Everything you need to know about vaccines

VACCINATION AND THE IMMUNE SYSTEM

Vaccination, simply put, can be considered an “exercise” or “school” for our immune system (IMS). It involves exposing the organism to non-virulent forms of microorganisms or their immunogen components (forms of microorganisms that will not cause disease but will stimulate the immune system to respond = immunogenicity), followed by the primary immune response (upon the first encounter with the immunogen) which is always slower and weaker than the secondary one (a repeated encounter with the immunogen). Following the immune reaction to the vaccine, a supply of cells and antibodies are created in the organism at the expense of which the IMS “remembers” and which will, upon reencounter with such a pathogen, react faster and much stronger as well as prevent the development of the disease. The described process is called active immunization, and almost all vaccines work on this principle.

It is very important to keep in mind that vaccination actually mimics natural processes that take place in our body amid encounter with a particular pathogen, but in a safe and controlled way in order to protect our health. Edward Jenner noticed, as early as the 18th century, that milkmaids did not get smallpox due to prior exposure to cowpox or having already overcome it. Therefore, Jenner decided that it would be good to inoculate the people, i.e., “infect” them with cowpox and thus protect them against smallpox which had significantly graver consequences to one’s health. It was then that the foundation for the development of vaccination was laid. Today, what Jenner did, thanks to the development of science and a number of new technologies, we do much faster and safer.

There is also passive immunization, which involves injecting specific antibodies against a specific antigen. Such an immunoglobulin fraction is prepared from a pool of donor plasma (1000 voluntary donors are taken for each plasma pool) and is used in certain situations when an extremely rapid, life-saving reaction is needed. Today, passive immunization is used relatively rarely.

Immunity can be divided into innate and acquired, and vaccination, although it includes some components of innate immunity, is based primarily on the mechanisms of acquired immunity because precisely they provide a highly specific defense against a particular pathogen. Therefore, vaccination against one pathogen does not protect against another.

On the other hand, the innate immune response is extremely fast and non-specific, i.e., directed against a larger number of pathogens, so it is of extreme importance for the body’s defense when acquired immunity has not yet been triggered. Acquired immunity has a better ability to “remember”, so with each following contact with what it remembers, it will react much faster and stronger, which is why it is certainly more important and effective in terms of vaccination. Consequently, every reencounter with the same pathogen will enhance the IMS response to it, so this principle is also used in vaccination when we are repeatedly vaccinated against the same pathogen (for example, against diphtheria, tetanus, measles, etc.).

The most important components of the mechanisms of acquired immunity are T-lymphocytes, B-lymphocytes, and antibodies (immunoglobulins, Ig). Initially, B-lymphocytes are developed in the bone marrow and leave it as naive mature B-cells and move to the periphery, to secondary lymphoid organs such as lymph nodes and spleen. On the surface, B-lymphocytes contain Ig class M (IgM) which can recognize certain epitopes (recognition site on the antigen) and in secondary lymphoid tissues wait for “their 5 minutes of glory”, i.e., the moment when they will recognize something, activate, and start producing antibodies. Some antigens, such as lipopolysaccharides and bacterial polysaccharides, can stimulate the activation and differentiation of B-lymphocytes into antibody-producing plasma cells without the T-lymphocytes giving them the “green light”. Therefore, we call them T-independent antigens. This response is extremely rapid but moderately long-lasting (lasts up to several months), does not stimulate the formation of memory cells, and the produced antibodies are of a relatively low affinity and specificity to the antigen.

The response to T-dependent antigens involves the active participation of T-lymphocytes and is much more important from the aspect of vaccination, given that certain memory cells are also produced, so the organism stays protected for much longer (lasts for years). In addition, antibodies produced during the IMS reaction to the T-dependent antigens have a much higher affinity for the antigen, and the overall response is somewhat slower than that to T-independent antigens (it takes up to several weeks). After entering the body, T-dependent antigens are processed by antigen-presenting cells (APC; these are macrophages, dendritic cells, and activated B-lymphocytes) which present them in a highly immunogenic form on their surface with the help of major histocompatibility class (MHC) molecules by T-helper cells (Th). After the Th recognizes the antigen that is presenting the APC, T-lymphocytes interact with APC. If the APC is a B-cell, it will activate and differentiate into a plasma cell that produces large amounts of antibodies. Th cells also secrete certain cytokines that stimulate B-cells to undergo somatic hypermutation and many other processes. The most important thing to know about cytokines is that their ultimate goal is to produce antibodies of as high affinity as possible, i.e., an affinity for the antigen to which IMS reacts at that moment.

Now that we are familiar with the basics of the immune response and acquired immunity, we can repeat the concrete process of immunization, i.e., what happens in our body after we receive the vaccine. The first task of our IMS is to recognize that something foreign has entered our body, and that part of the job is usually done by the components of innate immunity, which will then “mark” or through opsonization help with phagocytosis of that foreign component in the organism. After phagocytosis, APC will present this antigen on its surface to T-lymphocytes via the MHC molecule, which will, following the aforementioned process, stimulate B-lymphocytes to differentiate into antibody-producing plasma cells, but also to differentiate into memory B-cells which will wait until they reencounter the antigen. This whole process will result, as mentioned above, in a primary immune response that occurs somewhat more slowly and is relatively weak, but is of extreme importance because it prepares the IMS for the re-encounter with the pathogen which will be, due to the presence of antibodies and memory B-cells, significantly faster and stronger and will protect the body from the development of the disease.

The last sentence of the previous paragraph contains the exact essence of vaccination: by exposing ourselves to a non-dangerous pathogen that has been stripped of its virulent properties (those that cause disease), we prepare our IMS for any subsequent contact with that pathogen, but this time with one that has virulent properties and is dangerous for us and thus we defend ourselves against it.

