The SARS-CoV-2 vaccines. Part 1: The mechanisms of vaccines

Is the end of the pandemic near? Is SARS-CoV-2, the same one that has shown how fragile the egomaniacal Homo sapiens and his civilization are, giving its last breath? Will the longed-for vaccines end with this science-fiction situation? The vaccines are being presented as the definitive solution to our problems, the definitive weapon that will return us to the “old” normality. Is it true? Will they be able to achieve the long-awaited herd immunity worldwide? It is possible, but one thing must be clear: it will not be immediately. There is still much we don’t know about these potential remedies, in part because we haven’t yet completed the biography of SARS-CoV-2. This uncertainty feeds fear and anxiety in many people and generates emotional fractures that many take advantage of to spread fear, lies, hoaxes, and the marketing of vain hopes. Amidst so much confusion and idiocy, we want to throw a glimmer of clarity with a couple of articles in which we will dispel doubts about vaccines. Because, many times, fear comes from ignorance. We will talk about their types, the phases they must go through to be approved, the functioning of that marvel of nature that is our immune system, the vaccine candidates that will try to nullify the pandemic and its mechanisms of action, and many other things. Let’s get to it…

To better understand the goals of vaccines and their mode of action, we need to take a synthesized look at how our immune system works, a fundamental and complex component of our organisms that evolution has adapted and modified over millions of years to protect us from external and internal hazards.

After all this time of tedious pandemic, many of you will have heard of innate and adaptive immune systems or non-specific and specific immune systems. These are the broad categories into which our defense system and that of many other animal groups can be divided. These categories form a complementary whole that, acting collaboratively, protect us against infections and intrinsic dangers, such as cancer (the immune system inhibits thousands of potentially dangerous cancer cells every day, and we don’t even know it). However, as in all areas, the immune system is not perfect and can sometimes fail.

We must consider two issues: for our defense system to respond adequately against threats it must meet two requirements, namely:

  • Distinguishing the proper from the strange.

  • Distinguishing between a harmless element and a dangerous one.

If, for example, the immune system becomes confused and considers a harmless element of its own as threatening, the person will suffer from an autoimmune disease, such as lupus. If it considers a foreign body that is actually harmless as dangerous, such as pollen or dust, an allergenic response is triggered. On the other hand, if it does not detect a threatening body such as a pathogen intensely enough, an immunodeficiency is produced. These are all failures that the immune system can sometimes make and whose prevalence is increased by unhealthy lifestyles and pollution.

Okay, but when it works properly, how does the immune system protect us from the health hazards to which we are constantly exposed? Since the context of this article is the SARS-CoV-2 pandemic, we will discuss how our defense system behaves in the case of a viral infection.

When a pathogenic virus (or any other infectious agent) tries to invade the body, the first thing that acts is the innate immune system, characterized by triggering a rapid and non-specific response. It does not care about the type of pathogen, it always executes similar responses to all of them. Do not think that this system is activated once the virus has already penetrated the body. The skin or the mucous membranes, in fact, are part of the innate immune system since they function as the first barriers against the entry of pathogens. The muco-cutaneous barrier, that is, the one constituted by the skin and the mucous membranes, is non-specific, since it hinders the entry of any foreign body regardless of its nature.

However, as we go deeper into our bodies, other elements of the innate immune system appear, organized in a kind of contiguous walls. We have, for example, a group of cells composed of phagocytes (the macrophage being the main protagonist of this group) and dendritic cells among others that are literally in charge of gobbling up and digesting those suspicious agents that have penetrated the body. These types of cells are widely distributed throughout the body, but they also form a barricade immediately under the skin, so that they thicken and reinforce that first physical barrier. Other cells to highlight are the natural killer cells (NK), whose function is to “kill” and eliminate the partners that have been infected by the pathogens.

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Phagocytes are part of the innate immune system because they execute very similar responses regardless of the type of pathogen. Eosinofilos

Another essential element is the complement system, a set of plasma proteins that are born in the liver and that stimulate and complement the immune response. They are constantly fused and broken down to form different complexes with different functions: they excite the inflammatory process; they opsonize (i.e., cover) the surface of pathogens either to signal them or to destroy them by generating pores in their cell membranes or capsules (in the case of viruses); they generate chemotaxis, i.e., they emit chemical signals that attract leukocytes to the site where the pathogens are located…

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Some protein subunits of the complement system are responsible for directly destroying the pathogen. To do this, they pierce its cell membrane (in the case of bacteria) or its capsid (in the case of viruses) to destabilize and neutralize them. Wikipedia

We cannot ignore the other essential component of the non-specific immune response: the inflammation. Although annoying and painful, inflammation is a safety measure that prevents infections from going any further. Through different vascular modifications, the organism tries to attack and eliminate or, in its defect, to isolate the infected region and to recover the tissues damaged by the infection. Broadly speaking, what happens during the inflammatory phenomenon is the vasodilation of blood vessels near the infected area and the permeability of capillaries. This favors the filtration of blood plasma with the phagocytes and white blood cells that are immersed in it into the infected area to remove the pathogens. All these physiological reactions are stimulated by chemicals that synthesize various cell types. All these elements of the immune system, from phagocytes to inflammation, are interconnected and collaborate together during an immune response.

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The inflammation, although painful, is a physiological phenomenon that defends us against infections. Mi sistema inmune

Although we have already given some examples, it is clear that without its arsenal of chemical weaponry, the innate immune system would be defenseless. This division of the immune system (present in both the innate and adaptive systems) is known as the humoral immune system, and is made up of macromolecules, such as antibodies/immunoglobulins. Its counterpart would be the cellular immune system, composed of leukocytes or white blood cells, phagocytes and other cellular types.

