(1) Residual symptoms continuing after acute infection recovery;

After the WHO declared a pandemic ^[7] on 11th March 2020, most countries, to avoid the pandemic spread and limit the number of casualties, introduced several strict nonpharmaceutical interventions ^[8], namely (1) improved diagnostic testing and contact tracing; (2) isolation and quarantine for infected people; and (3) measures aimed at reducing mobility and creating social distancing (containment, mitigation, and suppression).

Most countries decided on the following containment measures:

• Physical distances > 1.5 m;
• Wearing masks and gloves;
• Stay-at-home orders;
• School and workplace closures and activation of distance learning and smart working;
• Closure of museums, commercial parks, gyms, and swimming pools;
• Cancellation of public events;
• Restrictions on size of crowds;
• Seat limitation on public transport to ensure the right distance between passengers;
• Restrictions on internal and international travel;
• Measurements of body temperature at the entrance of closed areas (<37.5 °C);
• Ensuring disinfection rules are followed in public areas such as public transport, shopping areas, schools, and universities;
• Protecting healthcare workers with appropriate personal protection equipment (PPE).

The main objective of these interventions is to reduce the reproduction number (Rt) of the virus. The Rt is defined “as the mean number of secondary cases generated by a typical primary case at time t in a population calculated for the whole period over a 5-day moving average” ^[9]. Thus, Rt is an indicator measuring the transmission of SARS-CoV-2 before and after the interventions.

In the past, against some infectious diseases, medicine was ineffective ^[10] Thucydides in his History of Peloponnesian War (II, vii3-5) wrote “The doctors were unable to cope, since they were treating the disease for the first time and in ignorance: indeed, the more they came into contact with sufferers, the more liable they were to lose their own lives.” Indeed, the only possible way to escape the plague was to avoid any contact with infected persons and contaminated objects. The Italian poet Giovanni Boccaccio (1313–1375), in his book The Decameron (1349–1353), tells the story of ten people, seven women and three men, who entertain themselves with novels while in isolation from the plague of Florence in a villa in the countryside. In the first chapter, Boccaccio describes how the plague struck the city of Florence, how people reacted, and the staggering death toll. Boccaccio, echoing Thucydides, also wrote: “Neither a doctor’s advice nor the strength of medicine could do anything to cure this illness”.

Accordingly, the procedure of obligatory quarantine was introduced as a measure to isolate and separate people, animals, foods, and objects that may have been exposed to a contagious disease. Quarantine is from the Italian “quaranta”, meaning forty. For millennia, contagious diseases were believed to be a divine punishment for sinners. Thus, in the Old Testament, God destroyed the earth with water for 40 days (בראשית: Genesis 7:4); Noah waited for forty days after the tops of mountains were seen after the flood (בראשית: Genesis 8:5–7). Moses was on Mount Sinai for 40 days (Exodus שְׁמֹות 24:18): “Then Moses entered the cloud as he went on up the mountain. And he stayed on the mountain forty days and forty nights”. In the New Testament, Jesus was tempted for 40 days (Matthew 4:2, Mark 1:13, Luke 4:2). There were 40 days between Jesus’ resurrection and ascension (Acts 1:3). Eugenia Tognotti ^[11] and Gensini et al. ^[12] reviewed the origin of quarantine from the time of the Bible to nowadays.