TYPES OF VACCINES

During the process of vaccine development, each team of scientists considers how best to stimulate the immune system to “remember” an antigen. The type of vaccine depends on the immune response to a particular pathogen, which population needs to be protected (children, the elderly, immunocompromised), and on the possibilities of producing that vaccine. The latter complicates the production process the most because sometimes pathogens, especially viruses or insufficiently studied parasites are not easy to cultivate. That can be due to insufficient knowledge of the life cycle, or due to extremely specific survival conditions that are sometimes impossible to ensure in wholesale production. Knowledge of antigenic properties and virulence also plays an important role; for example, whether the antigenic component is an integral part of the bacterium (flagellum, capsule, lipoproteins), whether the bacterium produces a toxin, disruption of human cell function, or something else. Another problem is the rapid mutation of some pathogens, such as the influenza virus, which requires the vaccine to be perfected every year in order to be effective against the newly formed genotype.

LIVE VACCINES

Vaccines are divided according to type into two main subgroups: live and non-live vaccines. Live vaccines contain a weakened, i.e., attenuated pathogen that has lost virulence but retained its immunogenicity, thereby stimulating a specific immune response (antibody production). The most common method of attenuating the virus involves breeding it in animal embryos, usually those of a chicken. In short, the virus is implanted in an embryo, incubated to promote its replication, and then transferred to another embryo. The procedure is repeated up to 200 times. This enhances replication within chicken cells, leading to the impossibility to replicate in human cells. Similar to that is the cultivation of the measles virus in the cell culture of a kidney of a green monkey, which causes the virus to lose its ability to bind to the receptors of human cells. Other methods of attenuation include heat treatment, chemical agents, or genetic modification. However, there is also the possibility of a mutation that can restore its original virulence or even increase it, although the chances of that are small. An example of this is the oral polio vaccine (OPV) which has been replaced with the inactivated poliovirus (IPV).

Attenuated vaccines in the routine vaccination schedule in the Republic of Croatia include the vaccine against tuberculosis or drought (BCG) which contains a weakened strain of the bacterium Mycobacterium bovis (protects only from more severe forms of TB) and MO-PA-RU (trivalent vaccine against measles, mumps, and rubella viruses). In addition to them, other live vaccines are available, including vaccines against anthrax, typhus, yellow fever, rotavirus, and chickenpox, none of which are mandatory in the Republic of Croatia but are advised when traveling to exotic countries (yellow fever), or for risk groups (veterinarians and ranchers for anthrax, immunocompromised for chickenpox).

Attenuated vaccines generally provide better protection than inactivated ones because they mimic a natural infection and stimulate mucosal immunity (specific IgA) and their action lasts longer than that of killed, inactivated vaccines. However, they are not recommended for use in pregnant women or immunocompromised people (people with HIV, chemotherapy, immunosuppressants, autoimmune diseases) because unforeseen reactions of the immune system are possible. A weakened system can easily develop a disease that one should be protected against, while vaccination in combination with an autoimmune disease leads to severe reactions of hypersensitivity that can even cause death. Because of patients with these types of conditions, the vaccination of healthy individuals is suggested in order to suppress the spread of diseases that, as can be seen, can have a fatal outcome.

NON-LIVE VACCINES

Non-live vaccines are again divided into two categories: whole killed and vaccines made from microbial subunits. Microbes are inactivated thermally (1 h at a temperature of 56-60˚C) or by treatment with antimicrobials (formaldehyde, ethanol). Due to the absence of infection, protection with this type is usually shorter and does not provide lifelong immunity, therefore it is suggested that booster doses be given throughout one’s lifetime. In the Republic of Croatia, the obligatory vaccine of this type is the before mentioned IPV.

This group also includes the cellular vaccine against pertussis or whooping cough (due to its pronounced reactogenicity, it is no longer used in our country). Among optional inactivated vaccines we should point out the influenza virus vaccine, which is advised each year before the start of the flu season, especially for at-risk groups (e.g. the elderly, the chronically ill, medical staff). Other available killed vaccines include rabies, tick-borne encephalitis (TBE), and hepatitis A vaccines.

Microbial subunits

Vaccines with subunits of viruses and bacteria also require booster vaccines to maintain immunity because memory T cells do not get activated. The most common antigenic components are proteins and polysaccharides specific to exactly that pathogen. Examples of this are pertussis and Haemophylus influenzae vaccines. The first acellular vaccine contains pertussis toxin, pertactin, pili, and filamentous hemagglutinins, proteins, and parts of the capsule characteristic for Bordetella pertussis. The polysaccharide capsular antigen PRP is part of the type b Haemophylus influenzae and is also found in the routine vaccination schedule in the Republic of Croatia. However, sometimes the part of the pathogen alone is not enough to activate the immune response, so conjugate vaccines are used – antigenic protein is bound to a carrier (polysaccharide, protein, lipid, alkaloid) and provokes a greater reaction, but again insufficient to cause disease. In the Republic of Croatia, the conjugate vaccine is precisely the one against H. influenzae type b where PRP binds to 4 different carriers: diphtheria toxoid, N. meningitidis outer shell protein, tetanus toxoid, and mutated diphtheria carrier protein CRM197. Two additional vaccines are also conjugated – meningococcal and pneumococcal. Since 2019, vaccination against pneumococci has been mandatory in Croatia, and it contains purified capsule polysaccharides of 7 different capsular subtypes of the causative agents of pneumococcal disease (Streptococcus pneumoniae) related to diphtheria toxoid. It exhibits better performance than the conventional polyvalent vaccine, which once contained capsule polysaccharides of as many as 23 capsular subtypes of S. pneumoniae, and then as well its versions that contained fewer capsule polysaccharides of different capsular subtypes (10- and 13-valent). The meningococcal vaccine, currently optional in our country, also contains purified polysaccharides of 4 different subcapsular subtypes of the Neisseria meningitidis pathogen bound to tetanus toxoid. The most widespread in our area is the Meningococcal type B, but there are A, C, Y, and W135 types as well.