The most obvious humoral defense of the innate immune system is mucus. Yes, nasal mucus is very annoying and uncomfortable, especially when there is an excess during a cold, but it is very important to deal with pathogens that colonize the body through the nasopharyngeal mucosa. Mucus hosts a large amount and diversity of antimicrobial enzymes that inhibit and destroy pathogens, but there is also an antibody or immunoglobulin A. These are capable of neutralizing the pathogen. How? By binding to the surface proteins it uses to contact the host cells. It is as if a key is added to an element that prevents its proper entry into the lock. The mucus in the airways also acts as a physical barrier that prevents contact between pathogens and cells. It also helps in the mechanical expulsion of pathogens: the mucus and the pathogens that are immersed in it are pushed out thanks to the movement of the cilia of the cells of the nasal epithelium.

We mentioned earlier a set of cells that have a voracious appetite for foreign particles entering the body. Well, these cells, and specifically dendritic cells, constitute the bridge between the innate and adaptive immune systems. When dendritic cells digest, for example, a virus (which happens in compartments called vesicles or phagosomes, which later fuse with another organelle, the lysosomes, which contain enzymes capable of digesting and hydrolyzing the foreign particle), they do not do so completely. Let’s say that they degrade it into smaller subunits of protein or peptide nature.

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This tentacle cell is one of the cells responsible for triggering the specific immune system. Dreamstime

These protein crumbs are installed by the dendritic cell on their own membrane surface. To do this, cells manufacture from a family of genes, known as the major histocompatibility complex (MHC), homonymous glycoproteins that will bind to the degraded viral peptides. All this complex will be arranged in the external face of the cell, inserted in the cell membrane. What the MHC molecules present are really the famous antigens, that is, the molecules that, after being recognized and studied by the immune system, trigger the immune response. For this reason, these cells of the innate immune system are also known as antigen-presenting cells (APCs). And to whom are they presented? To their adaptive immune system colleagues, the lymphocytes. Dendritic cells enter the bloodstream and travel to the secondary lymphoid organs, where the lymphocytes are waiting.

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There are two types of proteins synthesized from the genes of the major histocompatibility complex that are responsible for presenting the antigenic peptides (which are located in the groove at the top) to the lymphocytes. Ciencia y salud

We have a labyrinthine diversity of types and subtypes of lymphocytes. The two best known groups are T and B lymphocytes, which in turn are subdivided into several families. T cells have a problem: they cannot recognize the pathogen in its native state, i.e. complete (whereas B cells can). They need it processed and digested. Therefore they require the invaluable work of phagocytes and dendritic cells. With their surface proteins as sensors, they are able to recognize the MHC antigen-molecule complex that these phagocytes have arranged on their surface. This is how the adaptive immune system awakens, characterized by triggering a very specific and slow response, at least at the first contact. Unlike the innate system, which begins to develop in the fetal stage of each individual (hence its name), the adaptive system matures and develops a profile as it is exposed to the different particles that surround us throughout our lives.

When we say that the lymphocytes are activated, we are really talking about a series of induced genetic changes that these cells undergo and which generate many modifications in them. Firstly, the lymphocytes, which are undifferentiated, that is, they do not have a specific function, suffer a differentiation and mature into some type of specific lymphocyte with very specific functions. These already mature lymphocytes, with an established “personality”, begin to multiply clonally to form the army of the cellular adaptive immune system. Furthermore, these mature cells have their main objectives written in their genetic code: they know which cells they have to communicate with, what types of cytokines and antibodies they have to produce, where they have to go, etc. This is what the specific response of the immune system consists of, and building it is a slow process that sometimes takes several days. The adaptive immune system is so slow during the first contact because it has to make sure to fulfill the two requirements that we have already mentioned: to distinguish and to establish the origin and the dangerousness of the particle that it is scrutinizing in order not to make a mistake and not to attack elements of the own organism or harmless foreign bodies.

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Representation of the maturation pathways followed by an undifferentiated T CD4+ cell, a process highly dependent on the famous cytokines. Lane et al. (2010)

During the antigenic recognition by the lymphocytes, many things happen. For example, the antigen-presenting cell emits several types of cytokines that inform the lymphocytes of the type of pathogen that has infiltrated the body (whether it is a virus, a bacterium, a worm…). There is a type of T cells, the CD4+, which, depending on the cytokine they detect, will be transformed into a certain type of effector cell to adequately and specifically deal with the pathogen (for example, to emit certain antibodies). Its other function is to emit other cytokines to activate the rest of the lymphocytes (i.e. to induce their differentiation), inducing their proliferation and the initiation of their mechanisms. For example, it induces the manufacture of CD8+ or cytotoxic T cells (although they can also be activated by antigen-presenting cells), cells that are essential during virus infection or cancer cell proliferation, because like NK cells, they also kill damaged and diseased cells, although in a much more specific way. Thus, CD4+ T cells are a kind of adaptive immune response coordinators, which is why they are also called helper T cells.

B cells are the other major weapon of the adaptive immune system. They can be activated either by directly detecting the antigen through their surface receptors or indirectly by detecting the molecular signals released by CD4+ T cells. In response, they undergo a process of differentiation or maturation in plasma cells. As such, they will begin to clone, expand, and produce various types of antibodies or immunoglobulins, which generally have two functions: they adhere to the pathogen surface to neutralize it and prevent it from coming into contact with the cells, and opsonization, that is, they cover the surface of the pathogen to mark it, to add a signaling target that will intensify the innate and adaptive cellular immune response against the infectious agent, so that the cells can find it faster and attack it as soon as possible.