Nevertheless, 40 days may derive from the Pythagorean theory of numbers; according to Pythagoreans, the number 40 was considered to be sacred. Hippocratic teaching in the 5th century BCE established that an acute illness only manifested itself within forty days ^[12]. Only during the epidemic of 1347–1352 was an organized institutional response to control disease set up. Quarantine was introduced, for the first time, in 1377 by the Rector of the seaport of Ragusa (Dubrovnik, Croatia), and the first stable plague hospital (lazaretto or quarantine station) was built by the Republic of Venice in 1423 on the island of Santa Maria di Nazareth ^[12]. The term lazaretto, usually referred to as Nazarethum or Lazarethum, is related to Lazarus, who was brought back to life by Jesus (John 11:1–45) and/or to the Order of Saint Lazarus of Jerusalem, a Catholic military order founded by crusaders around 1119 at a leper hospital in Jerusalem, as a hospital and military order of chivalry ^[13]. The Venetian system became a model for other European countries: in 1467, Genoa adopted the Venetian system, and in 1476, in Marseille, France, a hospital for persons with leprosy was converted into a lazaretto ^[14]. Afterward, quarantine became the foundation of a coordinated disease-control strategy that included different measures such as isolation, sanitary cordons, bills of health issued to ships (certification assuring the absence of disease), sanification (i.e., fumigation), and disinfection. Girolamo Fracastoro, Latin Hieronymus Fracastorius (1478–1553, Verona), physician, poet, astronomer, and geologist, was the first to propose, in 1546, a scientific germ theory of disease. In his book “On Contagion and Contagious Diseases”, he affirmed that each disease is caused by a different type of rapidly multiplying minute body and that these bodies are transferred from the infector to the infected in three ways: by direct contact; by carriers such as soiled clothing and linen; and through the air.

In the millennial history of mankind, vaccination is a relatively young intervention of primary prevention. For about 200 years, vaccination strategies have had a profound effect in shaping the natural history of infectious diseases. Smallpox eradication represents the most impressive success of a vaccination strategy. As discussed above, smallpox represented a dreadful menace throughout the centuries ^[15]. It was common knowledge that smallpox survivors acquired immunity to the disease, so the practice of variolation, consisting in having healthy individuals inhale dust from smallpox lesions, become common in Europe and in North America. At the end of the 18th century, there were anecdotes regarding immunity to smallpox in people previously infected with cowpox, a zoonotic pathogen ^[16]. In 1798, Edward Jenner published his first observations on the benefits of inoculating biological material from cowpox lesions in humans, to protect from smallpox, and for the first time, the term “vaccination” (from Latin vacca, English cow) was used ^[17]. Initially, vaccination was perpetuated by transferring fluids from individual to individual, but this practice reduced the strength and the duration of protection. The next step was to deliberately infect cows to mass-produce sufficient material (“lymph”) for vaccination ^[15]. However, this practice led to an increase in the frequency of transmission of secondary infections, including syphilis. This issue was resolved after the observation of bacterial inactivation by glycerin made by Robert Koch ^[18], so lymph was treated with glycerin before inoculation. At the end of the 19th century, Louis Pasteur made observations that strongly enhanced the development of vaccines. Studying chicken cholera, he noticed that chickens inoculated with cultures left out over a prolonged period were protected from subsequent infection with fresh material, suggesting the existence of protective immunity induced by the inoculation of “aged” material. These observations would lead Louis Pasteur to the production of antirabic vaccination, using material from an infected dog’s brain, exposed to dry air ^[19].

In the meantime, studies on the immune system contributed to unravel the mechanisms of host defense from pathogens. In particular, studies by Elie Metchnikoff in 1884 ^[20] introduced the concept of cellular immunity, and Paul Ehrlich published his theory of receptor of immunity in 1897, paving the way for the development of antitoxins against pathogens such as diphtheria. At the end of the 19th century, five human vaccines were in use: two live virus vaccines (smallpox and rabies) and three dead bacterial vaccines (typhoid, cholera, and plague.).

