which affected several fields, such as the medical one. Immunoglobulins can be separated by electrophoresis in a particular fraction of the protein present in the sera (albumin which is the most abundant, the globulins, such as alfa1, alfa2, beta1, beta2; in the gamma-fraction the immunoglobulins are present quite diffused). The electrophoretic separation may also be represented in a histogram [C], in which each peak is higher when the band is more intense (which means that the represented protein is more abundant). [A] represents the electrophoretic separation using the serum belonging to a healthy individual. In this case, the antibody production is unspecific (meaning that several isotypes are produced at the same time) and very limited because there’s no pathogen invading the organism. If the analysed serum derived from an infected patient or cancer patient, the production of Ab is higher and it’s very specific (depending on the intruder, a specific isotype is mainly produced). The band representing that particular type of antibody, as a consequence, will be wider and more intense. The same thing happens with monoclonal antibodies. When their presence is represented on a histogram, they will be pictured by a very high and thin peak [D]. 1 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 When a MULTIVALENT pathogen (it’s characterized by several epitopes that may be identified by the B cells) is recognised, the immune cells activate and expand themselves. During the activation, these cells perform the germinal centre reaction, during which some of them change the affinity of the receptor. Taking the image on the right as an example, the B cells named as “c”, during the activation, expansion, but mostly during the germinal centre reaction, will differentiate into c1, c2… These differentiated cells all derive from the same naïve B cell, however they develop a different BCR with different affinity. This process is due to the hypermutation: physiologically, after this reaction, different antibodies are expressed against the same pathogen, but also against the same epitope (however, the affinity is different). Usually, an immune serum (it’s obtained by healed people from an infection) contains a mixture of antibodies with different affinity and specificity. These antibodies change from one individual to another and change also over time. When the immune serum is collected from different individuals at different time, they are different because they are randomly produced. The isolation of antibodies from immune sera was initially performed in order to fight against several infections (i.e., those caused by tetanus or diphtheria), however it’s not possible to know if the patients receive the same concentration of antibodies with the same affinity because of the diversity inside the same immune serum. All these variations (regarding the titer, affinity and isotype) compromise the exploitation of monoclonal antibodies in clinical practice, formulation of a diagnosis and research practice. The main consequence of the different affinity of the immune is that it’s detectable. For example, if a Western Blot is performed using two different samples having different affinity for a particular protein, it’s possible to differentiate them. The sample with a higher affinity will give strong bands as results, whereas the one with a lower affinity will produce faint bands. The amount of the protein may be the same, however the antibodies with a lower affinity are able to detach faster. MONOCLONAL ANTIBODIES are monospecific, or monovalent (they all act against the same epitope of the protein). They are all identical, coming from the same parental cell and the same differentiated plasma cells. They’re not able to change, because their production is stable (nowadays, for instance, we are still able to use monoclonal antibodies belonging to the ‘80s). Their obtainment was discovered by G. Koeler and C. Milstein (Nobel Prize in Physiology and Medicine in 1984). 2 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 WHAT DID THEY DO? They immunized normal mice with a specific antigen (protein X). They then obtained the mice’s spleen, which contains a mixture of B cells, among which there are also those specifically produced against protein X. These cells have to be isolated. B cells aren’t easy to be maintained in culture because they tend to die when they’re not specifically activated and when they don’t receive survival signals through their BCR or cytokines (this feature is shared by T cells). Koeler and Milstein decided to put them in culture together with peculiar neoplastic plasma cells (myeloma) that are deprived of the genes necessary for the antibodies production. As a result, these neoplastic cells, even if they’d be able to produce antibodies, don’t generate them. Cancer cells are immortalized, which means that, unlike B cells, they are able to grow and proliferate without any survival stimulus. The main idea of the experiment was to fuse these two types of cells. The fusion and the co-culture of these cells needed to be performed in a specific calcium-medium, known as HAT, which allows the survival of the fused cells only. As it’s explained in the graph down below, the fusion cannot be performed in other ways: The fusion of two B cells isn’t possible because the obtained products will die both in normal culture and in HAT because they don’t receive the needed survival signals. The fusion of two myeloma cells isn’t possible since the obtained products wouldn’t grow in HAT because one of the components of this special medium blocks the nucleotides biosynthesis of plasma cells. Consequently, the cells wouldn’t be able to grow. The only solution is the fusion between a B cell and a plasma neoplastic cell, which will produce cells that are able to grow in HAT and that will eventually produce antibodies. 3 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 HAT This particular medium is made up by three main components: Hypoxanthine, Aminopterin and Thymidine. Plasma cells exploit dihydrofolate reductase for synthetising nucleotides. This enzyme is blocked by the Aminopterin. The effect is that tumoral cells’ growth is blocked in this medium. B cells have an alternative pathway to synthetize nucleotides, involving Thymidine and Hypoxanthine, which are the other two components of HAT. Thymidine and Hypoxanthine are necessary for the activity of Thymidine Kinase (TK) and Hypoxanthine Guanine Phosphoribosyl Transferases (HGPRT). Thanks to them, the synthesis of nucleotides takes place. The fused cells (B cell + myeloma cell) are called hybridoma, which are immortalized cells producing a specific monoclonal antibody. Recalling the previous experiment, the initial pool of B cells was heterogeneous: there were different types of B cells, among which there were those specific for protein X (the one used to immunize the mice). How is it possible to isolate those producing Ab against protein X? The hybridoma has to be cloned in a microwell plate. Each hybridoma cell is cultured in a different well: when inside the well, if the cells don’t grow it means that the fusion hasn’t taken place; when the hybridoma survives, the medium of well changes its colour because the nutrients of the medium are consumed by the growing cells. After letting the cells grow, the medium is obtained and then used against protein X to check the different affinities of the cells coming from different wells. The best assay to evaluate the affinity may