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Recreation of the typical Y-shaped structure of an antibody or immunoglobulin. The colors indicate the different protein subunits that compose it. Wikipedia

When does the immune battle stop? It is a process partly mediated by molecular signals (involving the aforementioned cytokines) emitted by the immune system cells themselves, but other issues must also be considered. Many lymphocytes are antigen-dependent, that is, the greater the presence of antigens, which depends on the number of pathogens that are infecting the body, the greater the lymphocyte response. As the immune counter-attack advances, the density of pathogens and antigens in the body is reduced and, therefore, also the activation of lymphocytes. The immune system is utilitarian: many of its cells are created expressly for these situations, so that when the infections are over, the cells are no longer needed and end up dying and being recycled… except for some lymphocytes.

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This figure is typically shown to represent the activation of the immune system upon a first and second contact with a given antigen. The time in days is represented regarding the antibodies concentration. The left half shows the primary response executed before a first contact. We see a smooth increase in the production of antibodies that can take a week to reach the maximum concentration. On the other hand, when the antigen is detected for the second time after a lapse of time, the production of antibodies goes out of control. It reaches and exceeds the maximum concentration of antibodies achieved during the primary response in less than a week. The magnitude of the secondary response is enormous in order to avoid reinfection by the pathogen. Google Sites

Of all the undifferentiated lymphocytes, some of them will mature and be “hired” as sentinels. Both T and B cells can generate these memory cells which, after infection, will remain vigilant, travelling through the bloodstream, waiting for the pathogen they once fought to reappear and, this time, triggering an immediate response, in which there is hardly any waiting. The pathogen, if it has not changed significantly, will not stand a chance. The memory T cells will have prepared the specific cytokines that they synthesized against the invader during the primary response to launch them and quickly activate the pathways and mechanisms of action that were used during the first time. On their side, the memory B cells will have their arsenal of highly specific antibodies ready to avoid reinfection (although the specific antibodies that were manufactured during the first contact will also be circulating through the blood). As soon as the pathogen reappears, antibodies and cells will remember it when they detect its antigens and will go after it at lightning speed.

This is the long awaited immune memory, which protects us against future interferences of the same pathogen. That is the objective of every vaccine: to generate a specific immunity as complete as possible that includes a lasting memory. The duration of the immune memory is very difficult to predict as it depends on many factors: the mutation rate of the pathogen, the intensity of the immune response triggered by the antigens, the intensity of the infection, the immunocompetence, age, etc. Depending on all this, it can last months, years, or even a lifetime. That is why there are vaccines that need booster doses, because one is not enough to generate a lasting memory.

With these brief brushstrokes, I hope I have put the reader in context about how the immune system works. From here, the reason for the existence of vaccines will be better understood.

What is a vaccine?

So we know what a vaccine is for. But what is it? Let’s take a look at history. Let’s travel to late 18th century England. We can imagine a middle-aged doctor named Edward Jenner racking his brains, trying to figure out an experiment that will change history forever. Jenner and his contemporaries realized one thing: cowgirls were immune to the much-feared smallpox. At best, they developed the characteristic pustules on their skin (that is, precisely what “smallpox” means: “small pustule”), but nothing else. They were saved from the fierce attack of smallpox that often ended up taking the lives of the sick. It was known that cows could also get smallpox (although it was a different variety than the one that affected humans). What if those cowgirls, when coming into contact with the pus in the milk from cows affected by cowpox, acquired protection against the human variant? It should be noted that cowpox does not have the necessary adaptations to effectively infect people, so it does not trigger the disease in humans.

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Edward Jenner vaccinates young James Phipps against smallpox. This is how vaccination, the most effective prophylactic method in history, was born. Ernest Board-Wikipedia

In Jenner’s time, it was not known who was behind the infectious diseases. It would be just a century later when another doctor, Robert Koch, would discover the etiological agents of infections and would postulate the famous Koch’s postulates. At a time when ethics in science was conspicuous by its absence, Jenner inoculated one of those pustules of a person exposed to cowpox to the son of his gardener, the 8-year-old James Phipps. He did this with the idea of, subsequently, inoculating the child with the smallpox virus obtained from pustules of infected people. James would develop a few degrees of fever, some discomfort, and… nothing else. He had been immunized and protected against smallpox.

Jenner had found a way to beat that damn disease that had caused so many millions of deaths so far. He had discovered cross-reactivity, that is, how by inoculating a phylogenetically similar variant of the pathogen of interest and with hardly any virulence in people, immunity against it can be achieved. Since then he would be considered the father of immunology. He had discovered the vaccine (which, as the reader will have guessed, its name comes from the cowpox virus), although, the physician John Fewster had already described this phenomenon in 1768 actually. In fact, at that time, the inoculation of smallpox-diseased people’s pustules into healthy people was already widely practiced, a procedure known as variolization, which was actually known since ancient times. Hindus, Chinese, and Arabs were already using it as a prophylactic strategy, and not only against smallpox outbreaks, but also against leishmania. Jenner himself experienced it firsthand at the age of 8, curiously at the same age at which James Phipps was immunized. It consisted in inoculating pus from a smallpox pustule through an incision made in the skin. This method was certainly dangerous, since sometimes the immunization against the disease was achieved, but on other occasions the disease ended up developing in the patient, sometimes with dire consequences.

In any case, it was Jenner who solidly established the principles of vaccination. 200 years later, the World Health Organization (WHO) would celebrate the total eradication of smallpox thanks to Jenner’s early work. It is rightly said that his work has saved more lives than anyone else’s.

