Live-cell imaging in vaccine research: taking your 2D cell culture models to another dimension

Welcome to the first of a two-part series on the use of live-cell imaging in vaccine research. The initial article provides a succinct review of history and immunology as they relate to the development of vaccines, while the second will highlight state-of-the-art techniques in live-cell imaging and 3D cell culture models that are used to make vaccine research faster, more reproducible, and cost-effective. By the end of this series, you will have a solid foundation on the prospects of generating innovative, cutting-edge vaccines.


A brief history of vaccines

Immunization by vaccination is widely recognized as one of the world’s greatest public health achievements. Historical evidence suggests that early forms of vaccination were used since at least 1000 AD in China, India, Turkey, and likely other areas1. However, English country doctor Edward Jenner is often credited for performing the world’s first vaccination in 17962. Jenner inoculated an eight-year-old boy with pus from a cowpox lesion on a milkmaid’s hand and discovered that the boy was unaffected by smallpox even after multiple exposures. Nearly 100 years later in 1879, French chemist and microbiologist Louis Pasteur spearheaded central principles in modern vaccinology, including germ theory and the use of attenuated pathogens for immunization. These events laid the foundation for what we consider traditional vaccines – a “suspension of live (usually attenuated) or inactivated microorganisms (e.g., bacteria or viruses) or fractions thereof administered to induce immunity and prevent infectious disease or its sequelae”3. In the decades to follow, scientists from all over the globe produced additional vaccines against some of the world’s deadliest diseases, including diphtheria (1923), tuberculosis (1927), tetanus (1937), polio (1955), and measles (1963)4.

There is no doubt that vaccines have saved millions of lives since their inception, nonetheless the exact number is difficult to quantify. The smallpox vaccination alone is estimated to have saved roughly 150 to 200 million lives between 1980 and 20185. Similarly, in 2021, a study assessing disease burden estimated that for 10 major pathogens, including measles, hepatitis B virus, Haemophilus influenzae type B, and others, corresponding vaccines will have prevented 69 million deaths between 2000 and 2030, mostly in children under the age of five6. Although many diseases are nearly eliminated because of vaccines, their eradication has proven to be challenging. Only two diseases have been completely eradicated to date: smallpox and rinderpest7, largely due to both stringent surveillance and heavy vaccination efforts. There are two necessary conditions for eradication 1) the disease must be infectious and 2) measures to fight the disease must exist8. However, if the agent cannot be easily diagnosed, has a non-human reservoir, or has inconsistent community support, as was the case with the recent novel coronavirus, SARS-Cov29, this can seem almost impossible. Today, there are several classes of vaccines available, including whole pathogen vaccines, subunit vaccines, nucleic acid vaccines, and viral-vectored vaccines, all of which have the same core purpose: to stimulate the body’s defenses and provide protection against one or more diseases. Figure 1, taken from Andrew J. Pullard and Else M. Bijker10, gives an excellent overview of the various vaccination technologies currently in use or being developed.

A list of the various types of vaccines currently in use or being developed

Figure 1 | A list of the various types of vaccines currently in use or being developed. Some experimental vaccines, such as antigen-presenting cell vaccines, are still being developed for human applications. Figure obtained from Andrew J. Pullard and Else M. Bijker10.


A brief refresher on immunology

To select the right model system for your vaccine research, it is essential to understand how the adaptive immune system works. In the past, most vaccines were designed and tested empirically without a prior understanding of how they elicited long-term immunity11. This was often a long and inefficient process. Rational vaccine design, on the other hand, is much more efficient since the immune correlates of protection are known a priori. However, the researcher must experimentally define the immune response that a vaccine would need to trigger in order to confer protection against an infectious agent.

The human body has numerous ways to protect itself against pathogens via the immune system, which is made up of two parts: the innate immune system and the adaptive immune system. The innate immune system provides immediate protection against microbial invasion and is non-specific, while adaptive immunity is specific and results in immunologic memory after a primary infection. Consequently, when a host encounters the same pathogen after initial infection, the immune system mounts a potent secondary immune response that is typically much faster and more efficient in clearing the pathogen upon subsequent exposure.

