Embedded Files
You may click on the link below to access the paper. Make sure to also check its supplemental material.
B. J. Ward, P. Gobeil, A. Seguin, J. Atkins, I. Boulay, P. Y. Charbonneau, et al.
Nat Med 2021 Vol. 27 Issue 6 Pages 1071-1078 Accession Number: 34007070 PMCID: PMC8205852 DOI: 10.1038/s41591-021-01370-1
https://www.ncbi.nlm.nih.gov/pubmed/34007070
This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Overexpressing spike proteins using the transient expression method in tobacco is an effective strategy to produce virus-like particles, paving the way for potential vaccines. The second paper, published in Nature Medicine (Show the paper first page on slide), explored this in an FDA Phase 1 clinical trial. This initial phase aims to evaluate the vaccine’s safety, dosage, and potential side effects on a select group of volunteers.
Being a clinical trial, the researchers made several bioengineering modifications to enhance spike protein expression and stability. As displayed in the figure ((Pointing to Figure in Slide), this is the N-terminus (Pointing to Figure in Slide) and this is the C-terminus of the spike protein (Pointing to Figure in Slide). The authors altered the signal peptide at the N-terminus, directing the expressed protein to the endoplasmic reticulum in the cells. They also performed point mutations to mutate the furin protease recognition site to prevent S1/S2 cleavage (Point to the Figure on Slide), and incorporated additional mutations to improve VLP assembly and budding (Point to the Figure on Slide). They also used the electron microscopy to show the purified CoVLPs (Point to the figure on slide).
In this second study, the researchers clinically tested the efficacy of a tobacco-produced COVID vaccine. Following FDA guidelines, they conducted the trial in an observer-blinded, randomized controlled and dose escalating manner to assess the vaccine's safety, efficacy, and immune response.
The trial started by July 2020, and examined three dosages of virus-like particle COVID vaccines: 3.75 ug, 7.5 ug, and 15 ug. Two adjuvants, AS03 and CpG1018, were also included in the test to enhance the vaccine's effectiveness and longevity. (Point to the Slide) AS03 is an oil-in-water emulsion composed of squalene, di-alpha-tocopherol, and a surfactant, while CpG1018 is a short DNA molecule that stimulates the immune system. Hence, with three dosage levels, 3.75ug, 7.5ug and 15ug, and three adjuvant options, no adjuvant, AS03 and CpG1018), in total nine different groups were formed.
(Point to slide, a Canada map) The study recruited 180 Canadian participants, aged between 18-65, from two cities Montreal and Quebec. With these volunteers, they considered female/male ratio, Race groups, Ethinicity and Age groups. Therefore, 180 people, 9 research groups, this resulted in exactly 20 people per group. Each participant received two intramuscular doses 21 days apart, and blood samples were collected for further examination.
Figure 3
The researchers used the blood samples from nine groups to test the immune response to the CoVLP vaccine. (Fig. 3a, Y axis) They measured levels of Immunoglobulin G (IgG), which targets the Spike proteins. A higher IgG level indicates a stronger immune response.
(Fig 3a X-axis) You could see the three dosage levels of the vaccine: 3.75ug, 7.5ug, and 15ug. Each dose level also has three groups: those without an adjuvant, shown as in green, those with the AS03 adjuvant, shown as in red, and those with CpG1080 adjuvant, shown as in blue. The IgG levels were measured at three points: Day 0 at the start date, Day 21 after injecting the first dose, and Day 42 after injecting the second dose.
For example, (Fig. 3a) in the 3.75ug dose group, the green group who only received CoVLPs without adjuvants showed a slight increase in IgG levels from Day 0 to Day 21, then a 6-7 times increase at Day 42. While the red and blue groups who received the same dose of CoVLPs but with adjuvants had a similar starting IgG levels to the green group on Day 0, but showed a dramatic increase in IgG levels for later days, especially the red group with AS03 adjuvant. It had about a 70 times increase from Day 0 to Day 21, and another 68 times increase from Day 21 to Day 42.
The same trend was observed in the other two dosage groups, suggesting that, first, the adjuvants work and are needed with these COVID vaccines, second, it looks like that the 3.75ug dose with AS03 adjuvant is the most effective vaccine option.
(Figs. 3b and c) The authors also utilized two other assays to measure the humoral responses: the Pseudovirus neutralizing antibody titers and the live virus neutralizing antibody titers. In both assays, a higher value indicates a more effective antibody. All data suggest that the 3.75ug dosage level with AS03 adjuvant is the most effective combination.
In Figure 3a, in the context of a SARS-CoV-2 anti-spike IgG titer, the "anti-spike IgG value" refers to the level of Immunoglobulin G (IgG) antibodies in the blood that are specific for the SARS-CoV-2 spike protein. If a person has a high anti-spike IgG value, it typically means their immune system has produced a substantial number of antibodies against the SARS-CoV-2 spike protein.