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Vaccines are, without a doubt, one of the best inventions of humanity to improve their quality of life and prolong their survival. These graphs show how the reported cases of diphtheria, polio, measles and subacute sclerosing panencephalitis (SSPE), a serious brain disease that appears late in some measles patients, have been reduced to almost disappearance after the respective vaccination campaigns. Murphy et al. (2010)

Vaccines have been one of the most life-saving tools in history and have most improved and prolonged human well-being, no matter what anti-vaxxers say. The cost/benefit and risk/benefit ratios of vaccines demonstrate that they are arguably the most effective prophylactic measure in history, something that science has demonstrated countless times and will continue to do so, including with the SARS-CoV-2 pandemic.

The eradication of smallpox has been the most resounding victory for vaccines, the only pathology eradicated worldwide so far. However, many other diseases, such as polio, diphtheria, tetanus, rubella, measles…, are now anecdotal in many countries thanks to them. And let us give them time, because it is possible that, in the future, cancer or allergies will swell this oblivion list thanks to them too.

But what is a vaccine? It is a preventive or prophylactic method. It does not seek, therefore, to cure diseases, but to avoid them. For this, vaccines seek to replicate the functioning of the immune system in terms of creating a memory and an improved and faster reaction to pathogens. Vaccines are thus presented as a complement, a support for our defense system. In other words, vaccines are nothing more than the application of the basic principles of immunology to improve health.

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Vaccines greatly accelerate the achievement of the herd immunity, which is essential to control the spread of an infection and prevent it from getting out of control. Ingeniero del montón

The goal of vaccines in the short and medium term is to reduce the incidence and prevalence of diseases, mainly infectious diseases (although they are already being applied against other types of pathologies), and to avoid uncontrolled episodes of them (epidemics and pandemics). For this, it is necessary to achieve the herd immunity, through which the dilution of the spread of infections will be achieved. This is logical, because if the number of susceptible individuals in a population decreases, the pathogen will have a smaller number of reservoirs from which it can spread. If we had to wait until the pathogen naturally infected a significant percentage of the population to reach that goal, it would be disastrous. It would take multiple generations until the population acquired the herd immunity (provided that the pathogen did not change), with all the deaths and pain that this would bring. However, thanks to vaccines, this process is significantly accelerated, saving thousands of people, since it is not necessary to go through the disease to obtain the immunity against it.

It is not necessary to vaccinate the entire population to achieve the herd immunity, only a certain percentage. When the herd immunity is achieved, even people without immunity may be protected from the disease. They would be shielded by the people who are immunized, which would serve as a barrier against the progression of the infection. The percentage of the population immunized to achieve the herd immunity depends on the ability of the pathogen to spread: if it is high, as with SARS-CoV-2, a high percentage of the population will need to be immunized and vice versa. To stop SARS-CoV-2, it is estimated that at least 70-75% of the population must be immunized.

Whole virus vaccines

The good thing is that, thanks to the great progress made in many fields of science (genomics, immunology, proteomics, etc.), we have a wide range of approaches and technologies to treat infections.

There are many types of vaccines that can deal with viral infections (and many of them are also applied against other pathogens, such as bacteria or fungi). So many that scientists must answer a number of questions to choose which approach they believe will be most effective against an specific virus. Depending on how the immune system responds to the virus, who needs the vaccine against the pathogen, and the best technology to create the vaccine, experts will select one type of vaccine or another. Logically, each research team will reach its own conclusions, so that for the same pathology different types of vaccines can be developed, which is precisely what is happening for SARS-CoV-2. But this is good. If different effective strategies against the disease are achieved, it can be attacked from more sides, and victory will be more assured. In addition, some of the candidate vaccines will be more affordable for certain countries than others, or will be more effective in certain age groups than others. In other words, a plural and diversified strategy ensures the solution of a wide range of problems.

That said, let’s explore the diversity of antiviral vaccines that are used or still under study. All of them, in fact, are currently being tested against SARS-CoV-2. We can classify them into two large groups according to whether the vaccine uses the whole virus or a fraction of it.

Whole virus vaccines are the traditional ones, the ones that have been used since the times of Edward Jenner. There are two strategies:

  1. Using the inactivated or “dead” virus: the virus is inoculated intact, but without the ability to replicate in host cells. The great Louis Pasteur already used this method to develop the vaccine against the rabies virus, which, by the way, is very effective, just like the Salk polio vaccine. Other vaccines, such as those directed against some strains of flu or cholera, are also based on this principle, but show less effectiveness than those of rabies or polio. However, nowadays they are increasingly in disuse and other alternatives are being chosen. Basically because a virus that cannot reproduce an infection (i.e., cannot replicate or invade host cells) generates a weak immune response, since many immune pathways are not activated by lack of stimulation. In fact, several doses are usually needed to maintain a persistent immunity. However, it is a totally harmless vaccination technique, even for immunosuppressed people, because the virus cannot multiply. For this reason, there are countries that, for several cases, such as polio, usually use this alternative because it is not dangerous.

  2. Using the “live” virus, but attenuated or weakened. This strategy was discovered by Louis Pasteur and Émile Roux while looking into the rabies vaccine. Of the complete virus vaccines, this is the one mostly used nowadays. The virus still possesses its infective capacity, but it is very diminished, so it is very unlikely for it to generate the disease. They are more powerful than those based on dead viruses since they trigger more intense responses by reproducing the infective process. For example, these vaccines are capable of stimulating cytotoxic CD8+ T cells which, as explained, are essential in the fight against intracellular pathogens, since their mission is to exterminate the  infected cells to prevent the pathogen from spreading. This happens because the virus, as it retains its ability to proliferate, continues to generate antigens in the cytoplasm of the infected cells. These antigens will be presented later to these lymphocytes to trigger the relevant response. In contrast, inactivated viruses cannot reproduce and cannot maintain the antigen manufacture.