Vaccines prime the adaptive immune system against a weakened or inactive form of the whole pathogen or derivative antigenic fragments without the host acquiring the disease. This results in immunologic memory and a rapid secondary immune response when the host encounters the actual pathogen.


An introduction to adaptive immunity

The adaptive immune system consists of cell-mediated responses and humoral-mediated responses. Cell-mediated responses that kill infected or cancerous host cells are primarily directed by cytotoxic T lymphocytes and are particularly important for killing cells infected with viruses such as the novel coronavirus, SARS-Cov2. Helper T lymphocytes are arguably the most important immune cells, since they are needed for virtually all adaptive immune responses12. They stimulate B lymphocytes to start producing antibodies, and direct macrophages and neutrophils to destroy pathogens. They can even stimulate the activity of cytotoxic T lymphocytes through the release of important signaling molecules called cytokines12. The activation of both types of T lymphocytes is similar but differs slightly. T helper lymphocyte activation begins when a pathogen (such as a bacterium, virus, or fungus) first enters the host, and tissue-resident professional antigen-presenting cells, such as dendritic cells (DCs), digest the pathogen and display the resulting antigens on their cell surface using a structure called the Major Histocompatibility Complex class II (MHC-II). Antigens can also come from an administered vaccine, but the process of uptake by antigen-presenting cells is basically the same. Typically, after maturing and migrating to a lymph node, the DCs stimulate the activation of naïve T helper lymphocytes by recognition of the antigen-loaded MHC-II structure on the DC membrane surface, and through the binding of co-receptors and co-stimulators. Binding of the antigen alone is not enough to promote T cell activation. It also requires important signals released by DCs and other immune cells, called cytokines. Figure 2 provides an overview of the process.

A diagram illustrating the capture, transport, and presentation of antigens by dendritic cells

Figure 2 | A diagram illustrating the capture, transport, and presentation of antigens by dendritic cells (DCs). After foreign antigens (either from a pathogen or a vaccine) are taken up by the DC, it displays the antigen payload on the cell surface using a major histocompatibility complex type II molecule. Presentation of the antigen, along with other various cofactors, stimulates the activation of T lymphocytes. Figure obtained from Basic Immunology, by Abul K. Abbas13.

In the case of cytotoxic T lymphocyte activation, antigen presentation to the naïve cytotoxic T cell happens in almost the same way as it does for helper T lymphocyte activation, except it is done using a slightly different structure called the Major Histocompatibility Complex class I (MHC-I) in a process called antigen cross-presentation. Typically, these antigens are derived from cancerous or infected cells, which are taken up by dendritic cells. After a cytotoxic T lymphocyte is activated, it can begin killing infected cells. Cell killing begins when an activated cytotoxic T lymphocyte encounters an infected or cancerous cell that is presenting specific antigen-bound Major Histocompatibility Complex class I (MHC-I) proteins on its surface. The antigens can either be derived from tumor proteins if it is a cancer cell, or pieces of viral, bacterial, or fungal proteins or polysaccharides if it is an infected cell. The compromised cell presents these antigens on its own surface to tell the cytotoxic T cell that it needs to be killed. Each activated cytotoxic T lymphocyte has a unique T cell receptor that can bind a particular antigen. If it recognizes the antigen, in combination with other important co-stimulatory receptors and molecules, including cytokines, it triggers apoptosis of the infected or cancerous cell.

Helper T lymphocytes also play an important role in cell-mediated immune responses by directing other immune cells to kill pathogens or aid in wound healing, and they have different subtypes depending on their function. For instance, Th1 helper lymphocytes recruit macrophages to digest and kill pathogens, while Th17 helper lymphocytes promote inflammation and recruit neutrophils to sites of infection, and are especially important in combating extracellular bacterial and fungal infections14,15. The roles of both helper and cytotoxic T lymphocytes in cell-mediated immunity are summarized in Figure 3 below.