However, it's important to note that while a high level of anti-spike IgG usually suggests some degree of immune protection against SARS-CoV-2, it doesn't guarantee complete immunity. Other factors also play crucial roles in immune defense, including T cell responses and the presence of other types of antibodies. It is also still under research how exactly these antibody levels correlate with immunity or protection against COVID-19.
In Figure 3b, researchers utilized a pseudovirus neutralizing antibody (PsVNA) assay to measure the ability of antibodies to prevent a "pseudovirus" from entering cells. A pseudovirus is a viral particle that has been engineered to express a different virus's protein on its surface, which allows researchers to safely study dangerous viruses under less stringent biosafety level conditions. In this case of SARS-CoV-2, the virus that causes COVID-19, pseudoviruses often express the SARS-CoV-2 spike protein.
In the Y-axis, the "PsVNA50" value refers to the dilution of serum (the component of blood that contains antibodies) at which 50% of the pseudovirus is neutralized. For example, if a PsVNA50 titer is 1:640, it means that the serum can be diluted 640 times and still inhibit 50% of the pseudovirus particles. So a higher titers indicate a greater concentration of neutralizing antibodies in the serum, and thus, usually signify a stronger immune response.
In Figure 3c, the Y-axis is PRNT50 stands for Plaque Reduction Neutralization Test (50%). It's a measure used in virology to determine the concentration of antibodies that can neutralize a virus and prevent it from forming plaques (areas of infection) in cell cultures. It's often used as a measure of immune response to a vaccine or as a way of confirming a past infection. The "50" in PRNT50 refers to the dilution of serum (the component of blood that contains antibodies) that results in a 50% reduction in plaque formation. For example, if a PRNT50 titer is reported as 1:160, that means that the serum could be diluted 160 times and still reduce the number of plaques formed by the virus by 50%. In general, higher PRNT50 titers indicate a higher concentration of neutralizing antibodies, which suggests a stronger immune response.
Q: Figs. 3b and 3c tested Neutralizing Antibodies, what is the diffrence between "Antibodies" and "Neutralizing Antibodies"?
A: "Antibodies" is a broad term referring to a class of proteins produced by B cells (a type of white blood cell) in the body's immune system in response to foreign invaders, such as bacteria, viruses, or other foreign substances, collectively called antigens. Once produced, antibodies can recognize, bind to, and help neutralize or destroy these invaders. On the other hand, "neutralizing antibodies" is a more specific term that refers to a subset of antibodies that directly defend against infectious agents, particularly viruses. Neutralizing antibodies work by binding to specific parts of a virus, typically the parts that a virus uses to enter a host cell (like the spike protein on the coronavirus, for instance). By binding to these areas, neutralizing antibodies can block the virus's ability to infect host cells.
Therefore, all neutralizing antibodies are antibodies, but not all antibodies are neutralizing antibodies. Some antibodies don't neutralize the pathogen directly but instead tag it for destruction by other immune cells or help in other ways, like complement activation or stimulating inflammation.
In the context of disease and vaccines, much focus is often given to the level of neutralizing antibodies, because these are often a key part of preventing infection or reducing the severity of disease. However, it's important to note that they are just one part of a complex immune response.
Figure 4.
In the preceding analysis, the authors evaluated the humoral immunity responses of nine different groups. Humoral immunity, which generates antibodies, is a crucial component of adaptive immunity. Its counterpart, cellular immunity, doesn't directly produce antibodies but still plays a significant role in the comprehensive immune response, including antibody production.
To illustrate this, the authors utilized two assays to assess and compare the cellular immunity responses across the nine research groups. They focused on two types of immune responses: the interferon (IFN)-gamma and interleukin (IL)-4 responses. (Zoom in to Fig. 4a) The first assay involves specific immune cells that produce the cytokine IFN-gamma to regulate immune defense, while the latter includes immune cells that produce the cytokine IL-4, central to humoral and allergic responses.
In Fig. 4a, IFN-gamma (Interferon gamma) is a type of cytokine, a small protein released by cells that has a specific effect on the interactions between cells, communications between cells or the behavior of cells. IFN-gamma plays a crucial role in mediating the immune response, particularly against intracellular pathogens such as viruses. In an IFN-gamma cellular immune response assay, researchers measure the production of IFN-gamma as a marker of immune cell activation in response to a specific stimulus, like a pathogen or a vaccine.
Please let me explain this figure. The X-axis is the same as in the previous figure. While in the Y-axis, "SFC" stands for Spot Forming Cells. These are the individual cells that are producing IFN-gamma, as visualized on the assay plate. Each spot corresponds to one cell that's producing the cytokine. And the "PBMCs" refers to Peripheral Blood Mononuclear Cells, a type of blood cells that includes lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. These cells are crucial for immune function. So the "IFN-gamma SFC per million PBMCs" provides a specific quantification of the immune response. For each million PBMCs, how many cells are actively producing IFN-gamma in response to the stimulus? This can give researchers an indication of how robust the immune response is. Higher numbers would typically indicate a stronger cellular immune response.