It is understandable that some people are wary of these type of vaccine. What if, suddenly, the virus mutates and triggers the disease? Are attenuated viruses really dangerous? How are they obtained? The most widely used method since cell culture techniques were perfected consists, first, of extracting the pathogen in its native state from an infected human cell. This sample is inoculated into cultured cells from an animal, such as a primate, although human cell cultures have also been used and positive results have been obtained. The aim is to force the virus to adapt to the new host, for which it has to undergo a combination of mutations that, at random, will give the necessary skills to infect and reproduce in the new host. However, during this process a trade-off, an exchange, takes place: at the same time that the virus acquires the necessary faculties to reproduce in the new cells, those that allowed it to do so in human cells are weakened. Immunologists are therefore looking for a variant of the virus that clumsy reproduces in us and that, at the same time, maintains its antigenic elements to function effectively as a prophylactic tool. Before the rise of cell cultures, pathogens were often inactivated by doses of radiation, chemicals, or heat.

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By inoculating the virus into animal cells, it is forced to mitigate its ability to invade human cells. This is one of the methods used to obtain attenuated virus vaccines. Murphy et al. (2010)

Many will ask, “Is it possible that this attenuated virus could undergo some change that may give its original virulence back? Can the vaccine cause the disease that is trying to prevent? Scientists know that this risk is possible but also very unlikely, although it must be explained. Immunosuppressed people run the risk that this weakened virus could become an opportunistic pathogen, since it will hardly find barriers that will prevent it from proliferating and infecting even if its capacity to use human cells is impoverished. In fact, its use in immunosuppressed people and pregnant women is contraindicated.

Therefore, natural selection stands as an added risk factor in these vaccines. However, science has developed very efficient stratagems to prevent the attenuated viruses in the vaccines from causing disease or from suffering a genetic reversion that would return them to their initial or native state. For example, using recombinant DNA techniques (which we will discuss in more detail later), scientists can either induce mutations in vitro in the specific genes responsible for the virulence of the virus or directly cut out that part of the genome and keep only the part that stimulates the immune response. In this way, an immunogenic and non-virulent strain is obtained that will hardly be able to become dangerous again.

There are variations with respect to the base material used to manufacture these vaccines. Sometimes a different virus than the one of interest is used. This virus will act as a platform or vector, in whose genome the genes of the antigens of the virus of interest will have been included through genetic engineering techniques. The platform virus will be, logically, properly attenuated. Some vaccines against the coronavirus that are being studied contain genes of this virus embedded in the genome of attenuated viruses of influenza or polio. With this approach, it is intended to boost the immune response, which will be induced not only by the antigens of the coronavirus, but also by those of the platform virus.

Other times it is interesting to use a chimera virus, that is, a virus composed of a mixture of different viruses related to each other (heterologous viruses). This viral Frankenstein will have the characteristics of the viruses that compose it, ergo it will be able to express antigens corresponding to all those viruses. Its antigenic profile will be diverse and this will help trigger a more powerful immune response with a more intense and persistent memory than if only one virus was used.

Many of you may wonder: why not get vaccinated before an epidemic or pandemic emerges to prevent it in advance? Why not be more preventive? I bring this up because we have referred to recombinations of viruses that, predictably, were already “invented” by nature billions of years ago. We must bear in mind that the strains of many viruses are capable of recombining with each other. In other words, different strains of the same virus (let’s say influenza A) that infect the same cell could intermingle during the infection process and generate completely new strains, some of them even more dangerous and deadly. That is, if we also deliberately introduce attenuated strains of the virus obtained by genetic engineering for prophylactic purposes, we would be providing an ideal breeding ground for wild viruses to recombine with the attenuated ones and generate potentially dangerous strains. Therefore, in the event of a pandemic or epidemic outbreak, action must be taken at the time of its appearance, to avoid additional risks. As a curiosity, it should be noted that viral recombination is one of the causes that explain the jump between species of some viruses, such as influenza A, which can jump from waterfowl to pigs and people, for example.

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The H7N9 virus (a type of influenza A virus) occasionally causes outbreaks in humans. The jump between species occurs because different variants from different birds that cohabit in the same places can recombine in the same host. Thus a strain with the necessary skills to infect humans can be formed randomly. Naukas

Subunit vaccines

Whole virus vaccines have had unquestionable success, but for various reasons they are being replaced by new generation vaccines. Like everything in this life, nothing is perfect, and the whole pathogen vaccines have a series of disadvantages that have resulted in their progressive disuse (although they still have an obvious role). For example, for their acceptance, they require very long and expensive clinical trials (the current vaccine quality and research protocols are based on all previous experiences with these vaccines); they are expensive, which plays against their availability and diffusion; they can hardly cope with hypervariable pathogens, such as seasonal influenza viruses or HIV; they are difficult to preserve because they are very sensitive to thermal changes, therefore they need a very strict cold chain; the increasing number of infectious diseases forces the development of more versatile prophylactic technologies that can be manufactured more quickly.