The first half of the 20th century saw the development of passive immunization, with the production of antitoxins for diphtheria and tetanus. At the same time, new vaccines against tuberculosis, bacillus Calmette-Guérin (BCG), yellow fever, typhus, influenza A, and pertussis were developed, and the first combination vaccine, against diphtheria, tetanus, and pertussis was produced in 1948. Progresses in cell culture lead the way to the techniques of virus attenuation through passages on tissues and cellular monolayers, thanks to the studies of Hugh and Mary Maitland in 1928 and Ernest William Goodpasture in 1931, who first used the chorioallantoic membranes of a fertile hen’s egg as a culture medium for sterile passage of viruses. In the 1900s, poliomyelitis (caused by the poliovirus) represented another threat to public health. The virus spreads from person to person and can invade an infected person’s brain and spinal cord, causing paralysis. Better hygiene conditions led to an increase in the age of infected children, which in the previous centuries were breastfed, protected by maternal antibodies. The older age of infected children led to frequent polio outbreaks. The first effective antipolio vaccine was a formaldehyde-inactivated (or “killed”) PV vaccine (IPV) developed by Jonas Salk in 1955. A second vaccine which was demonstrated to be both safe and effective was the oral (or “live”) PV vaccine (OPV) was developed by Albert Sabin in 1963 ^[21]. Jonas Salk and Albert Sabin decided not to patent their vaccines and therefore sacrificed billions of dollars in potential royalties, approximately USD 500 million for Salk and approximately USD 1.2 billion for Sabin. Nowadays, thanks to the vaccines, the virus remains endemic only in Afghanistan and Pakistan.

In more recent years, research started to focus on multiple vaccines, starting from live viruses attenuated by multiple passages on cultured cells, such as the vaccine against measles, mumps, and rubella. Exploiting the newly available techniques of molecular biology, newly designed vaccines started to be produced. Japanese researchers developed an acellular pertussis vaccine based on two of the main protective antigens of Bordetella pertussis. Research on polysaccharide vaccines led to the development of new vaccines against pneumococcus, meningococcus, and Haemophilus Influenzae type B. Recombinant DNA technology and the possibility to produce recombinant protein in vitro paved the way for the release of the anti-hepatitis B vaccine. Under the urgent need to battle COVID-19, different SARS-CoV-2 vaccines, including the inactivated virus vaccine, nucleic acid vaccine, adenovirus vector vaccine, and viral subunit vaccine, have been developed ^[22]. In the history of vaccines, COVID-19 vaccines are unique for the extraordinary rapidity of their production. In recent years, mRNA vaccines have started to attract great attention thanks to their potential to:

(1) Speed up vaccine development;

(2) Simplify vaccine production, scale-up, and quality control;

(3) Be produced and scaled up in a predictable and consistent fashion regardless of the antigen;

(4) Have improved safety and efficacy;

(5) Challenge diseases impossible to prevent with other approaches;

(6) Enable precise antigen design;

(7) Generate proteins with a “native-like” presentation;

(8) Express proteins stabilized in a more immunogenic conformation or expose key antigenic sites;

(9) Deliver multiple mRNAs to the same cell;

(10) Allow the generation of multiprotein complexes or protein antigens from different pathogens, thus creating a single vaccine against several targets.

Moreover, mRNA is characterized as:

(11) Being noninfectious;

(12) Being nonintegrating;

(13) Being degradable by normal cellular processes soon after injection;

(14) Decreasing the risk of toxicity and long-term side effects;

(15) Not inducing vector-specific immunity;

(16) Not competing with pre-existing or newly raised vector immunity that could interfere with subsequent vaccinations.

An mRNA vaccine is based on the principle that mRNA is an intermediate messenger to be translated to an antigen after the delivery into host cells via various routes. The mRNA is synthesized in the laboratory by transcribing a DNA template of the genetic sequence encoding the immunogen. In the case of SARS-CoV-2, the spike (S) protein is identified as the immunodominant antigen of the virus. The most important problem is that mRNA is unstable, easily recognized by the immune system, and rapidly degraded by nucleases after entering the body. mRNA vaccines do not enter the nucleus but need to pass through the cell membrane, a negatively charged phospholipid bilayer, to enter the cytoplasm and then be translated into the target protein. Different delivery systems for mRNA vaccines, such as viral and nonviral vector delivery systems, may be utilized. Vectors based upon lipids or lipid-like compounds are the most common nonviral gene carriers.