A summary of the various cell-mediated immune responses orchestrated by T lymphocytes

Figure 3 | A summary of the various cell-mediated immune responses orchestrated by T lymphocytes. Figure obtained from Basic Immunology, by Abul K. Abbas13.

In contrast to cell-mediated immune responses, which typically eliminate intracellular pathogens, such as viruses and parasites, humoral-mediated immune responses mainly neutralize extracellular or freely circulating pathogens, and are primarily directed by antibody-secreting B lymphocytes. Each B lymphocyte, through genetic recombination, has its own membrane-bound antibodies with unique variable portions. When the antibody produced by a B lymphocyte binds to an antigen, it can activate the cell to start producing target-specific antibodies, but in most cases, a strong response requires additional stimulation by an activated T helper lymphocyte. Before this happens, the B lymphocyte must encounter the antigen either in the lymph tissues or in the systemic circulatory system. It imports the antibody-bound antigen, digests it, and displays it on the cell surface using its MHC-II complex in a similar manner to DCs and other professional antigen-presenting cells. When an activated helper T lymphocyte recognizes the same antigen displayed on the B lymphocyte’s MHC-II complex, it activates the B lymphocyte via the release of signaling molecules called cytokines. The B lymphocyte begins rapidly differentiating and cloning itself into antibody-producing cells, called plasma cells. However, not all B lymphocytes will differentiate into plasma cells. Some remain in the body as long-lived memory cells, which will rapidly proliferate and produce antibodies if the host re-encounters the pathogen. Both the T cell-dependent and T cell-independent activation of B lymphocytes are illustrated in Figure 4.

Examples of T lymphocyte dependent and independent activation of B lymphocytes

Figure 4 | Examples of T lymphocyte dependent and independent activation of B lymphocytes. Figure obtained from Basic Immunology, by Abul K. Abbas13.

In addition to stimulating B lymphocytes to produce antibodies, T lymphocytes can also induce B lymphocytes to undergo antibody class switching, depending upon the type of cytokines that are released by the T lymphocyte. Different antibody types perform different functions. Figure 5 gives a summary of the different antibody classes which are possible, and their principle functions in humoral-mediated immunity. In general, IgG antibodies help defend against extracellular bacteria or viruses by coating the pathogen and marking it for destruction by macrophages and neutrophils, which have receptors for the constant region of the IgG antibody. IgA antibodies are primarily involved in mucosal immunity and are the first line of defense against viruses like SARS-Cov2. Their primary role is to directly neutralize microbes, viruses, and toxins, usually by preventing them from adhering to cell surfaces, or facilitating IgA receptor-mediated phagocytosis or killing of pathogens. Interestingly, a recent study in 2022 by Sheikh-Mohamed et al. found that individuals who experienced breakthrough infections with SARS-Cov2 despite being vaccinated had lower concentrations of virus-specific IgA antibodies 2-4 weeks post vaccination16, suggesting that IgA plays an important role in long-term immunity against the virus.

An illustration of antibody class switching of B lymphocytes in response to activation by T lymphocytes

Figure 5 | An illustration of antibody class switching of B lymphocytes in response to activation by T lymphocytes. Figure obtained from Basic Immunology, by Abul K. Abbas13.

As shown in Figure 5, the antibodies produced by plasma cells can neutralize pathogens in a variety of ways. They can bind to certain antigens and block their activity (neutralization), prevent their movement, form antigen-antibody complexes which are destroyed by phagocytes and other immune cells (agglutination), or they can activate the complement pathway (complement activation), which leads to the formation of a membrane attack complex that punctures the cell membrane of the pathogen and leads to its death. Knowing which type of antibodies are produced post-vaccination can help you assess the efficacy of your vaccine. For example, since IgG antibodies are generally longer-lived than IgM, their presence is usually an indication of long-term immunity, as opposed to IgM, which is usually short-lived and less efficient at neutralizing pathogens17.

In the next article, I will cover the technical and economic challenges of vaccine development and familiarize you with the state-of-the-art techniques in the field of vaccine research. The article will explore how live-cell imaging, in combination with in vitro and ex vivo cell culture models, can rapidly advance your vaccine research while saving you time and money.

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