The vaccine - type of vaccine, correlation between vaccine and immune system

vaccine

1. The Vaccine

2. WHAT IS VACCINATION ? Vaccination is a prophylactic measure that aims to confer a specific active immunity against a specific infectious disease, comparable to that given by the disease itself Immunity : acquisition by the human organism of specific defensive abilities versus different infectious agents in order to preserve the integrity of the organism Some vaccinations: induced immune protection lasts a lifetime in other cases periodic reminders are needed to achieve prolonged immunity

3. VACCINE The vaccine is a substance capable of stimulating the immune system against viruses or bacteria . “A preparation of killed microorganisms, living attenuated organisms, or living fully avirulent organisms that is administered to produce or artificially increase immunity to a particular disease”. In some cases the vaccine alone is not sufficient to produce a good immune response and therefore a substance that enhances the effect called adjuvant is needed Adjuvant is a substance that is often added to a vaccine and has the function of increasing the immune response versus antigen

4. Disease prevention through vaccination has proven to be extremely beneficial reducing human morbidity , reducing mortality and suffering decreasing direct and indirect health costs. Today, vaccines protect against more than 25 debilitating or life-threatening diseases, including measles, polio, tetanus, diphtheria, meningitis, flu, typhus, uterian cervical cancer, and Sars CoV2 . For these reasons, interest in developing new vaccines and improving existing ones is growing rapidly around the world.

5. A vaccine is a biological preparation with high antigenic power that is able to induce an active immunity state against certain pathogens microorganisms . The components of the vaccine interact with the human immune system producing a specific humoral response (antibodies ) without exposing the subject to the pathology and its potentials complications . Induce immunity and immunological memory similar to those of natural infection but without the risk of disease . A vaccine typically contains a pathogen, or its part, and is often made up of: weakened or killed forms of microbe microbial toxins surface proteins of the microbe microorganism genome

6. Purpose of the vaccines prophylaxis : to prevent or improve the effects of a future infection by a natural or "wild" pathogen. antigen is introduced into a person's body and aims to create antibodies to this antigen. therapeutic used to help the body fight a disease that has already occurred, such as eg . the cancer. Vaccines contain dead or inactivated microorganisms or their purified products. There are different types of vaccine: Whole microorganism Parts of microorganism : Toxoids , Virus like-particles (VLP), Nucleic acids , Transgenic vectors of recombinant antigens

7. Type of vaccine BACTERIC Living attenuated : BCG, oral anti typhus Killed : anti pertussis , cholera Anatoxins : anti- diphtheria , anti- tetanus Surface antigens : Hib , antipneumus and meningococcus VIRAL Living attenuated : oral polio, anti- rubella , anti- measles , anti- mumps , Complete inactivated : anti- flu , injectable polio, anti- rabies Antigens surface : anti HBV Nucleic acid: Sars-Cov2

8. Correlation between vaccine and immune system The immune system is a host defense system that includes many biological structures and processes within an organism that protects against disease . To function properly, an immune system must detect a wide variety of pathogens (bacteria, viruses, parasites) and distinguish them from healthy body tissue . Distinguish between “self” and “non self” The “self” molecules are those that make up our body, and recognized as an integral part of it, “non-self” molecules are instead identified as foreign and then fought

9. In many animal species, there are two major subsystems of the immune system: the innate immune system the adaptive ( or acquired) immune system. Both subsystems use humoral immunity, antibody, and cell-mediated immunity to perform their functions . Innate immunity is ancestral immunity, or that immunity that has developed and perfected over the course of evolution and which represents the first line of defense of our army. It is also capable of recognizing a very limited number of receptors, using sensors encoded by genes already present in germ cells and expressed in all cell types. Adaptive immunity, present only in vertebrates, acts as a second line of defense as it requires a lag period of time to act from the moment of contact with the infecting agent. Unlike innate immunity, it maintains a memory and is able to recognize a large number of molecular configurations present in the agents of infection. This results in a better response to subsequent contact with the same pathogen. This process of acquired immunity is the basis of vaccination