be the ELISA: the protein is anchored on a plastic plate, and then different antibodies are tested. Those with a favourable affinity will bind the protein. For this experiment, the supernatant of each well is used to test the eventual presence of specific antibodies against protein X. After all the washes, an anti-mouse antibody (i.e., against a specific isotype such as the IgG or IgM) is used and the ELISA plate is developed: the only wells that are coloured are those containing the antibody. After this selection, the production of mAb takes place. The hybridoma producing the monoclonal antibody can be maintained for a long time: the culture may be expanded, otherwise they may be frozen with nitrogen liquid for several years. The Western Blot would be too expensive and time-costing. The hybridoma will never change its isotype, as well as its specificity and affinity because the plasma cells in this case never perform the germinal centre reaction. Another advantage is that it’s possible to produce an unlimited amount. mAb are used for research purposes, diagnostic purposes and 4 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 therapeutic approaches: they’re very useful improve the tumour diagnosis, but also other pathologies, such as the autoimmune diseases (they’ve also been tested against Covid). Hence, nowadays many small and large companies produce and sell monoclonal antibodies. Monoclonal antibodies have also been very useful for the definition of CD markers. CD stands for “Cluster of differentiation”, however they are also known as “Cluster Designation” or “Classification Determinant” and they are all the molecules expressed on the surface of the immune cells (however, nowadays other cells are taken into account too) specifically recognized by clusters of mAb. When at least two monoclonal antibodies recognise the same protein, the latter is called “CD” followed by a number (i.e., CD4; CD8; CD25…). Each CD defines a specific subtype of cells (i.e., CD4 or CD8) or a specific stage of differentiation (i.e., CD25+ or CD25− ) or a specific stage of development (i.e., double positive CD4 or CD8 are very immature T cells when compared to single positive CD4 and CD8). CD are conventionally used in immunology, for example, for obtaining the immunophenotype of the circulating cells or those infiltrating the tissues (i.e., tumour cells, but also cells involved in other pathologies). The antibodies against the CD are also useful to isolate and purify a specific type of cells, a specific subtype or a specific stage of activation/maturation. For example, we want to compare the process of CD4 activation against a specific antigen between patients and healthy people: the presence of monoclonal antibodies against the CD4 allows the isolation of these cells (otherwise, the isolation may be performed with a sorter or magnetic beads conjugated with the antibodies). Another application is performed in vivo to kill a specific type of cells: for example, we want to study the functioning of CD4 T cells during the development of a specific pathology (i.e., cancer). In this case, instead of using a transgenic mouse lacking the CD4 T cells, it’s possible to use specific antibodies to kill CD4 T cells, which is a cheaper technique. This process is known as depletion. Nowadays more than 400 CD have been identified and at the beginning some misunderstanding happened, for example the same molecule was designed with two different numbers. That’s why, in 1982 in Paris, the first International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA) was held, and it was decided that the CD designation was allowed only when two different monoclonal antibodies were able to recognise a specific molecule. On the other hand, when the molecule is recognised only by one type of monoclonal antibodies, a precise indicator is used: “w”, which is put between “CD” and the number (i.e., CDw186). This indicator underlines that the molecule still needs to be confirmed with a second type of mAb in order to be a “real CD molecule”. The official nomenclature also defines whether a certain cell population expresses or lacks a CD molecule thanks to the presence of some symbols: “+” for the presence and “-” for the absence of the CD. When, instead of the definition of the presence/absence, it’s important to underline the amount of CD, the name of that particular CD is followed by “low” or “high”. For example, the names “CD80low” or “CD80high”, when referred to the dendritic cells, specify if they are fully activated and they are able to properly behave as antigen presenting cells (CD80high), or if they’re not activated yet (CD80low). In this case it’s wrong to used the symbols “+” and “-“, because the dendritic cells constitutively express the CD80, however its concentration is variable. 5 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 The following table lists several types of CD molecules, the cells on which they’re expressed and how they function: TUMOUR DIAGNOSIS AND THERAPY The therapeutic use of murine monoclonal antibodies faced several issues: - Because murine monoclonal antibodies are quite big proteins, once they’ve introduced in the human organism, they’re characterized by a high immunogenicity (which means that they induce an immune response), a short half-life in blood (which means that repeated boosts are required). Both these two features don’t have any pathogenic effect, however they both lead to the neutralization of the therapy because of the immune response developed by the receiving organism against the introduced mAb. - Because they belong to another specie, murine monoclonal antibodies aren’t able to induce truly efficient human effector mechanisms. Antibodies have to be bound by the Fcγ, Fcε or Fcα receptors in order to induce phagocytosis, killing or degranulation. Because the introduced antibodies belong to a different species, this binding will be characterized by a different affinity. - Repeated boosts have also a high risk of inducing strong allergic response. 6 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 - There’s a lack of specific interaction among the murine mAb constant regions with human effector molecules (i.e., the complement). HOW TO SOLVE THESE PROBLEMS? Researchers tried different approaches to solve these issues: 1. The first intuition was to create fully human mAb fusing murine myeloma and human B cells together. Again, the myeloma cells weren’t able to produce immunoglobulins but they’re able to survive, whereas B cells weren’t able to survive but they induced the production of immunoglobulins. The obtained product was unstable because of the loss of human chromosomes by murine myeloma. 2. When both the used myeloma and the B cells derived from human, there were several ethic problems for the in vivo immunization, which involved humans in this case. 3. SCID mice were then used. These mice were immunodeficient, lacking T cells, NK cells and they were transferred with human thymocytes and lymphocytes from fetal lymphonodes in order to reconstitute the lymphocyte production in these mice with human cells. The aim was to immunize the mice and not the human being, however the technique was too expensive requiring an SPF environment because of the immunodeficiency of the mice. 4. Some researchers tried to immortalize the B cells without creating the hybridoma, but through the infection with EB virus, which naturally targets B cells very well. However, there was the production of low-affinity IgM. The final solution was the production of chimeric mAB: the mAB is engineered through cloning approaches. The variable genes coming from the murine antibodies are inserted in human cells and fused together with the constant portion of the human antibodies. In a mature B cell, the variable portion is completely rearranged. Through PCR, the genes of the variable region of the light and heavy chains are isolated in the murine thanks to specific primers. They are then fused with the human constant portions isolated from human B cells (again, thanks to PCR). The obtained product is called chimeric mAb, which are normal antibodies made up by the murine variable regions and human constant portion (so, they mostly derive from human antibodies). 