The other major category of vaccines also has a long history behind it. Subunit vaccines inoculate a part of the germ, some of its purified pieces with immunogenic potential (that is, with capacity to stimulate the immune system): surface proteins, nucleic acids, polysaccharides, or toxins (in the case of bacteria)… For viruses, it is common to use, for example, their surface proteins. Another viral subunit could be the gene or genes that encode some immunogenic element of the virus.

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The SARS-CoV-2 Spike protein, which coats its surface and gives it a crown-like appearance, is the most widely used antigen to create many vaccine candidates against the virus. Wikipedia

The most obvious advantage of these vaccines is that, logically, there is no risk of  an accidental infection (a risk, we insist, that is risible in the case of vaccines with attenuated pathogens). However, there is a worrying problem that lies in the very nature of the vaccine: it is based on a specifiv part of the pathogen. The immunogenic power of these vaccines is often weak. Among other reasons because many cells of the adaptive immune system only react actively when they detect molecular patterns (known as PAMPs, acronym for Pathogen Associated Molecular Patterns), common to several groups of germs. If those patterns are not represented, the immune response will be incomplete and weak. Therefore, these vaccines usually need an extra ingredient: adjuvants. They are organic or inorganic chemical compounds that intensify the immunogenicity of antigens. They promote a more powerful, durable, and effective immune response.

Adjuvant substances deceive the immune system. They imitate an active infection. Consequently, the immune system executes various responses, as it does during a true infection. It is normal to add several adjuvants to a vaccine to generate a wide diversity of immune responses in various parts of the body. Frequently, the aim of adjuvants is to stimulate the secretion of cytokines, the activation of dendritic cells, and the recognition of antigens by T cells. Aluminum salts are among the substances that have been used the most in this respect, and have been since the 1920s, although they are being replaced by other more effective adjuvants. Logically, these ingredients must also pass their corresponding test phases to evaluate their safety and effectiveness. It is necessary to clarify that these immune response enhancers are not only included in the subunit vaccines, they can also be added as a complement in whole pathogen vaccines or in those we will see below.

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Use of adjuvants in different vaccines over time. Garçon et al. (2011)

The constituent subunits of these vaccines are relatively easy to obtain, which favors their large-scale production and low cost. Science is developing increasingly sophisticated methods to produce powerful antigens more easily and quickly. Thanks to the advance of bioinformatics and genomics among other sciences, it is possible to discern which antigens will produce a more potent immune response, and this is achieved in the following way. Let’s imagine that we want to obtain the most immunogenic surface protein from SARS-CoV-2. First, the genome of the germ is decoded. This information, which has to be as complete as possible, is inserted into a computer so that, through complex calculations, the machine dictates which genes produce the most immunogenic surface proteins. Finally, they are synthesized and purified to test their potency in animals. This procedure, which has incredibly accelerated the manufacture of vaccines, is known as reverse vaccinology. Its methodology is, precisely, the inverse of the classical vaccinology, by which, through a tedious process of trial and error tests, it was possible to determine the most recommended antigen.

As if this wasn’t enough, the development of synthetic biology allows us to synthetically manufacture these antigenic subunits in the laboratory. Its production on a large scale is also relatively simple. In fact, some vaccine candidates against SARS-CoV-2 are based on this biotechnology, such as that of Covaxx (the UB-612), which attempts to reproduce the Spike protein of the coronavirus conjugated with other elements. The function of these additives is to optimize the immunogenicity of the antigen. The Spike surface protein is the candidate most selected by scientists to produce their subunit vaccines, because it is one of the elements that the immune system recognizes most clearly.

However, it is not even necessary to purify the entire protein (or the subunit in question). It is sufficient to produce its epitopes, that is, the regions or sequences of amino acids that are specifically detected by the immune system. The search for these regions is carried out by structural vaccinology, and obviously, reverse vaccinology plays a great role in this sense.

Unfortunately, as we have already commented, many subunit vaccines are, by themselves, weak. In the end, adding one or more adjuvants only makes the testing phases more difficult and prolonged, since the safety and efficacy of these additives must also be checked. In addition, the added difficulty of storage must also be taken into account. For this reason, scientists have continued to look for more effective alternatives, and have found them in nucleic acids: DNA and RNA.

DNA vaccines are one of the most recent and promising advances in the field of prophylaxis. They are based on the amazing technology of recombinant DNA, which consists of conjugating DNA molecules from different origins or species. One of the first DNA vaccines was that of hepatitis B, a good example of how this technology is handled. What Maurice Hilleman (1919-2005), the creator of this vaccine, did was to insert the genes responsible for antigen synthesis (a protein of the hepatitis virus) into bacterial plasmids, circular and short DNA molecules that constitute a molecule independent of the bacterium’s genome. In this way, the plasmid acts as an expression vector. Subsequently, he inoculated the plasmids into yeast cells of Saccharomyces cerevisiae so that, with their natural mechanisms of transcription and translation of the genome, they synthesized the antigens of the virus. Finally, it only remained to purify those proteins to manufacture the vaccines in industrial quantities.

This is, broadly speaking, the basis of DNA vaccines: transfection, that is, the introduction of exogenous genes into the cells. It could be said that they make possible to create vaccines à la carte. There are several ways to use them. One has just been described: recombinant DNA is a means of obtaining antigenic subunits. Thanks to recombinant DNA, therefore, now more than ever it is easy (because the manufacturing of plasmids is standardized), economic, and fast to manufacture antigens in a massive way. Before, production was laborious and expensive. From the blood of an infected patient, the desired antigen was purified. Now it is a matter of cloning the gene or genes encoding the pathogen’s immunogenic antigen into an expression vector and introducing it into a producing microorganism (such as yeast or bacteria). And not only this, but in the same expression vector can be inserted antigen genes from several different pathogens, so that with a vaccine could be generated immunity against a wide range of infectious diseases.