10. HUMORAL IMMUNE RESPONSE A gainst microroganism (virus, bacteria, fungi..) Only the B lymphocytes to bind the antigen are activated Clonal selection in which the activated B lymphocyte proliferates the microbial antigens bind to the B lymphocytes that presenting the specific receptor Two types of cells are produced: EFFECTIVE CELLS or PLASMACELLS (Primary immune response) they eliminate the antigen, after die . These cells produce ANTIBODY MEMORY‘S CELLS (Secondary immune response) they are long-lived, and when a new attack by the same antigen occurs, they multiply and produce: New plasmacells New memory’s cells

11. THE LYMPHOCYTES B RESPONSE Memory cells Antibody Antigens

12. The ANTIBODY The Ab are protein produced by the plasma cells of B lymphocytes when activated they have the same composition as specific antigen receptors they are secreted into the blood, lymph and intercellular fluids have the task of blocking and neutralizing the antigen, forming an Ag-Ab complex which will then be recognized by the macrophages IMMUNOGLOBULIN there are 5 different types of immunoglobulins, but all characterized by 4 Y-shaped polypeptide chains, 2 light chains (LC) and 2 heavy chains (HC). Each chain is made up of a: Constant region: determines the Ab class Variable region: the amino acid sequences determine the different Ab specificity.

13. The components of the vaccine are absorbed by phagocytic cells, macrophages and dendritic cells (DCs), peripheral tissues and by expressed receptors that recognize pathogen-associated molecular patterns (PAMP). Professional antigen presenting cells (APCs) that have absorbed the Ags become activated and migrate to nearby lymph nodes. Inside of them, the Ag processed by the APCs is presented to the lymphocytes which, recognizing the Ag and receiving the appropriate costimulatory signals, are activated. These antigen-specific B and T lymphocytes clonally expand to produce more progenitors that recognize the same Ag . In addition, memory B and T cells are formed which provide long-term (or life-long) protection against infection with the pathogen.

14. TYPES OF VACCINE Whole micro- organism It can be inactivated , so it is not able to replicate it self: the pathogen is first cultivated and then inactivated by physical agents ( i.e.heat ) or chemical agents ( i.e.formaldehyde ), or it can belive attenuated : it is used an attenuated mutant of the pathogenic microorganism ( Bacillus of Calmette-Guerrin ). Bovine-type tuberculosis mycobacteria ( M.bovis ) was attenuated by 230 passages (13 years) on growing medium These types of vaccines are very effective , and generally a second administration or the use of adjuvants is not required . But , in particular , attenuated vaccines present a risk at the level of manufacture or administration since they can revert to a pathogenic form . Newer generation vaccines improve safety by removing the whole microorganism from the formulation

15. Parts of microorganism : The vaccine contains only certain Ags that can be obtained by purification from microbial cultures ( subunit -vaccine),or the Ags can be produced through DNA recombinant techniques ( recombinant vaccine),or also through chemical synthesis ; toxoids : bacterial toxins ( usually exotoxins ) due to their highly antigenic property are inactivated and used to prepare vaccines ( toxoid vaccine); virus- like particles (VLP): nano structures that resemble the structures of viruses , composed by structural proteins that can be arranged in several layers and can also contain a lipid outer envelope . VLPs trigger a high humoral and cellular immune response and they are safe because of their lack of viral genomic material , which enhances safety during both manufacture and administration ; Nucleic acids : the vaccine is composed by DNA or RNA molecule that encodes for a microorganism antigen . Typically the DNA vaccine are combinant plasmid purified cultures ( for eg . by E.coli ). This recombinant plasmid contains components of bacterial origin , the DNA sequence that codifies for the Ag and other additional sequences that can guide the Ag to sub cellular compartments or that have immuno stimulatory effects . More recently a new type of vaccine is composed of only nucleic acid.

16. The material traditionally used as vaccines is the whole microorganism or part of it , but this approach is sometimes limited by the fact that the material for vaccination is not efficient , not available , or generating deleterious side effects . Today there are many innovative strategies for developing new vaccines because there are many molecular biology and immunology technologies and procedures that allow us to do this . Consideration….