7 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 There are several types of mAb: - Fully murine - Fully human - Chimeric (variable portions derive from murine and the rest is human). They’re less immunogenic when compared to the fully murine mAb and they can also bind the complement (but also the Fc-γ receptors, or other Fc receptors) through the different domains of the constant portion and activate the immune cells. Because the variable portions are murine, these antibodies are able to induce an immune response once they’ve been injected in the organism, that’s why the humanized mAb have been created - Humanized: instead of using all the murine variable regions, only the three CDR sequences of the variable portion (which are hypervariable regions) are cloned in the frame sequence of a variable region encoded by human genes. 95% of the mAb derives from human, which means that these antibodies are less immunogenic than the chimeric ones Nowadays, the fully murine monoclonal antibodies are mainly used for research purposes and for immunodiagnosis (for example, they’re exploited by anatomic pathology through an immunochemistry approach to diagnose the different subtypes of cancer; or their used to create ELISA kits). On the other hand fully- human, chimeric and humanized monoclonal antibodies are mainly used for immunotherapy. 8 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 HOW TO RECOGNISE THEM? It’s possible to distinguish all the different types of monoclonal antibodies thanks to their name: - Murine: they’re characterized by the suffix -omab - Human: they’re characterized by suffix -umab - Chimeric: they’re characterized by suffix -ximab - Humanized: they’re characterized by suffix -zumab PHAGE DISPLAY It’s the process allowing the obtainment of fully human monoclonal antibodies without immunizing an individual. Genes encoding Ab variable regions are isolated through PCR. This process is possible, for example, after obtaining B cells from a patient after an infection or after the development of a tumour (in general, after the organism has encountered an antigen inducing a humoral response against it). The isolated genes are then fused with those encoding phage tegument, so that phages expressing the variable region are created. A phage library is created, containing many different phages expressing all the variable regions of the immunoglobulin obtained from the patient. Inside the library, the phages expressing a specific variable region are isolated. This selection is performed using bacteria in which genes expressing a specific antigen have been transduced. All the phages expressing the variable region that is able to recognise that particular antigen will kill the colony of bacteria. 9 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 The pages that are able to kill the bacteria are isolated and the variable regions are cloned fused with the constant portion of the immunoglobulin. The fused genes are then transfected in myeloma cells, which are immortalized, enabling them to produce the mAb. The produced mAb are characterized by both variable and constant regions deriving from human. Another way to produce mAb is by exploiting plants. Vegetal cells, unlike the bacteria, allow the post-translational modifications leading to the production of proteins that are more similar to those that are produced by the mammalian cells. In general, plants are very useful for research purposes (i.e., they are also exploited for the production of antigens in order to create new vacc ines). 10 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 The table in the previous page shows different approaches to produce mAb using plants (whole plants; cell/tissue cultures; moss/microalgae cultures). Some advantages of these methods are: - They allow a cheaper production of mAb, when compared to the hybridoma approach - The purification of the proteins is easier when compared to the one performed using hybridoma - They allow a long-term storage - The transportation is easier: As a matter of fact, for these methods it’s no longer necessary to transport frozen cell, because it’s sufficient to sell the seeds containing the genes that are necessary for the mAb production The main disadvantage is that it’s possible to use these plants in an open environment leading to ethical issues (the seeds may diffuse). However, researchers are trying to solve this problem. The following table lists several mAb for Cancer Therapy approved by the FDA. 11 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 It’s possible to observe several types of monoclonal antibodies: - Trastuzumab is an example of humanized monoclonal antibody and it is mostly used against breast cancer, but also against other types of cancer involving EGF - Ipilimumab is an example of fully human monoclonal antibody and it targets CTLA-4 - Rituximab is an example of chimeric monoclonal antibody In the list there is no fully murine mAb underlying the fact that this type of monoclonal antibody is no longer used for therapeutical purposes. mAb MECHANISMS OF ACTION 12 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 mAb are able to act: - Directly, for example, they may neutralize the cells, or change their behaviour by binding specific molecules expressed on the cellular surface. They’re not able to bind internal molecules, but they can only bind external pathogens, or the epitope expressed by the infected cells - Indirectly, for example they may mediate cytotoxicity by the NK cells, the macrophages, the activation of the complement or by the degranulation of the neutrophils mAb IN CANCER THERAPY There are several approaches in cancer therapy, for example: 1. Increase of immunogenicity Rituximab and Cetuximab are able to bind tumour cells inducing the activation of the immune cells (NK cells or macrophages). Rituximab acts against the CD20 of mostly non-Hodgkin’s B-cell lymphoma, whereas Cetuximab binds the EGFR expressed by solid tumours inducing the activation of NK cells and macrophages. 2. Activation of intracellular pathways In this second method, the therapy directly affects the survival of the tumour cells. Trastuzumab binds Her2 receptors on breast cancer cells inducing their internalization, so that Her2 (an oncogene) isn’t able to properly work. This mAb is also able to induce the apoptotic pathway blocking the transduction of the survival signals. A second generation of Rituximab has been generated, targeting the CD20 just like it has been previously explained, however this modified molecule presents a modified Fc-γ part which doesn’t bind the Fc- γ receptor directly inducing the apoptosis masking the CD20 on the lymphoma cells. Because of the lack of survival signals, the apoptotic pathway is induced. 