Subunit vaccines are also possible for viral diseases, the most well-known being the flu vaccine. Since recently, the tetravalent influenza vaccine is also available in our country, and it contains hemagglutinins and neuraminidases of influenza virus strains type A and B (type A H1N1, type A-H3N2, and two types of strain B), the most common causes of the disease. Subjects vaccinated against HPV received virus-like L 1 particles, isolated from viral capsids of different strains. The quadrivalent vaccine most commonly contains strains 6, 11, 16, and 18, and the 9-valent vaccine includes strains 31, 33, 45, 52, and 58. Strains 16 and 18 have been associated with cervical cancer, anal cancer, and various genital and perianal neoplasms.

Toxoids

In addition to carriers in vaccines, toxoids also serve as antigenic components of certain pathogens. Clostridium tetani cause its (eponymous) disease of the same name by producing a toxin called tetanospasmin. Its effect on choline receptors leads to horrific, lethal muscle spasms. Corynebacterium diphtheriae causes upper respiratory tract infection by secreting the diphtheria toxin. All of these proteins can be weakened, more accurately, treated with formaldehyde, and retain their antigenicity but not virulence. Botulinum, a neurotoxic protein from Clostridium botulinum, has the opposite effect of tetanospasmin, i.e., it relaxes smooth muscles and eventually leads to the cessation of breathing. Vaccines against diphtheria and tetanus are mandatory, while vaccination against botulism is recommended for people who work with it and members of the military.

Recombinant vaccines

With the development of genetic engineering, the possibility of new, recombinant vaccines has also developed. The best known and most important is the one against hepatitis B. The outcome of the recombinant technology is the HBsAg protein, from the envelope of the virus. The gene from the virus is embedded into yeast (more commonly) or E. coli. Thanks to the strong transcription promoter in the yeast genome, the protein is produced in huge quantities in a relatively short time. Therefore, the yeast is grown in a bioreactor under suitable conditions, and the expressed HBsAg is eventually isolated, purified, and placed in a vaccine. In addition to hepB, recombination also produces the B subunit of cholera toxin (in combination with the oral vaccine against V. cholerae) and the lipoprotein OspA from the surface of Borrelia burgdorferi, the causative agent of Lyme borreliosis. LYMErix, however, was nowhere near perfection: complete protection was not proven; it was not determined whether booster doses were required or not; children, who are most susceptible to this disease, were not allowed to receive the vaccine; the anti-vaccination movement was just gaining momentum; the vaccine was associated with the development of autoimmune arthritis. Although no correlation was ever established with the last side effect, the vaccine became less and less sought after, only to finally disappear from the U.S. market in 2002.

FUTURE?

The most important vaccines we want to see in the future are those against malaria and HIV due to their frequency, as well as the increasing resistance of Plasmodium, i.e. HIV, to existing therapy, which again increases mortality. At the beginning of 2019, in three African countries, where the prevalence of malaria is extremely high, the 4th phase of clinical trials is conducted with Mosquirix vaccine, a recombinant vaccine with CSP, circumsporozoite protein. Currently, that is the only vaccine that has shown some effectiveness against malaria. Circumsporozoite protein is located on the surface of the parasite Plasmodium falciparum which causes malaria. In the vaccine, this protein is fragmented (split into pieces) and fused with RTS and S surface antigens of hepatitis B. After receiving the vaccine, antibodies are formed that, upon coming in contact with P. falciparum, protect against disease. This vaccine also protects against hepatitis B but is not recommended if the protection against hepatitis B is the primary reason for vaccination. As for the vaccine against HIV infection, so far, in the last 25 years, none has shown any progress towards eradication of the disease “thanks” to the extremely complex structure of the virus, many potential targets, insufficient knowledge of its mechanism of disease, and the number of strains. Rapid mutations are also not helpful. The RV144 vaccine (avian measles virus as an inert vector + gag-pol-env genes) provided protection in only 30% of cases, while its modified version of HVTN 702 is effective only against the strain dominant in South Africa.

DNA vaccines are also under development, the idea of ​​which, as the name suggests, is to inject naked DNA with a gene that causes a virulent component in the body. The gene for a particular pathogen is incorporated into a plasmid, which in turn is incorporated into a bacterial cell for faster replication. DNA is isolated and applied directly. In theory, it should be able to activate both cellular and humoral immunity without any risk of developing the disease. However, due to being limited to only protein antigens and a weaker immune response compared to the conventional human vaccines, as well as not knowing the effects of foreign DNA on human cells, this type of vaccine is currently used only in veterinary medicine. The most interesting example of this is the prevention of melanoma in dogs. By improving the administration of the vaccine using electroporation or a gene gun the effectiveness in humans could also increase in the future. For now, we have to be content with what we have, although it is already a huge progress, unfortunately, many do not know how to appreciate it. Scientists are still, to this day, crucified by people not understanding why something is not being done about every disease ever, not realizing how much effort, time, and will research requires, but unfortunately as well as money, which very often there is not as much of as there should be.

AUXILIARY SUBSTANCES IN VACCINES

Although antigens are most important for the performance of the vaccine, without the excipients the vaccine would be much less effective and poorly resistant to external influences. For example, although tetanus toxin causes disease, it is not immunogenic enough to stimulate antibody production and protect the organism. On the other hand, tetanus toxoid (deprived of its virulent properties) becomes more immunogenic with the adjuvant whilst not causing the disease. As is the case with other pharmaceutical forms (tablets, injections, infusions, syrups), so is with vaccines that by adding adjuvants, preservatives, co-solvents, and other excipients we achieve a better immune response to the vaccine, better stability, and longer shelf life, as well as sterility and compatibility with other ingredients of the vaccine and with the wrapping material in which they are packaged.