17. Recombinant DNA and Subunit vaccines The recombinant DNA technique allows to engineer the plasmid or genome of a microorganism (expression vector) that is able to express, in vitro, the protein of interest. This technique allows the production of large amount of a single protein (subunit) or of several proteins of an infectious agent, which can be used as subunit vaccines .

18. Innovative vaccines Dendritic cell vaccines combine dendritic cells with antigens to present them to the white blood cells of the body, thereby stimulating an immune reaction. These vaccines have shown some positive preliminary results for the treatment of brain tumors and are also tested in malignant melanoma . DNA vaccine is an alternative vaccination approach, created from an infectious agent's DNA. The proposed mechanism is the insertion of viral or bacterial DNA into human or animal cells. Some cells of the immune system that recognize the proteins expressed will attack against these proteins and cells expressing them. Because these cells live for a very long time, if the pathogen that normally expresses these proteins is encountered at a later time, they will be attacked instantly by the immune system. One potential advantage of DNA vaccines is that they are very easy to produce.  

19. Transgenic vectors of recombinant antigens: bacteria , viruses and plants can be engineered to produce vaccine Ags These recombinant antigens can be introduced into the host by the transgenic vector that produces them. In this way, non-pathogenic transgenic microorganisms that produce vaccine antigens can be introduced into the host. An example is the RVSV-ZEBOV vaccine which is used in 2018 to fight Ebola in Congo . RNA vaccine  is a novel type of vaccine which is composed of the nucleic acid RNA, packaged within a vector such as lipid nanoparticles . T-cell receptor peptide vaccines are under development for several diseases using models of Valley Fever, stomatitis, and atopic dermatitis. These peptides have been shown to modulate cytokine production and improve cell-mediated immunity .

20. RECOMBINANT PROTEINS AND SYNTHETIC PEPTIDES The production does not require the manipulation of the pathogen , avoiding the risk of accidental escape and the high bio-safety and bio-containment requirements ; Vaccine candidates can be designed even when there is limited information about the pathogen ; They can be used to overcome the natural immuno-dominance of highly variable epitopes and direct the immune responses against conserved and broadly protective epitopes . LIMIT: usually poor immunogens because they fail to be recognized as Pathogen-Associated Molecular Patterns (PAMPs) and activate innate immune responses, which is required for the full development of acquired immunity. To increase the responses against to conserved epitopes, they must be presented in an immunogenic conformation and/or accompanied by potent adjuvants.

21. Adjuvants for the enhancement of vaccines consisting of peptides or sub-units Due to the poor immunogenicity of peptide vaccines and the poor efficacy of sub-unit vaccines to induce a cell-mediated response, new technologies have been developed: (a) antigen-antibody complexes on a solid matrix that expose epitopes for both T and B cells, (b) the use of lipid vesicles (liposomes) or immuno-stimulants ( immuno-stimulating complex- iscom ). Liposomes are vesicles, with a structure that mimics that of cell membranes, which can incorporate protein subunits (but also nucleic acid) and deliver them directly to the cells of the immune system. Iscoms are prepared by mixing a glycoside, Quil A- saponin , derived from the bark of a plant with lipids and glycoproteins derived from viral pericapsids .

22. RECOMBINANT BACTERIA AS VACCINE VECTOR Thanks to advances in the fields of molecular biology and genetic engineering it is now possible to create live recombinant vectors capable of delivering heterologous antigens by the introduction of antigen-encoding genes In addition to being extensively used to produce recombinant sub- unit vaccines , bacteria can also serve as vectors for the in vivo delivery of antigens or DNA. They are useful systems for heterologous antigen presentation so they can elicit humoral and/or cellular immune responses . Several bacteria have been used as vectors , such as Mycobacterium bovis BCG, Listeria monocytogenes , Salmonellae spp and Shigellae spp . All these are capable of eliciting immune responses against important viral , bacterial , protozoan , and metazoan pathogens in animal models .

