Have you ever gone to take out a screw only to find that you don’t have the right screwdriver bit? Now think of all the different bacteria and viruses as having these bit patterns exhibited on them, and our antibodies functioning as screw bits to prevent the screw heads from binding to their other cellular targets. Now, if we didn’t have the appropriate screw bit pattern, normally, we could only hope that our body would generate one on its own, or we have one that would work reasonably well. But what happens if we could make sure that we have the required pattern ready for when the time comes?
Hi folks, my name is Cole and I have a Master’s of Immunology. Today on Investigate Explore Discover, we’re going to be looking at single-step B-cell receptor modification. So hang around with me throughout this whole video to get all the relevant background information so that way we can dive into some exciting experimental results. But to make sure that we are all on the same page, we need to review some basic immunology concepts.
Our immune systems are made up of many different cells that can be categorized based on the role they play in defense against pathogens. The adaptive immune response plays a key role in immunization and elicits long lasting robust activity. This activity is primarily mediated by B cells and T cells. Now B cells function to attack invaders or foreign pathogens outside of the cells. Today, we’re talking about things happening throughout the entirety of B cell development.
B cells first developed in the bone marrow in an antigen independent fashion. This is where B cells first generate their specific B cell receptors. Now, the B cell receptor or BCR is an antibody or immunoglobulin precursor made up of two paired heavy and light chains. These chains are composed of two regions, the variable and constant region. The variable regions are at the ends of the receptor and create a binding space, functioning as the screwdriver bit that combines pathogenic antigens, the screw head patterns. Now, the variable regions of these receptors are very diverse.
The light chain is further made up of either a kappa or lambda chain, which joins together two distinct V and J regions, while the heavy chain joins together V, D and J regions to be complete. By combining these germline genes together, this creates a unique screw head and through a process called somatic hypermutation, these newly created receptors then increase their diversity through multiple point mutations and edits, giving a greater possibility to identify more pathogenic variants. Somatic hypermutation occurs in the germinal centers, and are assessed by antigen presenting cells. Germinal centers are found in the lymph nodes or in the spleen, and are subdivided into a few zones where specific maturation steps occur.
It is in the white zone that B cell receptors are tested for their ability to bind pathogenic antigens presented to them. The cells that have higher affinity for foreign antigen end up maturing and exiting the germinal center in the form of antibody producing plasma cells, or long-lived memory B cells.
In the periphery, when B cells encounter antigen, they present it to helper T cells, which then stimulate the B cells to produce more antibodies against an antigen that they present to the T cell in the first place. These antibodies function to neutralize the targeted antigen or screw head. These broadly neutralizing antibodies have significant clinical relevance as they indicate a functional immune response and passive transfer of them can prevent infection in animal models.
Now, around the world, approximately 38 million people are infected with HIV, and millions more are continually infected per year. HIV infects CD4 T cells with its spike protein complex on its envelope, and continually kills CD4 T cells over time if left untreated. HIV also has great antigenic diversity, making it difficult to universally vaccinate against. Thus there is an urgent need to create an effective broad acting vaccine. Like all retroviruses, HIV undergoes a life cycle of invasion, replication and release only to be repeated again. Vaccination results in the creation of pathogen-specific antibodies and finding immunogens that effectively accomplish this can be tricky. Thus, why we use animal models to validate findings before we test them in humans.
Now, finding what works to induce targeted antibodies that bind the envelope spike protein has proven to be difficult. But we currently know of a few broadly neutralizing antibody classes that could bind to the HIV spike protein like PGT121 and VRC01. Now, a major hurdle in making HIV vaccines is that the unmutated precursors for broadly neutralizing antibodies typically lack specificity for most envelope isolates. Germline targeting vaccines aimed to engineer immunogens that target unmutated broadly neutralizing antibody precursors, generating robust, effective antibody responses. This is beneficial because it reduces the uncertainty when using inferred germline sequences.
Currently, there is an effective germline targeting immunogen called EODGT8 that is known to stimulate the release of VRC01 antibodies. This immunogen is also currently undergoing a phase one safety trial. However, previous studies utilizing this immunogen did not address VRC01 class responses at physiologically relevant B cell frequencies. This can be overcome by using congenic adoptive transfer models. This method transfers cells from one animal into another to assess the cell function. Accurate identification of target cells in recipient animals is done by looking at targeted cell markers.