Currently, more than 25 vaccines have been developed against diseases such as measles, polio, tetanus, meningitis, diphtheria, influenza, cervical cancer, etc. Many of these diseases have not appeared for years, thanks to vaccines. Most children receive vaccines against these diseases on a regular and timely basis, but almost 20 million of them worldwide are unable to be vaccinated (for various reasons), putting them at direct risk of infection.

We will discuss the main groups of excipients individually, explain their role in vaccines and provide evidence on the safety of their application.

Adjuvants

The first group of vaccine excipients are adjuvants. The function of adjuvants is to enhance the host’s immune response. Alexander T. Glenny showed in 1926 that binding antigens to tiny particles of aluminum salt significantly increased antibody production in the host. Since that discovery, the use of aluminum salts in vaccines has become commonplace. In the beginning, science did not invest time and money in researching the exact mechanism by which aluminum salts work, and the research that was conducted did not find a consistent, universal answer. Aluminum salts are not the only adjuvant used in vaccines, but they are certainly by far the most common. It is generally thought that, by forming depots at the injection site, adjuvants increase the activation of immune system cells and cytokines that are crucial for forming immunity. In addition, they stimulate the transfer of antigens to the lymph nodes. Research conducted in the last ten years has confirmed some of these hypotheses and suggested a mechanism by which the adjuvant works.

By forming depots, the contact of the immune system cells with the antigens is prolonged and antigen phagocytosis is promoted. Moreover, the presence of aluminum salts at the injection site creates a small inflammation that further stimulates and attracts the cells of the immune system. In addition to attracting immune system cells, adjuvants also increase the uptake of antigens into the cells that then present them, and those cells are also crucial in the process of “remembering” the antigen. Aluminum salts as adjuvants also have a protective role – they prevent the adsorption of proteins from the vaccine onto the walls of the packaging.

Aluminum and its salts are naturally present in our organism since birth. The body does not need it so it just eliminates it unchanged. Aluminum from food is eliminated via feces in the form of insoluble salts and the absorbed fraction is expelled through the kidneys. The amount of aluminum found in vaccines is extremely small, less than one milligram per dose of vaccine. A study conducted in 2011 compared the amount of aluminum absorbed from food and that from vaccines, and the results showed that the total amount of aluminum ingested and absorbed in that way was less than the allowable upper limit of intake in one week measuring 1 mg per kilogram of body weight. Daily exposure to aluminum from food is estimated at 10-15 mg for an adult, which is far more than the 1 mg we can find in one dose of vaccine. In the first 6 months of their life newborns receive 4.4 mg of aluminum via vaccine, which is also less than the amount they ingest by consuming food. Thus, in the first 6 months, the newborn receives 7 mg of aluminum from breast milk and 38 mg from the formula. A big contributor to the daily exposure to aluminum are also cosmetic products, especially antiperspirants that expose the body to an additional 2 mg of aluminum every day. These 2 mg stay on the body until the remaining antiperspirant is washed off; 24 hours on average. Regarding the long-term and short-term safety of the application of aluminum salts in vaccines, several studies have shown that aluminum in this dose does not affect the health of the individual. What is associated with aluminum salts in injections is itching and transient granuloma as a consequence of contact allergy to aluminum in a small number of children. Such a reaction is transient and harmless.

Preservatives

The next important group of vaccine excipients are preservatives. Preservatives are excipients that are also added to other pharmaceutical forms, especially those that are multi-dose or those at risk of contamination during each administration. Their purpose is to destroy pathogens that could potentially enter the vaccine and infect the person to whom it is administered. Although all vaccines are produced under sterile conditions, multi-dose forms are at risk of contamination due to multiple administration which increases the likelihood of contamination of the preparation. In the case of single-dose forms, there is no need for this because those are stored under sterile conditions and administered only once in a sterile manner. Initially, preservatives were not being added to the vaccines. As a result, in the early 20th century, about a hundred children contracted sepsis because the vaccine was contaminated with the bacterium Staphylococcus aureus. After that incident, since 1930, we have been smarter, so we add preservatives to multi-dose forms of vaccines (and some single-dose ones). The ones most commonly used are phenol and thiomersal.

Thiomersal is probably the most controversial vaccine ingredient. Therefore, it is important to emphasize a few facts about this preservative. Thiomersal and mercury are two different terms. Mercury (Hg) is a chemical element that can exist in nature as such, free; whereas thiomersal is a compound that contains mercury in its structure along with other elements. When it enters the body, thiomersal is metabolized into ethylmercury and thiosalicylate. The next important difference is between ethylmercury and methylmercury. Methylmercury, unlike ethylmercury, is not a product of thiomersal metabolism. It occurs in nature in the presence of mercury and can be taken into the body through food, most commonly through some species of fish. A vaccine containing 0.01% of thiomersal contains approximately 25 micrograms of mercury per 0.5 mL of dose, the same amount as the one contained in 85 g of canned tuna.

Methylmercury can cause neurological disorders even in much lower concentrations. The first knowledge about the neurotoxicity of methylmercury dates back to the 1950s, when mercury poisoning through seafood, especially fish, was discovered in Minamata Bay in Japan. The poisoning was caused by the consumption of seafood from the bay where large amounts of industrial waste were being disposed of. Symptoms of the disease included visual and hearing impairment, narrowing of the visual field, ataxia, and tremor, and the disease was called Minamata disease after the bay. In the 1970s, cereals treated with methylmercury as a fungicide were imported into Iraq. Bread produced from these cereals was consumed by a large number of pregnant women, and shortly afterward methylmercury poisoning was determined in a part of the population, especially in pregnant women. After the birth of children, intrauterine exposure to methylmercury was shown to affect the neurological development of the fetus, so children were born with symptoms similar to cerebral palsy in case of severe exposure, i.e., with sensory and motor disorders and developmental disorders in case of lower exposure. Mothers, on the other hand, showed almost no symptoms of poisoning at all. The control of the concentration of methylmercury has since tightened, and the permitted concentration limits are significantly lower.