23. RECOMBINANT VIRAL AS VACCINE VECTOR Recombinant viral vectors have potential for therapeutic use because they are enable intracellular antigen expression and induce a robust cytotoxic T lymphocyte (CTL) response , leading to the elimination of virus- infected cells . So, in general they don’t need adjuvant . In most cases , virus are genetically engineered to reduce or eliminate pathogenicity . For example , in adenovirus- based vectors , the E1A and E1B encoding regions , which are needed for replication in infected cells , are deleted and replaced with the target gene .

24. The gene encoding the immunogenic protein is inserted in a plasmid, downstream of a promoter (p) and within a non-essential gene of the virus (gene for thymidine-kinase-TK ). The plasmid containing the exogenous gene and the virus DNA are used to transform a host cell (animal or human cells), within which the recombination of plasmid and viral DNA takes place with the formation of a recombinant virus. The homology between the sequences present in the plasmid and the viral sequences of the vaccine virus allows for a specific homologous recombination. The foreign protein will be expressed by the recombinant virus produced by the transduced cell. Costruction of a vaccine vector

25. VIRUS-LIKE PARTICLES , NANOPARTICLES AND MULTIMERIC PEPTIDES In the virion , structural proteins are usually arranged in tight and well-ordered conformation , which is believed to be recognized as a Pathogen-Associated Molecular Patterns ( PAMP). So, one way to increase the immunogenicity of viral antigens is to deliver them in multimeric conformation and as virus- like particles ( VLPs ). Virus- like particles are artificial nano- structures that resemble a virus. They are composed of all or some of the proteins that form the viral capsid but lack genomic material , precluding any possibility of reversion mutations or pathogenic infection . VLP preparations do not require the use of inactivating agents ( i.e.formalin ) that might destroy immunologicaly relevant epitopes

26. Example of vaccines

27. Production of modified bacterial toxins , by the same pathogenic strain (after genetic mutation ) Pertussis vaccine Using the recombinant DNA technique a Bordetella pertussis strain was obtained capable of producing a toxin whooping cough completely identical , antigenically to that of the pathogenic strain, but absolutely free of toxicity. The PT toxin gene was cloned and expressed in bacteria to map B- and T-cell epitopes and identify amino acids important for enzyme activity and toxicity. Based on this information, the gene encoding PT was mutated to produce an inactive protein. This genetically modified PT is nontoxic, highly immunogenic, and able to protect against exposure with virulent B. pertussis. The mutated toxin, produced in the laboratory on large scale, is purified and used as a vaccine

28. Conjugated vaccines formed by polysaccharides Some pathogenic bacteria are covered with a polysaccharide capsule that primarily protect the bacteria from phagocytosis, or uptake of the bacteria by immune cells. Conjugate vaccines have been developed to induce a robust immune response against bacterial capsular polysaccharides  (CPSs ). Examples of polysaccharide vaccines include Meningococcal disease caused by Neisseria meningitidis groups A, C, W135 and Y, as well as Pneumococcal disease

29. The conjugate vaccine is synthesized directly in properly engineered E.coli cells. Polysaccharide vaccine: only the sugar part of the bacterium, the capsule, is included as an antigen to stimulate the immune response Conjugate vaccine the sugar is combined with the carrier protein. Polysaccharide vaccines stimulate B cell responses, thus leading to the production of type-specific antibodies that enhance the ingestion and killing of pathogens by phagocytes. Advantages of conjugate vaccines over polysaccharide vaccines are: 1 ) stimulate an effective response even in children under the age of two years, 2 ) induce immunological memory, 3 ) lead to the production of serum IgG with high affinity for polysaccharide antigens.

30. Conjugated vaccines formed by polysaccharides Vaccines ex against S.pneumoniae N. meningitidis (serogroups A, C, W and Y) or H. influenzae , bacteria that cause meningitis are based on polysaccharide antigens of the capsule conjugated with a “carrier” protein ( conjugate vaccines ). These antigens are not immunogens and must be associated with a carrier molecule which in this case consists of diphtheria anatoxin (CRM197) or tetanus anatoxin . The function of the "carrier" protein is to provide T epitopes, so that the immune response to the conjugate is not thymus-independent .