In Fig. 4b, IL-4 (Interleukin 4) is a type of cytokine that plays a significant role in regulating immune responses, specifically by stimulating the differentiation of naive helper T cells (Th0 cells) to Th2 cells. The Th2 cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 is particularly important in the response against extracellular parasites and is also crucial in promoting allergic reactions. In an IL-4 cellular immune response assay, the production of IL-4 is measured as an indication of immune cell activation in response to a specific stimulus, like a pathogen or a vaccine. "SFC" and "PBMCs" are the same in Fig. 4a. So, "IL-4 SFC per million PBMCs" provides a specific quantification of the immune response. It signifies the number of cells actively producing IL-4 per million PBMCs in response to the stimulus. This measure can help researchers understand the magnitude of the immune response, with higher numbers typically indicating a more robust cellular immune response.
Q: In Paper II, multiple terms related to the immune system were mentioned, such as "Innate Immunity", "Adaptive Immunity", "Humoral Immunity" and "Cellular Immunity", what are the relationships among these "Immunity" terms?
A: The immune system is traditionally divided into two main branches: the innate immune system and the adaptive immune system.
The innate immune system is the body's first line of defense against infections. It includes physical barriers (like skin and mucous membranes), chemical barriers (like stomach acid and antimicrobial peptides), and a variety of immune cells (like macrophages and neutrophils) that can respond quickly to infections but in a generic, non-specific way. The innate immune system does not have memory of previous infections.
The adaptive immune system, on the other hand, is more specialized and tailored to individual pathogens. It develops over time and adapts to the specific pathogens that the body encounters. This system is capable of "remembering" pathogens, so it can provide faster and stronger responses to repeat infections.
The adaptive immune system itself is further divided into two branches: the humoral immune response and the cellular immune response.
1. Humoral Immunity: This is mediated by B cells and the antibodies they produce, particularly Immunoglobulin G (IgG). It's called "humoral" because it involves components that are present in the "humors," or body fluids. The primary function of this branch is to neutralize and eliminate pathogens outside cells (extracellular pathogens), such as bacteria and viruses in the bloodstream or tissue fluids.
2. Cellular Immunity: This is mediated by T cells, which include helper T cells and cytotoxic T cells. It's named "cellular" because it involves the activation of cells to combat pathogens, particularly those that hide inside the host's cells (intracellular pathogens), such as viruses and some bacteria. Helper T cells release cytokines to regulate or assist in the active immune response, while cytotoxic T cells directly destroy infected cells.
Q: What is the differences between Humoral Responses to Vaccines and Cellular Responses to Vaccines?
A: In this Paper II, the authors tested Humoral Responses in Fig. 3 and further tested Cellular Responses in Fig. 4. Vaccines aim to stimulate the immune system to recognize and combat pathogens, particularly viruses and bacteria. They often aim to induce both humoral and cellular immune responses, as both are important for optimal protection. Here's a look at how they differ:
1. Humoral Response to Vaccines: The humoral response is primarily associated with the production of antibodies by B cells. When a vaccine is administered, it contains antigens (either in the form of inactivated/killed pathogens, parts of the pathogen such as proteins, or a harmless vector carrying the pathogen's genetic material). These antigens trigger the immune system, specifically activating the appropriate B cells. These B cells proliferate and differentiate into plasma cells, which produce antibodies specifically designed to bind to the antigen. These antibodies can neutralize the pathogen, preventing it from infecting cells, and can also 'tag' pathogens for destruction by other immune cells. In addition to plasma cells, memory B cells are also formed and can mount a quick response upon re-exposure to the same antigen.
2. Cellular Response to Vaccines: The cellular immune response involves T cells, specifically cytotoxic T cells and helper T cells. Cytotoxic T cells kill infected cells directly, preventing pathogens from using those cells to reproduce. Helper T cells produce cytokines that stimulate other immune cells, including B cells and cytotoxic T cells. Vaccines help to stimulate this response by presenting antigens to these T cells, causing them to activate and proliferate. Some of these will become memory T cells, which can respond rapidly upon future exposure to the same pathogen.
So now, you may have a basic understanding why the authors performd assays in Figs. 3 and 4 to test the adaptive immune responses to the CoVLP vaccines. It's important to note that the effectiveness of the humoral and cellular responses can depend on the type of vaccine and the pathogen it's designed to protect against. For some pathogens, a strong humoral response is sufficient for protection. For others, particularly those that hide inside cells (like viruses or some types of bacteria), a strong cellular response is also necessary. Therefore, modern vaccine design often aims to induce both humoral and cellular immunity for optimal protection.
Page updated
Report abuse