Ethylmercury is significantly less toxic than methylmercury. However, in the same period, the permitted amounts of ethyl mercury were also maximally reduced, among other things in vaccines. The main difference between ethylmercury and methylmercury is in their pharmacokinetics. The elimination half-life of ethylmercury is about 7 days, while the elimination half-life of methylmercury is as much as 50 days. We said that methylmercury is taken into the body through food, which means that this intake is continuous and that, due to such a long elimination half-life, it accumulates in our body. Ethylmercury in vaccines is in 1000 to 1,000,000 times lower concentrations than what toxic amounts are. Also, vaccination is interval and short-term, so with a short elimination half-life, there is no danger of ethylmercury accumulating in the body.

Thiomersal has been used in vaccines since 1930 when its safety was determined based on animal and human studies. Since then, a significant amount of research has been conducted to show whether vaccines play a role in the development of autism and other neurological diseases. However, the connection was never found. One of the largest studies on this topic was published in 2014 – a meta-analysis of all case-control and cohort studies conducted so far in the MEDLINE, PubMed, EMBASE, and Google Scholar databases. A total of 1,266,327 children participated in all studies, and it was shown that neither thiomersal, nor mercury, nor vaccines generally cause autism. The only side effect associated with thiomersal is that which occurs locally – redness and erythema at the injection site which are temporary. Back in 2007, the European Medicines Agency (EMA) published guidelines for the safe use of thiomersal in vaccines. These guidelines state that the EMA, based on all the evidence and documents gathered, does not see a link between vaccines and autism and other neurological disorders.

It is important to emphasize that, preventively, thiomersal is no longer used in vaccines, except in multi-dose containers of trivalent and tetravalent influenza vaccine which are not included in the mandatory vaccination schedule, and only in a few single-dose vaccines. In mid-1999, the Public Health Service (PHS) and the American Academy of Pediatrics (AAP) suggested, for the first time, the withdrawal of thiomersal from the vaccine for preventive purposes. From then until 2001, fewer and fewer manufacturers used thiomersal as a preservative in children’s vaccines, until the stocks of thiomersal vaccine were completely depleted in 2003. Since that year, advances in technology and production have made single-dose forms available for most vaccines, and thiomersal has again begun to be used almost exclusively in multi-dose forms of influenza vaccine which, for financial and economic viability, are formulated as multidose forms.

In Croatia, out of 39 available vaccines, thiomersal is used as a preservative in only 4 of them: tetanus vaccine 1 dose and 10 doses (adsorbed, suspension for injection) and diphtheria and tetanus vaccine with reduced antigen content 1 dose and 10 doses (adsorbed, suspension for injection).

Thiomersal is not only present in vaccines. It is found in cosmetic products (make-up removers, ophthalmic solutions, eyeshadows, mascaras), hygiene products (contact lens solutions, antiseptic sprays, soap-free cleansers), and biological products (antitoxins, immunoglobulin preparations, drops, and sprays for the nose, ear, and throat).

Stabilizers

Stabilizers are added to vaccines to protect them and their active substances (viruses, bacteria) during the lyophilization process. These can be sugars (sucrose, lactose), amino acids (glycine, glutamate), as well as proteins (gelatin and its derivatives, human serum albumin). The proteins used are highly purified, and the gelatin (usually obtained from pigs) is also hydrolyzed and contains no traces of pig DNA. Cases of the development of an acute allergic reaction to animal gelatin, one in every 2,000,000 doses of vaccine, have been documented.

Symptoms of an allergic reaction include rhinorrhea, rash, laryngotracheal edema, and hypotension. In all cases, the symptoms disappear after treatment with diphenhydramine and epinephrine. Today, such cases are becoming rarer – due to the stricter control of the production and purification of gelatin used in vaccines, but also because most people today know whether they are allergic to gelatin. Increased control also guarantees that beef gelatin does not contain the cause of spongiform encephalopathy (mad cow disease or Creutzfeld-Jakob disease). Similarly, human serum albumin is suspected to be infected and thus can transmit the infection to the person receiving the vaccine. The blood from which the serum albumin will be purified is selected and previously tested for the presence of pathogens, and there is no documented case of vaccine transmission.

Out of the vaccines available in Croatia, vaccines that include stabilizers are those against measles, rubella, tick-borne encephalitis, diphtheria, tetanus, pertussis, poliomyelitis, Haemophilus influenzae type b, whooping cough, mumps, rabies, and yellow fever.

Of the other excipients added for the purpose of formulation, surfactants and flavoring agents must be mentioned. Of the surfactants, polysorbate 80, a standard food industry emulsifier, is most commonly used and acts as a solubilizer. Its use is completely safe, especially in such low concentrations as there are in vaccines. Sucrose, also safe and omnipresent, is used as a flavoring agent in oral vaccines.

Substances in the production process

In vaccine production, substances that are not present in the vaccine itself or are present in barely measurable quantities at the end of the production process are often used. These are technically not excipients but are still listed as such. These include antibiotics, egg or yeast proteins, formaldehyde, and acidity regulators.

Antibiotics are added during vaccine production for the same purpose as preservatives – to ensure the sterility of the vaccine and prevent the growth of microorganisms. Penicillins, cephalosporins, or sulfonamides – groups of antibiotics to which allergic reactions are more common, are never used for this purposes. Approved for use for these purposes are neomycin, streptomycin, polymyxin B, gentamicin, and kanamycin.