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Once the plasmidic expression vector and the genes of the antigens of the virus in question have been recombined, it is time to inoculate them into the body, for which there are various techniques. Pinterest

These vaccines can also be directly inoculated via intramuscular syringe, but a much faster method is that of biolistics: through a gene gun, DNA particles wrapped in tungsten or gold metal projectiles are rapidly inoculated into the cells through an injection of compressed gas. The aim is that some of the millions of particles with recombinant plasmids reach the host cells randomly and are integrated into their genetic machinery so that they can synthesize the antigens and present them to the immune system. This is a particular way of simulating the reproduction of the virus in the cell.

Another very original way of applying these vaccines is to take advantage of natural processes. In other words, it is possible to use the natural infectious mechanism of the virus to introduce these vaccines in the body. For example, in the veterinary field it is common since the 1980s to use some viruses of the family Poxviridae (to which the viruses of smallpox belong) attenuated as a gene carrier that synthesize the antigens of the virus of interest. When this endogenous virus introduces its genetic material into the host cells during infection, it transfers at the same time the foreign genetic segment. The proteins encoded in those genes will be synthesized by the host cell, and we already have the antigen inside the body. In the case of SARS-CoV-2, several companies (Johnson & Johnson, CanSino Biological Inc.) are working on a similar option. By means of an attenuated adenovirus (an etiological agent of the common cold) they try to insert in the cells of the patients the genetic sequence with the antigen of interest. The adenovirus has been genetically modified to avoid a potential accidental infection. It is only capable of infecting cells by integrating its genome into the cell’s genome but cannot assemble itself or generate copies of itself after protein synthesis. Thus, infection by adenovirus is ruled out. There are many other viruses that can be used as vectors: some laboratories are analyzing the plausibility of using the H1N1 influenza virus (National Research Center of Egypt), the measles virus (Pasteur Institute and collaborators), the Newcastle disease virus (Dynavax), as vectors of the coronavirus antigens.

DNA vaccines have many advantages over other types. Besides lacking virulence, it has been proven that many of them stimulate very complete immune responses, including the activation of cytotoxic pathways. It is also very practical and inexpensive, partly because DNA is a molecule that is tolerant of thermal changes and can be stored without too many technological requirements at relatively high temperatures.

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Different methodologies to ensure the transfection of an exogenous nucleic acid. Bifi

Even with all the advantages of these vaccines, there are people concerned that they may cause harmful mutations in their genes, a fear we also compiled in our survey (although DNA was often confused with messenger RNA). The risk that plasmids integrate into our genome and cause some change, for example, that the host cell becomes cancerous, that they cause a chromosomal disorganization, or even that the immune system detects the foreing DNA as a target to be destroyed and generates anti-DNA antibodies that trigger an autoimmune disease, are already contemplated by experts. Although it is true that more information is still needed about the potential risks, there is nothing to worry about. In the preclinical (cultures of animal cells) and clinical (human patients) trials that have been conducted with these vaccines, no conclusive evidence has been obtained on any of these effects. In fact, the rate of permanent integration of these elements into the host cell genome is anecdotal, almost negligible. Already in the year 2000, B. J. Ledwith, from the Department of Genetic and Cellular Toxicology of Merck Research Laboratories, and collaborators reviewed the probability of permanent integration of a DNA plasmid into the host genome and they saw that this phenomenon has a frequency rate that is three orders of magnitude lower than the frequency of spontaneous mutations that inactivate some gene. Needless to say, these mutations are already anecdotal, so imagine the probability of integration of DNA plasmids… Other authors have obtained the same results since then. Actually, the plasmid transfection is transitory, lasting a few days. This, in fact, is a problem, especially in the field of gene therapy, where it is of interest that DNA plasmids are integrated into the patient’s genome permanently to control the instability of the altered target gene(s). In any case, the concerns of experts are others. As with other vaccines, the essential thing is that the vaccine should be sufficiently immunogenic. Furthermore, its application is not as easy as it may seem, because it is necessary for the expression vector to cross the cell membrane (for which it is helped by a method known as electroporation, which increases the permeability and transitory porosity of the cell membranes). Once inside the cytoplasm, the vector must penetrate the nucleus through one of the pores of the nuclear membrane, where its transcription will occur. The question is that it is not yet known how this process occurs. On the contrary, what B. J. Ledwith and many other scientists have observed is that most of the plasmidic load of these vaccines appears in the cytoplasm and not in the nucleus. To what extent does this limit the potency of the vaccine? Anyway, and as another example of how controlled and rigorous the vaccine research procedure is, there are already protocols to detect and avoid the unlikely cases of mutagenesis or autoimmune reactivity that may occur.

DNA is not the only nucleic acid that has been proposed for making SARS-CoV-2 vaccines. The pharmaceutical company Pfizer and many others (Moderna, BioNTech, or Arcturus) are working on the first messenger RNA vaccines (mRNA from now on) that will come out to the market. They are the last milestone in vaccines, to the point that there is none in the market yet. They are all in the testing phase.

The mRNA is the DNA transcript. It is like a DNA molecule but translated into a different language. It is the Hermes of the nucleic acids since it works as a carrier of the genetic information that the cell zealously stores in the DNA of its nucleus. The mRNA, once synthesized from the DNA, will leave the nucleus towards the cytoplasm, where it will go to the endoplasmic reticulum, a kind of ribosome storage. Ribosomes are responsible for translating the mRNA into amino acid chains, i.e. into proteins. Consequently, through the appropriate mRNA, the cell alone will be able to synthesize the SARS-CoV-2 antigens and present them to the immune system.