31. Peptide or synthetic vaccines I f epitopes or antigenic determinants of immunological interest are identified in the complete structure of a protein, their sequence can be reproduced by chemical synthesis and a synthetic peptide identical to that of the virus can be created. To be immunogenic, these peptides must in any case be conjugated with a vector molecule Synthetic vaccines, in particular long synthetic peptides of approximately 25–50 amino acids in length, are attractive for HIV vaccine development and for induction of therapeutic immune responses in patients with (pre-) malignant disorders

32. Recombinant Vaccine After identifying the protein of immunological interest of a pathogen and its sequence, it is possible to isolate the gene that codes for the protein and insert it into a plasmid (recombinant plasmid), which acts as a transfer molecule for insertion into a vector expression (the type of plasmid used will depend on the type of vector ). The most commonly used expression vectors are: 1 bacteria ( E.coli ) 2 yeasts ( S.cerevisiae ) 3 baculoviruses . Bacteria have problems in correctly glycosylating the polypeptides produced, for this reason the proteins obtained have a lower immunogenic capacity, while yeasts and baculoviruses can correctly glycosylate the glycoproteins.

33. Baculovirus expression Recently, due to its enormous capacity for expression in the production of subunit vaccines, baculovirus is being used. Baculovirus is an insect virus (e.g. silkworm) that can replicate in stable insect cell lines. A viral promoter regulates the polyhedrine gene which encodes approximately 60% of the total baculovirus proteins and can be replaced by foreign genes. Through this system, proteins from various viruses, including papillomaviruses, have been produced and expressed in insect cells. It was also possible to express proteins in a form similar to virions ("VLP " or particles that look like viruses) creating a product with a high immunogenic capacity. VLP of papillomavirus

34. Vaccine against Hepatitis B: 1st Authorized Recombinant Vaccine Hepatitis B represents a major health problem: the virus responsible (HBV) as well as causing cirrhosis liver is also a carcinogen (cancer al liver ) To date, the number of chronic carriers is estimated at 300 million and can transmit the virus to anyone who comes into contact with blood, infected semen, vaginal discharge, saliva, sweat or tears, thus perpetuating the maintenance of the infection.

35. HEPATITIS B VIRUS - HBV DNA Virus with envelope The virion contain 2 antigens associated to the internal nucleocapsid : HBc Ag (core) and HBeAg and 1 antigens presente on to envelope surface HBsAg ( Hepatitis B surface Antigen ) HBsAg is the receptor used by the virus to recognize and adhere to liver cells and therefore represents the molecule of choice for the preparation of the vaccine .

36. The viral gene encoding HBsAg was cloned into a vector and then transferred and expressed in S.cerevisiae yeast. The antigen produced by yeast presents all the characteristics of the HBsAg protein native (glycosylation and other post modifications translational) Ag is purified for ultracentrifugation , chromatography and fractional precipitation ( purity > 98 %) is adsorbed on Al(OH) 3 which acts as adjuvant

37. Subunit Vaccine ( recombinant ) of hepatitis B virus

38. HPV L1 Virus Like Particles Vaccine The discovery for the development of vaccines against Human Papilloma Virus (HPV) was that proteins of the viral capsid (L1 alone or L1 + L2), expressed in microorganisms , can assemble into VLP that maintain epitopes capable of inducing in humans the immune response VLPs from all types of HPV can be produced, specific to each viral genotype. Two approved vaccines have been developed FDA : Quadrivalent Gardasil (16, 18, 6 and 11) Bivalent Cervarix (16 and 18) VLPs are virus-resembling nanoparticles formed by viral structural proteins but do not have any core genetic material. Because VLPs are not infective, they are considered as one powerful tool for a broad spectrum of applications, including vaccines and antibody development