Egg proteins can be found in traces in the flu vaccine because the flu virus we use in the vaccine is produced in the egg, dating back 70 years. The vaccine is produced by inoculating three different strains into fertilized chicken eggs. At the end of the process, the strains are combined and provide protection from the strains present that season. This method of producing vaccines is fast and inexpensive, which, in this case, is important since the strains of the flu virus change every year, therefore new vaccines are produced every year. Although the vaccine is ultimately purified, a small amount of egg protein will likely remain in the final product. Egg proteins are potentially dangerous only for people who are allergic to eggs, only 0.5% of the population. As children are actually at the greatest risk of allergic reactions to eggs, vaccines routinely used in children are not produced in eggs, but in the cell culture of the chicken embryo fibroblasts. Residual proteins in such vaccines reach only 40 pg (picograms, 10-12 grams), which is not enough to cause an allergic reaction even in children.

Formaldehyde, an organic compound normally present in our body is used in the production of vaccines to inactivate the toxins from viruses and bacteria. Although a small amount of formaldehyde may remain in the vaccine, it is harmless because it is immediately transferred to the water upon entering the body. The amount of formaldehyde normally present in the blood of a two-month-old child is 1.1 mg which is ten times more than the amount of formaldehyde that can be left behind in vaccines (0.1 mg). In fact, one pear contains 50 times more formaldehyde than a vaccine. Our body needs formaldehyde for the synthesis of purines and amino acids at a concentration of 2.5 μg of formaldehyde for every 2.5 mL of blood. Which, again, is significantly more than the amount contained in the vaccine.

Vaccines are products of the pharmaceutical industry in which excipients are most (if not only) criticized and there exists the greatest (unjustified) fear of them. It is important to understand that the excipients used in vaccines, as in all other pharmaceutical forms, are of strictly controlled quality and that they have been in use for almost a century in which we have exclusively made progress. Numerous agencies and laboratories around the world care about what, how much, when, and to whom it is administered, and their safety of application and effectiveness are constantly checked and evaluated so that nothing would be left to chance. Their application has been repeatedly proven to be safe and necessary.

Q&A

1. Vaccines contain mercury which is neurotoxic.

Before we touch upon the vaccines it is necessary to resolve a few things relating to mercury. Namely, mercury can come in the form of organic and inorganic compounds. Inorganic mercury can be further divided into elemental (Hg0) and monovalent and divalent cationic mercury (Hg⁺ and Hg²⁺). It is known that small marine organisms lead to methylation of inorganic mercury, resulting in a whole range of various organic mercury compounds. The best known is methylmercury, which accumulates in the food chain if the environment is polluted and the initial methylation occurs in marine organisms. The toxicity of a particular form of mercury depends precisely on its physicochemical properties. Changes in the redox potential (more accurately, whether the mercury is in the elemental state or in the form of a cation) affect the chemical activity of the metal, and thus its distribution in the body, as well as the possibility of transport across the biological membrane [46].

Elemental mercury is toxic when its vapors are inhaled. Since it is lipophilic, it is rapidly absorbed in the lungs through the membranes of the alveoli and enters the circulation where it has two possible destinies: part will be oxidized in erythrocytes to divalent cation, and part will cross the blood-brain barrier, be oxidized by catalase to divalent cation and bind to the thiol groups of amino acids (-SH groups), resulting in neurotoxicity. Thus, for mercury to, in this case, even reach the brain at all, it must be lipophilic in order to cross all membranes and cause neurotoxicity. On the other hand, if a person comes in contact with cationic mercury, neurotoxicity will not occur because such a form of mercury cannot cross membranes. It will bind to plasma proteins [46].

When we talk about methylmercury, the cause of poisoning in Japan and Iraq, we must emphasize that it has a high affinity for the thiol groups of amino acids, and is in itself lipophilic due to the organic component. Scientists have proven that methylmercury will bind to thiol groups of molecules and, using membrane transporters for specifically those molecules, cross the blood-brain barrier and lead to neurotoxicity [46].

Hence, from the examples listed above, we can conclude that toxicity primarily depends on the physicochemical properties of each element and we cannot generalize by sticking a toxicity label to a whole number of different chemical compounds based on the knowledge of the action of a single compound.

Let us get back to vaccines. Certain vaccines contain a preservative called thiomersal. Thiomersal is an organometallic compound, more accurately an organic compound of mercury. Discussions about the harmful effects of thiomersal began after the disasters in Japan and Iraq where large numbers of people were poisoned by methylmercury through food. However, even the website of the World Health Organization (WHO) states that there is no scientific evidence of its use in vaccines, but that elimination or reduction of thiomersal in certain vaccines can not only cause microbiological contamination of the vaccine but can also affect the quality, safety, and efficacy of the vaccine [47]. Moreover, there are different stories about the connection between thiomersal and the development of autism, but studies have also confirmed that there is no connection between those two notions [48].

The conclusion is that we cannot compare two completely different chemical compounds based solely on the fact that they contain the same atom. By such an analogy, we can say that table salt is the most lethal agent we know: in its structure, it contains chlorine, which was used as a war poison in the First World War, and sodium, which self-ignites when in contact with water.

2. Vaccines contain aluminum which causes encephalopathies.

It is true, aluminum ions (Al³⁺) do act neurotoxic in a way that they bind to the negatively charged membranes of neurons and lead to oxidative stress in the brain. However, as we have been repeating all this time, when we talk about the toxicity of a compound we have to keep in mind several variables, and they are dose, duration of exposure, mode of administration and the chemical form of the substance applied. For a substance to have a toxic effect on the body, it must be taken in a dose that exceeds the toxicity limit, or it must be repeatedly taken into the body in lower doses in order to return to a state of toxic action. Furthermore, whether a person comes in contact with aluminum by drinking aluminum-contaminated water or by vaccination, is not the same because the route of administration is different, and thus the pharmacokinetics (more precisely, the toxicokinetics) of the compound. For the chemical form, we have already clarified in the previous statement on the example of mercury and mercury compounds.