13 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 The image on the left represents the binding between the Rituximab and the B cell. This interaction directly kills the cell, otherwise it leads to the activation of NK cells and macrophages. On the right, different mechanisms against the CTLA-4 or PD-1 are represented. CTLA-4 and PD-1 are two different receptors belonging to the CD28 family expressed on the T cells, however they mediate the opposite effect. They both bind co-stimulatory molecules (just like the CD28), however, instead of the increase of the kinases’ recruitment for the amplification of the TCR signal, they recruit the phosphatases. As a consequence, the TCR signal is inhibited switching off the activation of effector cells. Many tumour cells express the ligand of these two receptors. Anti-CTLA-4 and Anti-PD-1 monoclonal antibodies have been created (this discovery has been awarded with a Nobel prize) to block this interaction in vivo allowing the functioning of the effector cells against tumour cells. 14 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 APPLICATIONS OF MONOCLONAL ANTIBODIES 1. Serological Immunodiagnosis (ABO typization or serological HLA typization) Our red blood cells, based on the expressed enzyme, are able to add different sugars on their surface creating different blood types. If we use different mAb, the one inducing the agglutination is the one recognising the antigen expressed by the red blood cells. If there’s no agglutination, the individual is type 0 because neither the A nor the B antigens are expressed. When the agglutination takes place, the individual may be type A or type B (the definition is obtainable testing anti-A and anti-B antibodies singularly). The HLA typization is no longer performed using mAb (they have been replaced by PCR) because of the cost. When the test involved mAb, they were tested against different leukocytes antigens (i.e., HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP). Again, when only ONE mAb recognises the protein, the name of that protein also involves “w” (i.e., HLA- Cw6). After the typization, the haplotype of the individual is obtained. 2. Tumour diagnosis and therapy mAb may be used against specific tumour antigens in order to understand the subset of cancer during a diagnostic process. For example, the oncogene Her2 is expressed only in 30% of breast cancers, whereas others express oestrogen or progesterone receptors, others don’t express any of them. These three subsets require three different therapeutical approaches. For instance, Trastuzumab is completely useless for those patients whose cancer doesn’t express Her2 and they would only experience the side effects of the therapy. In immuno-radiotherapy, mAb are conjugated to radioactive drugs. For example, during the immunoscintigraphy, mAb are conjugated to γ or α emitting radioisotypes, thanks to which is possible to properly analyse specific organs of the patients. 15 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 mAb are also conjugated with chemotherapy drugs functioning as vehicles. In this way, it’s possible to create a more precise therapy avoiding the side effects of these drugs on the healthy organs. They can also be conjugated together with enzymes which are necessary for the metabolism of the drugs. This approach is able to reduce the resistance because cancer cells are able to fight against the therapy because they lack the enzyme that is necessary to catabolize the drug. Finally, they can be conjugated with toxins to directly kill specific cells. The image down below represents all the possible conjugations. The mAb are named affinity proteins because the review was discussing the different mAb that can be used, as well as just small portions of the antibody. For the conjugation it can be used the total antibody, or only the Fab (the fragment binding the antigen), only the Nanobody, only the scFv (single chain variable fragment), or only the Minibody (the variable region fused with a small part of the constant region), the Triabody (three variable regions complexed together) or the Diabody, or the Tetrabody. Each portion has its own advantages and disadvantages. For example, when it’s necessary to understand the cancer subset, it’s better to use the Fab only. Hence, because there’s no constant region, the cross-reaction with the potential Fc-γ receptors on the cancer cells is less potent. When a biopsy is obtained, hence, there’re also infiltrating cells that express many Fc-γ receptors which easily react with the constant portion of the antibodies just because of their chemical affinity (the binding isn’t specific immunologically speaking) leading to different staining occurring for the same biopsy. Another example is when it’s necessary to vehicle an enzyme or a toxin inside the cell. In this cases, smaller Ab are better because of their ability to enter the cells. 16 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 mAb have been considered when developing therapies against SARS-CoV and SARS-CoV2. NEW METHOD TO GENERATE HUMAN MONOCLONAL ANTIBODIES Antonio Lanzavecchia has discovered how to isolate the antibodies against the SARS-CoV (in the previous pandemic). Even during the SARS-CoV2 pandemic, one of the different approaches that have been used to fight the infection involves the hyperimmune serum, which derives from healed patient, which means that it contains the antibodies. The main problem regarding the exploitation of this kind of serum to cure other patients is that it’s not possible to be sure that every patient receives the same type or titer of the antibodies. Lanzavecchia tried to create a particular type of antibodies taking the serum from SARS patients, in which the IgG neutralized the virus, meaning that the production of memory B cells took place. The memory IgG+ B cells were isolated from the patient through monoclonal antibodies and flow cytometry, and then they were immortalized using the EBV. 17 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 The process is well summarized in the previous scheme, in which the CD22+ B cells are isolated using the magnetic beads and then they’re infected by EBV to become immortalized. This virus is integrated inside the genome of the cells inducing a tumoral transformation of the cells. The infected memory B cells are then stimulated by some PAMPs, such as the CPG, which bind the Toll-like receptors (otherwise some mixed bacterial oligonucleotides may be used instead of the PAPMs) to increase the immortalization and the proliferation of the cells. The memory B cells are then cloned just like the hybridoma (each cell is put in a different well) and then the different clones are checked together with their production of IgG against SARS-CoV through the ELISA assay. In this way, the clone producing the most reliable IgG (with the highest affinity and the best neutralization activity against the virus) is isolated. The IgG of the chosen clone are then used to immunize people who have been infected by the virus. The same idea has been applied to SARS-CoV2 as well. The main purpose is to avoid the development of the most severe symptoms of the infection (Severe Acute Respiratory Syndrome, SARS), which may lead to the death of the infected individuals due to the failure of several organs. 