In vaccinology, scientists work with two types of mRNAs that carry the information in which the antigens of the pathogen of interest are encoded. One does not have the capacity to replicate itself or to self-amplify. The other, however, does, as it is accompanied by the necessary equipment that will allow it to proliferate in the host cells, as would an RNA virus (such as coronavirus), but differing in that it has no infective capacity. It is usually used for this purpose the genome of some virus of the alphavirus group, in which the genes encoding its surface proteins are replaced by the genes of the virus of interest. In this way, it is not necessary to inject the vaccine repeatedly, but the mRNA itself becomes an automatic factory for the production of antigens. Logically, this second method is more complex and expensive than that of mRNAs without capacity to proliferate.

Obtaining these biomolecules is relatively simple. Basically, we need a plasmid obtained from some bacteria in which the genes that code for viral antigens are already included. Then, by means of a series of enzymes, the desired mRNA molecule is obtained from the DNA template. It is then purified and processed to eliminate impurities and undesirable elements that could affect the vaccine’s efficacy.

mRNA vaccines-messenger RNA-DNA-recombinant DNA-mRNA-mutations-vaccines
Messenger RNA vaccines are the next milestone in health prophylaxis. In this scheme we can observe how the mRNA that contains the genetic information of the antigens of a specific pathogen is included in a lipidic vesicle that acts as a release vector. Its mission is to ensure that the mRNA reaches its destination: the cytoplasm of the host cells, where it will be “read” by the ribosomes. The Conversation

The results obtained in pre-clinical and clinical trials are very encouraging, and not only against typical infectious diseases, but also against recurrent and difficult-to-treat infections, such as AIDS or and several types of cancer. They have many advantages: they can be manufactured quickly, at low cost and are usually powerful and versatile, as they produce strong lymphocyte and neutralizing antibodies responses and long-lasting immunity with very few doses. They could mark a turning point in vaccine manufacturing. As they are easy to obtain, they would help to significantly reduce the excessive time that often entails to develop and manufacture a vaccine. They also have fewer inherent risks than the other vaccines we have mentioned. Since mRNA is not inserted into the genome of host cells (contrary to what conspiracy theorists mistakenly claim), there is no danger of mutagenesis (although we insist that for DNA vaccines there is also no evidence of this risk). Nor are they infectious elements, so they cannot cause the disease.

Why then are there no longer any in use and why not invest more in this new modality of vaccines? Despite all their advantages, these vaccines pose a major obstacle that until recently has not been overcome: they are very unstable and inefficient when used in living organisms. This happens because the immune system attacks the nucleic acid. If we think about it, it makes sense. Over time and after multiple exposures to various pathogens and their antigens (including their nucleic acids), the innate immune system has developed defense mechanisms that attack foreign nucleic acids as soon as they are detected circulating in the body. The immune cells synthesize enzymes called proteases that are capable of destroying these biomolecules. But that’s not all. The cells to which the mRNAs are directed have mechanisms that naturally degrade them in their cytoplasm, so that these vaccines barely have time to act due to these cleaning mechanisms (whose mission is to avoid accumulations of potentially toxic metabolites in the cell). Such a challenge is being overcome by using release vectors (natural and synthetic), whose mission is to increase the stability and viability of mRNA in living systems by protecting it from degrading enzymes and promoting its penetration into cells. The important thing is that the mRNA should survive long enough to enter the cytoplasm of the host cells and be processed by the ribosomes. Subsequently, the cell itself will eliminate it by means of proteolytic mechanisms to avoid its toxic accumulation.

These would be the most frequent types of subunit vaccines. However, there is another one whose nature is somewhat ambiguous. It’s a kind of hybrid between a whole virus vaccine and a subunit vaccine, and we’re going to reference it because there are also candidates based on this type of vaccine to fight the pandemic. We’re talking about the virus-like particle vaccines. This technology attempts to replicate the carcass of the virus of interest. In other words, they are viruses but without genetic material and, consequently, without the capacity to infect or reproduce. They try to mimic the morphology, geometry, size, organization, and surface antigens of viruses or virions to recreate as accurately as possible an infectious episode and the subsequent immune counterattack. It is assumed that this will achieve the same intensity and immune power as if the virus itself had entered.

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Example of a virus-like particle. It replicates the geometry, morphology, distribution, and types of antigens of the virus of interest. The main difference is that this particle doesn’t have a genome. Crisci et al. (2013)

These structures are composed of structural proteins obtained from the virus in question with the ability to self-assemble. They can carry one or more antigens of the pathogen in order to achieve greater efficiency. The good thing about this technology is that it can replicate the Pathogen Associated Molecular Patterns, something that other subunit vaccines lack. Sometimes they need adjuvants, but many others their size and antigenic profile is sufficient to trigger a strong immune response.

As of November 25, 2020, virtually all vaccines in clinical phase 3 are subunit vaccines, either formed by proteins or nucleic acids. Inactivated or attenuated virus vaccines are all in the preclinical phase. The type of vaccine that is most clearly in demand are the subunit vaccines, which, as we mentioned at the beginning, are rendering the whole virus vaccines obsolete.

This first part is important to get into context and become familiar with vaccines. Taking all these data into account, what we will tell you in the second part will surely be much more understandable. In the next chapter we will detail the phases of development of a vaccine, the reasons why the time of these phases has been reduced to fight the pandemic, and the lights and shadows of the different vaccines against COVID-19 that have been approved or are about to be approved.


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