Aluminum in vaccines is found in the form of aluminum salts that act as adjuvants (explained in the section “Auxiliary substances in vaccines”) [49]. One study examined aluminum concentrations in infants receiving FDA-approved vaccines [50]. It has been proven that the amount of aluminum in the body due to vaccines is only slightly higher than the one obtained through food and breast milk and that the sum of these concentrations, i.e., the total amount of aluminum in the body is far below the MRL (minimal risk level). Another study explicitly stated that, despite the difference in bioavailability of aluminum ingested by water, inhalation, and vaccination, the total aluminum exposure is far less than the level that causes neurotoxicity [51].

Furthermore, if someone is interested in the proportion of aluminum salts in vaccines, they can also check this in the description of the properties of the drug available on the HALMED website.

After all, let us not forget to mention one more thing: pills used for elevated stomach acid levels contain precisely aluminum hydroxide as an active ingredient. Led by the premise that aluminum causes encephalopathies, the vast majority of the population (especially today due to a stressful lifestyle and frequent stomach problems) should suffer from the said disease.

3. Mercury, antifreeze, phenol, formaldehyde, aluminum are put in the vaccine.

As we already mentioned, vaccines do not contain elemental mercury or aluminum, but some vaccines contain their salts or organic compounds, whose pharmacological and toxicological profiles differ significantly. The claim that antifreeze is in the composition of the vaccine is simply incorrect. Namely, antifreeze is mostly made up of toxic ethylene glycol, whereas vaccines can contain propylene glycol, which is in concentrations that are harmless to the body, and is used to inactivate some viruses or as a preservative. Phenol, i.e., its derivatives are used as preservatives in vaccines and their concentration must meet pharmacopoeial requirements. Formaldehyde is utilized in vaccine production as an antigen-inactivating agent. For example, anatoxin is obtained by treating tetanus toxin with formaldehyde, which is immunogenic but will not cause a dangerous disease. Formaldehyde, in certain concentrations, indeed is toxic to humans. However, the European Pharmacopoeia clearly prescribes that the amount of formaldehyde in vaccines should not exceed 0.2 mg/mL. By comparison, there is approximately 2.5 mg/mL in our blood. The formaldehyde administered by the vaccine is rapidly metabolized and at that dose does not pose any risk of toxic effects.

4. Why do we get vaccinated against diseases that are rare anyway?

The diseases we are vaccinated against are rare precisely because of the systematic vaccination of society. Before the use of the vaccine, some diseases were relatively common, and mortality from these diseases was many times higher than today. Also, the health system continuously conducts assessments of the justification of vaccination against a certain disease. For example, the obligation to vaccinate against tuberculosis is usually lifted when the incidence of tuberculosis in a specific area falls below the prescribed agreed limit.

5. Why are we vaccinated against diseases if they have been eradicated?

We do not get vaccinated against diseases that have been eradicated. Such is the example of smallpox, the last case of which was recorded in 1977, and in 1980 the WHO declared them eradicated. Today, except in extreme situations, we do not vaccinate against smallpox, although the older generations have been systematically vaccinated.

6. Vaccines weaken children’s immunity.

Just as bodybuilders need to regularly exercise their muscles to maintain strength and fitness, so each person needs to exercise their immune system in order to be ready to defend themselves against potential infections. Exercise of the immune system is done in such a way that the person is in contact with antigens and that, as a consequence of contact, they develop antibodies that protect against any exposure to pathogens. The way in which we “help” our immune system to build is precisely through vaccination. As explained in more detail in the “Vaccination and the immune system” chapter, by ingesting a small amount of killed pathogens or fragments of pathogens, we serve, to our immune system, on a tray a tool with which it will build small soldiers called antibodies. In each following contact with the pathogen, these soldiers will be ready for war and defend their territory, i.e., the organism.

But let us get back to the premise of the weakening of the immune system. This is one of the common arguments of the opponents of vaccination which does not hold water. Evidence that it does not hold water is a study published in 2018 where they investigated the correlation between vaccines and infections (i.e. diseases) for which children aged 2 to 4 years are not vaccinated. Simply put, it was observed whether vaccinated children get sick more often from so-called non-vaccine-targeted infections. The result refuted this correlation [52].

7. Vaccination causes autism.

The claim that vaccines cause autism was popularized by Andrew Wakefield thanks to his 1998 scientific paper. However, shortly after that, some of the largest retrospective cohort studies to date showed that vaccines, especially those against measles, mumps, and rubella (MMR), do not cause autism, as well as the preservative thiomersal [53,54]. After that, a large number of scientists and investigative journalists led by Sunday Times journalist Brian Deer got involved in Wakefield’s case. In this way, it was revealed that Wakefield was in a great financial conflict of interest. He falsified data from his work and performed unethical procedures on children with autism, such as colonoscopy and lumbar puncture. Interestingly, he also bought blood samples for £5 from children at his son’s birthday party [55]. After a lengthy trial, Wakefield admitted to all counts of the indictment and was tried in 2010 at the UK General Medical Council before the members of the profession [56,57]. A few days later, his original scientific work was withdrawn, and he lost his medical license the same year [58]. Hundreds of papers refuting Wakefield’s claims have been published to date. One such recent study is the popular “Danish study” which, on a sample of more than 650 thousand children, thus covering the largest number of autism cases to date, showed that vaccines do not cause autism, even in children at the highest risk of developing it.

8. It is better to acquire immunity naturally than artificially, i.e., by vaccination.

While it is true that it is possible to acquire immunity against a particular disease after having overcome it, it is by no means better than being vaccinated against that disease. Namely, there is no case of a registered vaccine where the consequences of vaccination are worse for the body than the symptoms of the disease we are vaccinated against. Moreover, getting sick from certain diseases we get vaccinated against can be deadly. Furthermore, against some diseases, it is not possible to develop immunity by overcoming them. One such example is tetanus because tetanus toxin is simply not immunogenic enough. On the other hand, the tetanus toxoid, which is present in the vaccine alongside an adjuvant, is sufficiently immunogenic to create an immunity that usually lasts for decades.