18 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 The virus binds two different receptors ACE2 (Angiotensin-Converting Enzyme) or TMPRSS2 (Transmembrane Serine Protease) to enter the cell. After the replication and the production of proteins, the infection induces a hyperactivation of the immune system: the so-called “cytokines storm”. Many pro-inflammatory cytokines are produced by the immune system inducing not only the amplification of the activation of the immune cells, but also the normal epithelial cells are activated. The effects may be mild which, after a supportive care, then develop in a normal immune response. However, the effects may also be very severe, inducing huge tissue damages involving several organs (in this case, unfortunately, the damages may be fatal). Researchers have been trying to study every step of the infection in order to exploit mAb (that have been already created for other pathologies) against SARS-CoV2. For example, anti-(IL-1, or IL-6, or JAK) have been developed to block the cytokine storm. The pie chart on the right shows several therapeutic approaches that have been used in 2020 against SARS-CoV2. For each therapy, there’s always the combination of an under-investigation part and a previously approved one. The first one is referred to the aspect of the therapy that has been developed for the SARS-CoV2 only, whereas the second one underlines the use of approaches that have already been developed for other diseases but that can also be applied to SARS-CoV2. 19 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 These two pie charts on the left shows an analysis of targets and formats of the therapeutics under development of COVID- 19. All the new drugs have been created against the viral protein (i.e., S protein), whereas some of them target the pro- inflammatory cytokines (i.e., IL-6 or its receptor IL-6R). As for the formats, the mAb are the main products. These other two graphs show the development status of COVID-19 therapeutic antibodies. The anti-IL6, anti-IL1 or anti-JAK switch off the activation of all our innate cells, as well as the adaptive ones (T and B cells) in order to block the cytokine signal. 20 Laura Conte / Larisa Laois – Lesson 16 Immunology (Prof. Paola Cappello) – 02/12/2021 Several complement inhibitors have been created. For example, there’re inhibitors targeting the anaphylatoxin (i.e., anti-C5a). The idea is to neutralize the activation of the complement through the presence of antibodies, in order to downmodulate the tissue damages caused by the complement activation. Other approaches involve: - Tocilizumab, an IL-6 antagonist - Convalescent plasma - Anakinra, a IL-1 inhibitor Even though all these therapies have been developed, there’s still no cure against SARS-Cov2. Currently, in Italy (Siena) Rino Rappuoli is trying to produce mAb starting from infected people. 21 Larisa Laios/Michelle Guichardaz – Lezione 17- Immunology- Prof. Cappello – 7.12.2021 VACCINES A Vaccine is a biological preparation able to induce a protective immune memory against infectious diseases. In the past, vaccines had been able to eradicate smallpox (in 1980) or almost eradicate other diseases (i.e., polio, which is still endemic in the Middle Eastern countries). Vaccines are based on our immune memory: when a pathogen enters our organism, a specific T cell activates and begins its clonal expansion and differentiation. Once the pathogen has been cleared and the infection has been overcome, the healing process starts: the number of the clonal T cells decreases and only memory T cells remain. They can persist for a very long time (from months to decades). IMMUNE MEMORIES… The population of memory cells that survive: - are those selected from those that react with a greater affinity. For example, as seen, memory B cells are characterized by a higher affinity compared to the B cells they are generated from. - are more numerous (100-1000 units). - have some epigenetic changes, especially in those genes necessary for the proliferation, production of cytokines or weapons for the CTL cells. Overall, the genetic regions that code elements involved in the reaction have become more accessible and present the absence of methylation (epigenetic memory). For these reasons, the T cells do not require the binding of the co-stimulatory molecules to be fully activated. The anamnestic response activated by memory lymphocytes may be based on: Antibody production Activation of memory T killer cells Activation of different T helper subpopulation The strategy can vary based on the pathogen involved in the infection, the characteristics of the danger signals (cytokines) and the kind of initial inflammatory response and can lead to different genetic programs of differentiation. … AND THEIR CELLULAR FEATURES The memory B cells are follicular (FoB2) because the B1 and the marginal-zone B cells cannot differentiate into memory cells since they do not require the T cell contact. Without the CD40 binding (CD40 ligand is present on T cells) – what is called the formation of the T-B cells conjugate, memory cells differentiation cannot occur. The memory is a feature specific for the follicular B cells and all the other T cells. After an initial priming, a significant population of memory cells can persist for a long time, circulating or residential, in a more significant number compared to the virgin T or B cells they originated from. Memory cells have less complex activation requirements and can provide a fast and efficient secondary immune response without a co-stimulatory signal. Memory cells display antigen receptor reacting with the antigen at high affinity (different BCR affinity for the same epitope) – this regards mainly B cells since affinity maturation does not occur in T cells. The memory B cells, compared to the virgin T cells, not only present a higher affinity BCR but also present a different antibody isotype (they do not express IgM or IgD as mature naïve B cells, but they express IgG, IgA or IgE depending on the pathogen that has induced their activation). 1 Larisa Laios/Michelle Guichardaz – Lezione 17- Immunology- Prof. Cappello – 7.12.2021 Activated FoB2 B cells are ready to rapidly proliferate, undergo symmetric division and differentiate in plasma cells and produce large quantities of hyper mutated antibodies and memory B cells, which inherit the genetic changes occurred in the progenitor. Centroblast: BCR of switched isotype BCR of high affinity for the targeted antigen Those memory cells are mainly residential in lymphoid organs but can also be circulating. Memory T cells What happens after clonal expansion? The differentiation in both effector and memory T cells. We can distinguish at least two sub-populations of memory T cells: Central Memory T cells: these are mainly homed in the lymphoid organs (lymph nodes and spleen) and their traffic towards or exiting the infected tissue is guided by cytokines and chemokines. Effector Memory T cells: are more circulating than the central memory T cells and can be found in tissues – not only lymphoid tissue associated with mucosae – and blood. They are the first cells thar respond to an intruding pathogen. The memory B or T cells are maintained after the healing. When the second or third encounter with the same pathogen occurs, they can activate a very fast adaptive immune response, but only when present above a certain number (a threshold that has been estimated experimentally). When below threshold, no secondary immune response is registered and, just like virgin T cells, they require all the other steps (the antigen presentation, the co-stimulating process and so on). In this case, we’re talking about a loss of memory: the frequency of memory cells is too low to mount a protective reaction. History of the Immune Memory The suspicion of the existence of a memory immune system goes back to 430 BC, when Thucydides noticed that “a certain disease – the plague – did not take the same person twice, at least not in order to kill one”. In the Faroe Islands, reported as another example, a natural experiment occurred: in 1781 the inhabitants had been decimated by a measles epidemy. The same viral epidemy re-occurs 65 years later. Ludwing Panum observed that the inhabitants who were older than 65 years old did not contract the virus: this is because they already experienced or encountered the infection before, unlike those younger than 65 years old. THRESHOLD 2 Larisa Laios/Michelle Guichardaz – Lezione 17- Immunology- Prof. Cappello – 7.12.2021 Immunologists evaluated the number of T cells necessary to induce an immune response in case of a re-infection thus establishing the threshold under which T cells are considered too few to mount a secondary response. T cells were simply counted before, during and after infection. 