Now to generate these specifically mutated cells requires germline editing of mouse models. This germline DNA editing can be done reliably through CRISPR-Cas9. This method utilizes Guide RNA which directs the DNA splicing machinery, Cas9, to complementary DNA sequences. It is here that the targeted DNA is cut out and the desired programmed DNA is inserted for a functional mutation.
An issue with this method when using mice is that generating typical knock-in models expressing correct human B cell receptors requires many rounds of crossbreeding. And due to only being able to edit one gene at a time, this leads this process to be time-consuming, challenging work, and a faster method would be incredibly useful.
Now, I want to take a moment and really highlight why advancing models to enable easier research is so important. HIV is an antigenically diverse virus, and a broad acting vaccine is needed. Getting animals ready for these studies is an important but time-consuming, laborious process. Thus, the rapid generation of mouse models with immunoglobulin heavy and light chains knocked-in would allow generation of proper B cell receptor specificity and B cell responses under physiological conditions, giving us the ability to further investigate these treatments before giving them to humans.
This brings us to the paper that we’re focusing on today. This paper is called Multiplex CRISPR-Cas9-Mediated Engineering of Preclinical Mouse Models Bearing Native Human B cell Receptors – by Wang et al. from the Ragon Institute of Massachusetts General Hospital of MIT and Harvard University in Cambridge, Massachusetts, USA. In this paper, the authors assess the functional implications of using a rapidly acting protocol for inserting specific human B cell receptor pairs onto mouse B cells. Now to start out, the authors wanted to complement research that they’d already done. They had previously identified that by using CRISPR-Cas9, they could express pre-arranged human immunoglobulin or IG heavy chains in mice epi-native IgH chain locus. To complement this, they next wanted to see if it was possible to do this with the IG kappa chain, one of the light chain options.
Now, they first used the CRISPR design database to design specific Guide RNAs to efficiently cleave precise regions of the native mouse immunoglobulin kappa locus. They found two single Guide RNAs that fulfilled this condition and had minimal predicted off target effects. They then injected all of the components required for targeting and editing of the selected mouse germline DNA into fertilized mouse oocytes so that the insertion of the preassembled human antibody light chain for PGT121 could occur in the mouse germline DNA.
To test if this actually happened, though, the authors needed to look at the resulting mice that were born. From this experiment, there were 30 founder mice, of which 7 of them carried preassembled light chain mutations, but only 6 of them had intact wild-type complimentary IG kappa loci. Now, when looking at the B cell receptors of these mice, the authors identified that 90.5 of these mice had identical light chain sequences to be inserted PGT121 sequence and that these light chains were pairing up with native mouse heavy chains to form functional B cell receptors. To assess the stability of these edits, the authors crossed these founder mice with a wild type mouse. Even though the numbers were not large, 4 out of 7 founder mice transmitted the mutated preassembled light chain in a mendelian fashion. From this information the authors knew that they could successfully rapidly mutate the light and heavy chains of mouse BCRs independently. They next sought to find if they could modify both at the same time.
They tested this by editing CLK21, heavy and light chains. CLK21 is one of the native human germline VRC01 class BCRs, identified to generate broadly neutralizing antibodies to HIV. And when the authors injected all of the required components for DNA editing into fertilized mouse oocytes, this resulted in 14 founder mice being born, of which 4 of these mice expressed both the mutated heavy and light chains. To determine whether these inserted heavy and light chain sequences affect B cell development, the authors compared edited 2 wild type cells in the bone marrow and splenic germinal centers. And when looking at the B cell frequency, there were not any major differences, indicating that there was no major change of B cell maturation or development due to the inserted sequences.
Now, to get a further understanding of any possible changes, the authors also looked at the B cell repertoire in response to immunogens. It was observed that only these edited cells responded to stimulation with the EODGT8 probes, targeting VRC01 antibodies, thus showing the CLK21 mouse line displays antigen specific human B cell receptors in live mice.