9. Vaccines contain various toxins.

Vaccines simply do not contain toxins. Such a thing makes no sense medically or ethically. The complete qualitative and quantitative composition of all vaccines registered in the Republic of Croatia can be found on the official website of the Agency for Medicinal Products and Medical Devices of Croatia (HALMED) and in the databases of summaries of drug properties such as the Mediately drug database [59,60].

10. Most people get sick after vaccination.

It is not true that most people get sick after vaccination. It is possible to get sick after vaccination, but this does not mean that the vaccine is the cause of the disease, but that the person was exposed to the pathogen before they developed immunity to the disease they are being vaccinated against. Also, for most vaccines, there is not even a theoretical probability of contracting the disease we are being vaccinated against, since these vaccines do not contain the pathogen, merely a specific antigen.

11. Vaccines cause serious side effects, diseases, and can even lead to death. Not to mention the long-term harmful effects on our health.

It is true that vaccines, in some cases, can cause serious side effects, such as an anaphylactic reaction. However, the frequency of these side effects is several times less in magnitude than the frequency of the same symptoms in the diseases we are vaccinated against. For example, 1-3 people per 1000 suffering from measles develop encephalitis, a severe and potentially fatal complication of the disease. On the other hand, only 1 person per million vaccinated against measles develops the same side effect.

12. 2in1, 3in1, and other types of combination vaccines are far worse than ordinary ones and cause harmful consequences to human health.

Combination vaccines are vaccines that contain antigens needed to develop immunity against more than one disease. Such vaccines are in most cases better than those protecting only against one disease because they reduce the number of stings and visits to the doctor, which of course also carries its risks [61].

13. Vaccines cause allergies.

Several large clinical studies have been conducted to determine whether vaccines cause allergies, asthma, atopic dermatitis, and similar conditions. None of these studies showed an increased incidence of these diseases in vaccinated children. In fact, some studies have shown that, for example, asthma is less common in vaccinated children[62,63,64].

14. Diseases we are vaccinated against are not as dangerous as they say. What is the worst thing that can happen if a person gets infected with measles? They will get over them.

Even if the disease is not fatal, it is completely unreasonable and unethical to intentionally expose a child to a disease that causes certain symptoms and suffering. However, the diseases we vaccinate against are not harmless at all. Measles, considered by many to be a childhood disease, took 2.6 million lives in 1980 alone. That number, thanks to vaccination, has today been reduced to tens of thousands of deaths a year, which is still a devastating statistic. The WHO estimates that more than 20 million lives have been saved thanks to measles vaccination in this millennium alone. Most of the diseases we get vaccinated against are even more deadly than measles. For example, the mortality rate from tetanus even with all the modern medical care is about 10% [65,66].

15. No one should be forbidden to get vaccinated, but those who do not want to do so should not have to. Vaccination concerns only the health of the person being vaccinated and there should be a right to choose.

Vaccination should by no means concern only those being vaccinated. Namely, there are people in society who, due to being immunocompromised, are not allowed to be vaccinated for medical reasons, and such people are protected by the society’s collective immunity. Additionally, the health of young children who are not old enough to receive a particular vaccine according to the vaccination calendar depends on their environment. Finally, vaccines are not 100% effective in preventing diseases. Consequently, there is a small portion of non-reactors to the vaccine in our society that we are also protecting with collective immunity.

16. Vaccines turn our children into homosexuals. It is all part of the new world order and the gay lobby’s plan to have fewer of us in the world.

Vaccination has been carried out systematically for more than a hundred years. If the pharmaceutical industry really wants to depopulate the Earth or reduce the number of people using vaccines, then it is doing a very poor job of it. Namely, the population on the planet has increased fivefold since the beginning of the systematic vaccination of society and has been growing at a rate of over 1% per year for centuries [67].

Currently, there is no mechanism that could change a person’s sexual orientation on a biological basis. Consequently, such a thing is not achievable even via vaccines. After all, if this were true, then the share of people of homosexual orientation in the total population would correspond to the vaccination trends. However, the studies we have show that this is not the case [68].

17. Many vaccines today, including influenza vaccines, contain virus-infected animal tissues and cells infected with bacteria from dogs, pigs, chickens, cows and calves, eagles, African green monkeys, and human fetuses.

Unlike most bacteria, viruses cannot live on surfaces (substrates) that are not alive, i.e., that do not contain cells. For this reason, the viruses used in the process of vaccine production are grown on cell lines. It is important to emphasize that the cell lines used in vaccine production are not obtained from humans or animals, i.e, no one has ever been harmed in order for their cells to be used in vaccine production. Viruses obtained from cell lines are collected and then used in the technological process. Hence, the vaccine does not contain the cell line from which the virus was acquired. Of course, it, therefore, does not contain parts of animal organisms either.

18. Why are the media and health professionals still raging about measles when almost everyone has already been vaccinated?

As long as, in a certain region, there are cases of the disease that we had planned to eradicate by now, the media and health professionals have a right to “rage”. Even when the vaccination against a disease is high, so-called “pockets” are often present, i.e., parts of the population with extremely low vaccination coverage. Exactly those pockets are the source of new epidemics. For this reason, it is necessary to constantly point out the importance of vaccination.

19. Vaccines do not go through ‘rigorous safety tests’ at all.

Vaccines, like any other medication, are probably the most tested product of any industry at all. Namely, in order for any drug to be registered, it is necessary to perform extensive phases of clinical trials, which often takes years and costs hundreds of millions of dollars. The drug that is released to the market constantly undergoes processes that control the quality, efficacy, and safety of use and is monitored by regulatory authorities. In addition to all this, vaccines are also used in children and are accompanied by numerous controversies, which is why they are often tested even more than other drugs. Due to the extensive production process and quality and safety control, for some vaccines, it takes up to three years to be produced, and even the smallest error in a batch is enough to cause the entire batch to have to be destroyed [69].

Translated by: Josipa Radeljak