7 x10-5 is the minimal frequency of memory cells providing a protective memory reaction. What was interesting though is that the number of T cells seems, after the peak in the acute? infection phase, to fluctuate up and down even after the infection ended. This means, as following explained, that they can proliferate a bit sometimes. How can we maintain and re-stimulate this pool of cells without a re-infection? cytokine There are different theories explaining this process, but one thing is for sure: the cytokines (IL2, IL15 and especially IL7) produced during the antigen presentation – even not correlated to the pathogen linked to memory B cells – are enough to produce the suboptimal signal to keep the proliferation of cells rather active (not their expansion – so, not a huge proliferation as in the case of virgin T cells) and their survival. The persistence of a significant immune memory depends on the kind of antigen and the intensity of primary response and subsequent boosts. A few microbes induce a very persistent immune memory. This longer memory may be due to: a. Repeated antigen re-stimulations. Re-entry of the same microbe causes small inapparent infections sufficient to boost immune memory. In this case, the antigen coming from the microbe, which again enters our body, simply acts as a boost (stimulating T cells) and a re-infection will probably be asymptomatic. b. Persistent antigen re-stimulations. Residual microbial depots may persist for long-periods after the clearance of the primary infection. Otherwise, the antigen may be another protein similar to the one that previously stimulated memory cells generation. They cannot merely be harmful to our body but sufficient to keep our immune system alert. The definition of the persistence of a protective immune memory is critical for the planning of vaccination schedules. THE FIRST VACCINE Scientists, ever since early times, have tried to induce immune memories: magical practices in China (we don’t know when they started but around the 18th century – 1710 – variolization was also diffused in Europe) known as variolization, consisted in taking content from human smallpox vesicles and the later introduction of the dried content into the nose of another person. Nice idea, but wrong execution. 3 Larisa Laios/Michelle Guichardaz – Lezione 17- Immunology- Prof. Cappello – 7.12.2021 Later in 1798, Edward Jenner created the smallpox vaccine after the elaboration of diffuse epidemiological observations drawn from the folklore of the countryside. Smallpox, which affects both farm animals and humans, resulted less serious for those who lived in the countryside compared to those living, for example, in London. Jenner then theorized that this milder version of the disease was due to an early exposure to the virus. An eight-year-old child orphan, James Phipps, was inoculated with cowpox fluid and then, 6 weeks later, with human fluid coming from smallpox pustules. James displayed no symptoms of the disease. This non ethical experiment demonstrated that the use of attenuate live – since viruses in cowpox fluid were alive – virus was enough to induce the generation of a memory immunity able to fight the natural human virus. Louis Pasteur took on this approach and developed vaccines based on attenuated microbes (smallpox, carbuncle and rabies). This led to the development of smallpox vaccines and a slow but sure journey to complete elimination of the disease: in 1980, the WHO declared “smallpox is dead!”. Thanks to vaccines, the equation can turn into: that is to say that immune memory can be induced without passing through the disease. VACCINES TODAY Vaccines became essential weapons to defend infants (without a very potent immune system), the elderly (with advancing age, immune functioning decreases as all other cells functioning) and the immunocompromised. This social importance is better highlighted by the ability of vaccines to protect individuals who are not responsive to vaccines (no vaccine is 100% efficient) thanks to the ability of those responsive to destroy the pathogen and lower the probability of spreading out and reaching those sensitive (Herd Immunity). Each of us, because of polymorphism, different haplotypes…, responds differently to the antigen: with a standard vaccine, some people cannot acquire the immunization. The negative effect of vaccination on the virus spreading has a huge importance: reducing spreading means reducing proliferation and consequently reducing possible acquiring of mutations. We have heard about this concept over and over during Covid-19, but it wasn’t so hard, back in the day, to convince people to get vaccinated. On the opposite, in some people themselves were voluntarily funding the process and pushing vaccination. Talking about Polio, two vaccines were separately developed – one from Salk and the other from Sabine, the first from killed virus and the other from live attenuated virus. Salk vaccine managed to decrease the number of infected people, but only Sabine vaccine leads to virus eradication. Indeed, killed virus simply is phagocyted, processed and exposed through MHCII thus mainly activating T helper and antibodies production; instead, a live attenuated virus is able to enter by itself our cells (since it is attenuated, it has lost its pathogenic ability but not the infectious one) but not to proliferate. However, the presence of virus inside the cell is enough to allow its presentation (actually, not the whole virus presentation, but the presentation of some of its peptides) through MHCI. This leads to activation of different cells and different cytokines production. As we can see, Salk vaccine produced a peak in IgM production (which normally is the first type of antibody produced) that then decreases to be substituted by IgG. The problem was that the virus also had a tropism for the intestine. With Sabine vaccine, people also obtained nasal, intestine, duodenal… production of IgA. 4 Larisa Laios/Michelle Guichardaz – Lezione 17- Immunology- Prof. Cappello – 7.12.2021 This explains what follows: with Salk vaccine, people were cleared by viruses, but, since they still homed in faeces, these viruses still spread through water and food; with Sabine vaccine, which was orally administrated, IgA produced in gut assured viruses clearance and killing in the whole organism thus permitting a spreading reduction. Of the 3 strains of wild