Micro204 B cell actiavtion Flipped session organization 1:00 – 1:20pm – Questions about last year’s lecture (and replay of imaging videos from the lecture as needed) 1:20 – 1:50pm – Discuss the Immunity paper and associated questions. 1:50 – 2:15pm – Combine thinking from lecture and paper into thoughts about design of an Ebola vaccine. Break out into groups of 3-4 to consider in-class questions about how to design such a vaccine. 2:15 – 2:30 – Get back together as a group to go over each groups ideas on vaccine design.

Questions about the lecture?

Questions about the lecture What are the components of the BCR? What is BAFF? What signals are needed to maintain a mature B cell compartment? What is a key difference of a T-dependent antigen versus a T- independent one? Can B cells make a T-dependent antibody response against a non- protein antigen? What effect does CD40L deficiency have on the B cell response? Can you name some costimulatory signals for B cells? Can you name 3 things that are special about a plasma cell? What happens at the DNA level to the IgM constant region when the B cell undergoes isotype switch? Where does affinity maturation occur? What two processes are essential for affinity maturation to occur? What are 2 outputs of the germinal center?

Questions on Hou et al., 2011 Immunity 34, 375: 1. How does NP-Ficoll trigger an antibody response? Are there any costimulatory signals involved? 2. How does Myd88 in the DC augment the B cell response to OVA mixed with soluble CpG? 3. Linking CpG to OVA now reveals a partial role for Myd88 in the B cell. What type of effect is Myd88 having in the B cell that might be helping the antibody response? 4. What does a viral like particle (VLP) look like? Draw a rough diagram. 5. Most assays in the paper measure serum antibody by ELISA, but some (such as in Figure 5C) measure antibody secreting cell (plasma cell) numbers. Briefly explain (perhaps with a diagram) how these assays are performed and how they differ in what they are measuring. 6. Describe in simple terms what is being shown in Figure 5E. What effect is being revealed? 7. Draw a diagram, keeping it as simple as you can, containing a B cell, a DC and a T cell and showing where Myd88 is acting during the antibody response (1) to VLP mixed with soluble CpG and (2) to VLP containing CpG. The diagram should help explain why Myd88 function in the B cell is important only in context 2.

Ficoll Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Ficoll radii range from 2-7 nm. Activates the alternative pathway of complement

Antigen-C3d complexes cross-link BCR and CR2-CD19 -> increase sensitivity to antigen

VLPs (Q  phage)

Questions on Hou et al., 2011 Immunity 34, 375: 1. How does NP-Ficoll trigger an antibody response? Are there any costimulatory signals involved? (TI; Complement C3b/d-CR2) 2. How does Myd88 in the DC augment the B cell response to OVA mixed with soluble CpG? (Activate the DC via TLR9; increased costimulatory molecule expression likely augments helper T cell activation) 3. Linking CpG to OVA now reveals a partial role for Myd88 in the B cell. What type of effect is Myd88 having in the B cell that might be helping the antibody response? (Promoting costimulatory molecule expression and proliferation) 4. What does a viral like particle (VLP) look like? Draw a rough diagram. 5. Most assays in the paper measure serum antibody by ELISA, but some (such as in Figure 5C) measure antibody secreting cell (plasma cell) numbers. Briefly explain (perhaps with a diagram) how these assays are performed and how they differ in what they are measuring. (Diagram on board) 6. Describe in simple terms what is being shown in Figure 5E. What effect is being revealed? (Antigen binding B cells are being detected; a lack of expansion of these cells is shown in the Myd88 KO). 7. Draw a diagram, keeping it as simple as you can, containing a B cell, a DC and a T cell and showing where Myd88 is acting during the antibody response (1) to VLP mixed with soluble CpG and (2) to VLP containing CpG. The diagram should help explain why Myd88 function in the B cell is important only in context 2.

Ebola Virus EBOV carries a negative-sense ssRNA genome in virions that are cylindrical/tubular, and contain viral envelope glycoprotein (GP), matrix, and nucleocapsid (NP) components. The overall cylinders are ~80 nm in diameter, and having a GP projecting as 7-10 nm long spikes from its lipid bilayer surface. The cylinders are ~800 nm in length. Individual GP molecules appear with spacings of about 10 nm. Ebola is introduced into humans through contact with the blood, secretions, organs or other bodily fluids of infected animals. It spreads through human-to-human transmission via direct contact (through broken skin or mucous membranes) with the blood, secretions, organs or other bodily fluids of infected people, and with surfaces and materials (e.g. bedding, clothing) contaminated with these fluids.

What things might we think about in trying to design an Ebola vaccine that elicits a robust B cell response? In groups of 3-4, think over how you would design your vaccine. 1.Summarize what components it would contain and explain why you chose those components. 2.What cell types would you want it to engage and why? 3.What molecular pathways are you hoping it will trigger? Why? 4.What route of immunization will you favor and why?

Making an Ebola vaccine What have we learned from the paper that might influence vaccine design? 1. Glycoprotein with costimulatory agent (TLR ligand; C3b; ) 2. Make it one particle. Small enough to internalize but big enough to cross-link. Maybe a virus. 3. Properties of the sugar? How heavily glycosylated is the GP? Are Ab respones known to engage the protein. 4. Route of infection influences what isotype you might want to favor. If mucosal might want IgA versus systemic want IgG. 5.

Current state of Ebola vaccine development Inactivated Ebola virus vaccines were shown to not promote an adequate immune response to the real pathogen. Several promising vaccine candidates that integrate viral subunits have been shown to protect nonhuman primates (usually macaques) against lethal infection. These include: – replication-deficient adenovirus vectors – replication-competent vesicular stomatitis (VSV) – human parainfluenza (HPIV-3) vectors – virus-like particle preparations Phase I clinical trials involve the administration of the vaccine to healthy human subjects to evaluate the immune response, identify any side effects and determine the appropriate dosage. Phase I trials have been completed for the replication-competent VSV- EBOV vaccine and replication-deficient cAd3-EBO Z vaccine.

Adenoviruses (members of the family Adenoviridae) are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a dsDNA genome. cAd3-ZEBOV is an experimental vaccine for two ebolaviruses, Ebola virus and Sudan virus. This vaccine is derived from Chimp Adenovirus type 3 (ChAd3), genetically engineered to express glycoproteins from the Zaire and Sudan species of ebolavirus.

VSV member of the Rhabdoviridae family negative sense RNA genome enveloped virus with a surface glycoprotein (G)