Now, to assess the stability of these edits, the authors crossed these founder mice with a wild type mouse and found that 4 of the mice retained the desired mutations, indicating that the mutated germline sequence could be passed to offspring in a mendelian ratio. To convince themselves to the value and functionality of what they’ve just done, the authors repeated this procedure with other heavy and light chain pairs of additional VRC01 broadly neutralizing antibodies. They chose CLK09 and CLK19, which are germline BCRs that are capable of binding EODGT8. These other heavy and light chain mutations that the authors tested displayed similar results to the previously tested CLK21 in terms of B cell development and antigen specificity function. Since these three B cell receptors all bear functional EODGT8 tetramer binding, they wanted to verify that they could work in live animals. So they adopted and transferred these cells at varying low frequency into wild type mice. They determined that these cells were successfully transferred, albeit they had lower numbers than what were initially put in. They then tested the B cell responses of mice that were treated with EODGT8 immunogen and the authors found that at all levels tested, the mice that had human B cell receptors can be recruited to mouse germinal centers.
However, there was still more to look at in terms of how the B cells function after adoptive transfer. A time course experiment was used to identify how EODGT8 immunization affects the germinal center response kinetics, accumulation and somatic hypermutation of the edited cells. On day 8, these results showed that edited B cells are recruited to germinal centers. You can see here what this actually looks like. The red denotes the germinal centers and the white are the transplanted cells. Interestingly, CLK19 induced germinal center formation at a much lower frequency than other groups, perhaps due to its lower binding affinity to EODGT8. But by day 15, the percentage of B cells increased slightly from day 8, and by day 36, there were low but still detectable levels of the edited B cells in the germinal centers. Now, the authors also used a serum binding analysis to reveal their serum antibody responses from these cells. And at day 36, they also found that these edited B cells produced class-switch antibodies and could develop into memory B cells, indicating long-lived protection against the immunogen. It is interesting to note that all of these effects were magnitude dependent on the amount of cells that were initially transferred in.
Now, the authors also investigated whether these B cells undergo normal B cell somatic hypermutation. And when looking at the B cell receptor sequences after transfer, they found that the edited mouse B cells can undergo significant somatic hypermutation and accumulate broadly neutralizing antibody mutations after a single priming immunization, which are indicated by each of the branches on the evolutionary trees.
Now, to quickly summarize everything altogether, the authors of this paper found that they could co-inject fertilized zygotes with Cas9 and 2 donor plasmids to insert a predetermined heavy and light chain B cell receptor combination. They were able to successfully generate 3 combinations of germline antibody precursors for VRC01 antibodies, and after a single priming event with EODGT8 which is targeted to generate VRC01 antibodies, these edited B cells were able to be activated in the germinal centers. They were able to class which antibodies and mature into memory B cells and undergo somatic hypermutation, increasing the theoretical breadth of their neutralization capacity.
Now, not only do I think that these results are exciting to investigate and learn about, they’re also significant in a broader context. This information is significant because this protocol eliminates extra crosses between mice expressing heavy or light chains alone, saving lots of time. Now, these edited B cells allow for authentic vaccine response examination due to their normal B cell functions, which allows for further research to be done on germline targeting immunogen vaccines, giving us another possible avenue for eliminating HIV.
All science is basically a stepping stone for new knowledge. And these steps are driven by questions. And I had a few questions myself after reviewing this information. My first question revolves around influenza because it has such a high mutation rate. Can the type of approach used in this paper be used to create a broadly acting influenza vaccine negating the need for us to get a flu shot every year? The authors also found that they could modify two pieces of germline DNA. But how many more can they do at the same time? 3? 4? 5? And are B cells the only cell that can be edited this way? Or could T cell research also benefit from this rapid technique? As always, though, my final question revolves around you. What sort of ideas or questions popped into your head when hearing about this information? I would love to hear about them in the comment section below. Also, let me know if there are any topics that you’d like to hear about in the future.
Ultimately, I hope that you learned something. But more importantly, I hope that you enjoyed your time doing so. So if you did, give this video a like and subscribe for more in the future. Well, that’s everything for today. Thank you for watching, and I’ll see you next time.
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