poliovirus (type 1, type 2 and type 3), wild poliovirus type 2 was eradicated in 1999 and no case of wild poliovirus type 3 has been found since the last reported case in Nigeria in November 2012. Both strains have officially been certified as globally eradicated. As at 2020, wild poliovirus type 1 affects two countries: Pakistan and Afghanistan. VACCINES IN THE FUTURE An ideal vaccine must be: Safe: it should not induce the pathology for which we want vaccinate people. Stable: it should be easy to be maintained in order to assure its wide diffusion. Inexpensive. Effective in inducing the right adaptive immune response to the pathogen we want to vaccinate against (most of vaccines in use elicit Ab production). Able to elicit a long-lasting protection (long-lasting plasma cells, high affinity Ab after GC reaction). The development process usually takes 10-15 years, due to the number of stages the process must successfully go through; pre-clinical and clinical trials, carried out to assess vaccine safety, can take around 3 5 Larisa Laios/Michelle Guichardaz – Lezione 17- Immunology- Prof. Cappello – 7.12.2021 years themselves. Do not be fold by the “little” time (6-8 months) the Covid-19 vaccine has been developed: no stage has been skipped. During pre-clinical trials, animal tests are performed to prove the feasibility of vaccine concerning its ability to fight the disease. Then, the clinical trials (three phases to respectively prove if: the vaccine is safe, it is tolerated, it is able to induce the immune response) take different phases. The “end points” of phase II and III need to be evaluated in an increased number of people. Phase 4 is very critical: companies have to follow up the people tested to track adverse events and discuss the risk-benefit profile of the vaccine. E.g., the Sabine vaccine for polio, thanks to which polio was almost eradicated, is not used anymore for this specific reason: the use of a living virus can lead to the development of the disease we are trying to protect ourselves from, thus being more a risk than a benefit. It is important to note that this vaccine is still used, though, in underdeveloped countries, where a “risky” vaccine is better than no vaccine at all. CLASSIFICATION OF VACCINES A live virulent microbe administered in a different way to its natural entry (e.g., instead of airway pathway, you can inject it thus probably reducing its pathogenicity). A living microbe that has been modified thus losing its ability to cause disease (attenuated microbe). A dead microbe. A fragment or inactivated microbial toxin (i.e., toxoids: exotoxins engineered to become less or non- pathogenic. A strategy used for tetanus). A purified microbe antigen or epitope. Antigen pulsed dendritic cells. A DNA plasmid (naked DNA, inserted DNA in vectors) coding for microbial antigens. RNA from a virus. The table reports the pro and cons of each vaccine. Inactivated vaccine (polio, hepatitis A, rabies) 6 Larisa Laios/Michelle Guichardaz – Lezione 17- Immunology- Prof. Cappello – 7.12.2021 -Pros: easy to store and transport. Low risk of causing infection, since, being killed, it is less pathogenic for sure: as said, a killed virus is not able to enter a cell nor to proliferate. -Cons: on the other hand, the immune response induced is weak, thus it requires higher concentration, several doses or boosters. Subunit vaccine (hepatitis B, influenza) -Pros: low risk of adverse reactions, can be used for individuals with weak immune systems. -Cons: poorly immunogenic (you need high concentration or several boosts), difficult to manufacture and requires boosters, expensive. These can be sugars conjugated to proteins, which improves the B cell response and the consequent cascade of events (activation of T cells, of the germinal centre reaction, the production of high affinity antibodies). For example, talking about the purified portion of a microbe, in case of meningococcus or pneumococci it can be a sugar. The problem is that it is not so much immunogenic and only able to induce a B response. The strategy developed to overcome these limits is to conjugate the sugar with a carrier protein or other small peptides that must be inert, just useful as carrier. In this way, B cells recognize the sugar portion of the conjugate but, after the phagocytosis, they also perform the presentation of the peptides to T cell. This allows the Th cells and consequently the follicular B cells activation. So, instead of inducing only low affinity IgM, with this strategy, you can also obtain IgG, IgA… with higher affinity. In fact, thanks to T cells, germinal centre reactions can be performed. Live, attenuated vaccines (measles, mumps, rubella, varicella) -Pros: activates killer T cells (other than memory T cells, B cells thanks to Th cells); one or two doses can induce life-long protection. -Cons: difficult to store (they must be refrigerated), less safe for those with weak immune system. How were these attenuated vaccines produced (at the beginning, 15-20 years ago)? One strategy was to grow those viruses in the lab, and after many cycles of replication, they would lose spontaneously their pathogenicity. The other strategy was to culture the virus with other kinds of cells. For example, for human vaccine, one of the main strategies is to culture the virus with monkey kidney cells allowing the attenuation of pathogenicity. In fact, as demonstrated by Jenner, when a virus grows in different species cells type, a virus becomes milder in terms of pathogenicity. This last approach, though, came with the possibility of the virus to acquire new characteristics from different animal species, so, today, we use the DNA recombinant cloning method (cutting off the virus’ genes that make it pathogenic). Toxoid Vaccines (diphtheria, tetanus) -Pros: unable to cause disease or spread. Stable and easy to distribute. -Cons: require boosters. These are created starting from an exotoxin, usually inactivated in the lab with formalin fixation, unable to induce disease since they are no longer able to bind their receptors. 7 Larisa Laios/Michelle Guichardaz – Lezione 17- Immunology- Prof. Cappello – 7.12.2021 DNA Vaccine Naked DNA plasmid coding for one of the proteins of the pathogen, cloned by E. Coli, incapsulated in safe – not harmful and not pathogenic for us – vectors needed to mediate the entrance in our cells. (ma questo vale anche per I naked DNA plasmids?) Low immunogenicity is a limit of this strategy, together with the fact that sequence inserted cannot be so long. Strategies to help naked DNA entrance in our cells can be the electroporation to induce membrane potential changes and pores formation or the concomitant administration of adjuvants. The major advantage is that it cannot be integrated within our genome. Since any vector used is in any case a foreign object for our organism, during the first vaccination, our body responds with an immune reaction not only towards the foreign DNA, but also against the vector. This means that a second boost – which is therefore not possible - would result in our immune system neutralizing the vector and inactivating the vaccine. This is why a second boost is useless. HOW DOES A VACCINE WORK? After intradermal administration (more immunogenic way thanks to the high number of immature? dendritic cells), the antigen presenting cells can be transfected with the vaccine and, at lymph node, they can present the antigen to the T cells. Also B cells can recognize the antigen which can be then trasported through the lymphatic vessels. Then, the cascade of responses begins leading to the production of memory cells. Memory T cells can then reach the blood circulation through the lymphatic vessels to circulate through tissues or in the lymph nodes, ready to respond to a real threat. The SARS-CoV2 vaccine has been proven to induce the formation of this lymphoid structure (image on the side): B follicles surrounded by T cells like in the lymph nodes but not in the lymph nodes (e.g., even in the lymphoid tissues associated with mucosa), inducing the germinal centre reactions and the production of high affinity antibodies. New type of vaccine: transgenic plant A transgenic plant produced vaccine works sort of differently, exploiting a recombinant approach. For example, researchers can incorporate one envelope peptide (e.g., of rabies virus) gene in a plant virus that must act as vector. This virus is then used to infect a plant and, as a result, plant cells will express antigen whose gene was inserted in the virus. Then, the obtained plant can be used to feed animals (e.g., mouse). For example, a spinach infected with tobacco mosaic virus that previously incorporated DNA encoding a rabies virus peptide showed to be able to immunize mice that ate it. In fact, injecting rabies virus in these mice, they showed to be protected from infection. Anti-idiotype vaccine 8 Larisa Laios/Michelle Guichardaz – Lezione 17- Immunology- Prof. Cappello – 7.12.2021 This is a strategy not already used but that we can think to use in future. When researching more dangerous viruses, such as HIV, vaccines do not involve antigens (indeed, it would be dangerous to handle these viruses, risking to allow their spread), but antibodies: Niels Jerne demonstrated that (both in humans and mice), in each of us, there is a Network of Idiotypes and Anti-Idiotypes antibodies. As seen, the idiotype is the small portion in the antigen binding site different in each of us. When you are infected with an antigen, your organism develops antibody, against it, characterized by high complementarity. Jerne found out that our organism normally develop antibodies against the idiotype produced. As previously seen talking about idiotype, allotype and isotype, when you inject in a mouse A a serum coming from a different mouse A (different mouse, same strain, with identical B and T cells, MHC molecules…), you obtain the production of antibodies against the different portions of antibodies coming from the second mouse. In fact, if Fc, isotypes, frame regions…, are equal in the two mice, hypervariable regions are different. So, we develop antibodies against these portions. In fact, we usually develop antibodies complementary to antigens. But also, we produce antibodies against the different idiotypes, Jerne discovered. Take an example: you get infected with the flu virus You develop an antibody response to the virus. If these antibodies stay too long in the body, your immune cells recognize those antibodies as proteins with a different portion represented by the hypervariable region. This leads to the production of antibodies against your own antibodies, but also against a specific idiotype. This second round of antibodies (anti-idiotypes antibodies), therefore, is very similar to the original antigen (come fosse una proprietà transitiva). This similarity is the reason why we could use these antibodies to vaccine people. Moreover, the advantage is that they are not pathogenic at all. In HIV case, HIV induce the production of anti-HIV antibodies (Ab1) that stimulate the production of anti- idiotypes antibodies, which are similar to the antigen portion that induces the first wave of antibodies production. This can be an effective way to vaccine people from a virus without using the original antigen but using those antibodies which are very similar to it. It is absolute safe and can be produced in large quantities with a minimal dose of antigen. In addition, these antibodies are characterized by high immunogenicity (often higher than that of the original ag). THE JOURNEY OF THE SARS-COV2 VACCINE (just for your curiosity) At the beginning, the most diffuse ideas were: to insert DNA coding for envelope protein (Spike protein) in viral vectors; to synthetize Spike protein and use simply this to induce the immune response; to use the correspondent mRNA, which is administered thanks to lipid micro-vesicles to overcome the usual mRNA instability especially outside the cell. Liposome is able to fuse with our plasma-membrane allowing the mRNA entrance and the consequent translation. In this case, one limit is the conservation of both mRNA and liposomes. One alternative is the vaccine based on viruses (mainly adenoviruses) that vehicle RNA coding for Spike protein. Mainly in China and India, companies use the strategy of attenuated virus. Techniques to ameliorate the engulfment of vaccines from APCs 9 Larisa Laios/Michelle Guichardaz – Lezione 17- Immunology- Prof. Cappello – 7.12.2021 - Adjuvants: adjuvants are substances that enhance the immunogenicity of antigens. They cause a slow release of the antigen, aggregate soluble antigens and trigger a concomitant induction of danger signals and pro-inflammatory cytokines (thanks to the activation of the innate immunity, which includes APCs). Several adjuvants (killed microbes, constituent of microbes, mineral oils, cytokines…) are exploited to effectively vaccinate experimental animals. Aluminum salts – which is unfortunately also related to the redness and dolor in injection site – and squalene in water emulsion are the only adjuvants approved for human use. Mimicking and triggering a few alarm signals accompanying microbial infection, adjuvants induce a local inflammation and activate macrophage and dendritic cells to express costimulatory molecules and secrete pro-inflammatory cytokines. The risk lies in the ability of adjuvants to enhance the capacity of one antigen to elicit an immune response (antigen immunogenicity). Thus, adjuvants may overcome immune tolerance to self- antigens and induce autoimmunity. In fact, if you induce a too strong immune response, also autoreactive T and B cells may be activated and react against our molecules. In a few cases adjuvants may also elicit a systemic inflammation. - Combine ag with mannose to induce B cells activation - Combine ag with an antibody thus presenting it as an immunocomplex, since the latter can activate the Fcγ receptor on APCs enhancing phagocytosis - DNA expressing ag conjugated with CTLA-4 (which binds B7) or with chemokines that activate DC - Address the vaccine on a certain route of presentation (e.g., ag of human papilloma virus can be linked to signal peptide for lysosome membrane protein meaning that, when you administer the protein, it is directed to lysosome membrane and consequently presented through MHC class II) - ISCOM (MHC class I): if you want to induce presentation through MHCI Transport vectors: liposomes, virosomes SMAA: Solid Matrix complex – antibody – antigen. Considering two small vesicles, you can bind antibodies against T cells epitope and B cells epitope. When you administer these kinds of vesicles, you can obtain a simultaneous response by both T and B cells. ISCOM is the other strategy that can be used to drive the protein (the antigen) contained in this sort of liposome (it is not a real liposome). It will fuse with the plasma membrane and the protein entering the cytosol will undergo proteasome degradation and consequently MHCI presentation. 10