39. In the quadrivalent vaccine L1 protein is expressed in Saccharomyces cerevisiae and generate VLPs that mimic the HPV 16, 18, 6 and 11 capsid . The purified particles are adsorbed with aluminum amorphous hydroxyphosphate sulfate acting as adjuvant . In the bivalent vaccine to obtain the expression of L1 VLP of HPV 16 and 18 a vector is used recombinant baculovirus

40. Genetic vaccines or Naked DNA Naked DNA can be used as a gene therapy for preventive and therapeutic vaccines DNA not attached to another molecule or encapsulated by a liposome or virus. The proteins are not expressed. The gene of interest is inserted into a DNA plasmid that also contains short regulatory sequences The naked DNA plasmid is injected directly into muscle tissue that expresses the protein for a desired amount of time, stimulating the immune response (or inhibition of metastasis) Muscle cells are especially good for this technique because they are large cells that are multi-nucleated

41. Delivering of genetic vaccines DNA vaccines offer many advantages: greater stability, rapid and cost-effective production, flexibility to produce vaccines for a wide variety of infectious diseases. However, their immunogenicity is often weak due to DNA degradation by nucleases and inefficient release to immune cells. Delivery systems based on biomaterials are needed. For this, micro and nanoparticles are used that encapsulate the plasmid DNA and allow the administration of the DNA vaccine. Microparticles allow for passive targeting to APCs through size exclusion and can allow for prolonged presentation of DNA to cells through degradation and release of encapsulated vaccines. Nanoparticles increased internalization, increased transfection efficiency, and the ability to increase absorption across mucosal surfaces. The selection of the appropriate biomaterial can lead to enhanced immune stimulation and activation through activation of innate immune response receptors and target DNA for professional antigen presenting cells .

42. Micro- or nanoparticulates encapsulate plasmid DNA used for vaccine delivery. In one model, particulates can be taken up by muscle cells or epithelial cells and pathogen-derived antigens are then transcribed and translated from plasmid DNA and secreted into extracellular spaces where they can be taken up by B-cell receptor mediated endocytosis or by professional APCs such as macrophages or dendritic cells. (a) Alternatively, APCs can be directly transfected by uptake of particulate encapsulated DNA. (b) Professional APCs such as macrophages are important for uptake of larger micro particles by phagocytosis, while dendritic cells are more effective at uptake of nanoparticulates by macropinocytosis .

43. Reverse vaccinology represents the last frontier in the preparation of vaccines as it does not start from the pathogenic microorganism, but from its genome . The technique consists of: 1 ) computerized analysis, using particular algorithms, of the microorganims genome for the identification of hundreds (or thousands) of sequences (or ORFs ) capable of encoding proteins, or pathogenic factors that present structural elements typical of surface or secreted proteins, recognizable by the immune system. 2 ) The ORFs selected are then cloned and expressed in suitable microorganisms ( eg E.coli ). 3 ) The proteins produced are purified and used to immunize laboratory animals. 4 ) The immune sera obtained are then tested in vitro by studying their bactericidal power against the pathogenic bacterium and 5 ) in vivo by studying their ability to protect laboratory animals, inoculated with lethal doses of the pathogenic bacterium against which one wishes to put to point the vaccine. At the end, those proteins (usually 3 or 4) that show the ability to stimulate the production of protective Ab constitute the material for the preparation of the vaccine . Reverse Vaccinology

44. Compared to the development of conventional vaccines which takes about 10 years, with reverse vaccinology it can take approximately one third of the time to identify and express the most valid antigens for vaccine development . This technique has allowed the development of the vaccine against meningitis B and offers vaccine prevention prospects also for other pathogens such as group B and group A streptococcus, Streptococcus pneumoniae and antibiotic-resistant Staphylococcus aureus.

45. COVID -19

46. Coronaviruses are enveloped pleomorphic viruses a characteristic fringe of projections composed of the protein S on their surface. These viruses have a positive ssRNA genome , which is complexed with the helically-shaped nucleocapsid (N) protein nucleocapsids . SARS-CoV-2 virus contains 4 structural proteins: spike (S), nucleocapsid (N), envelope (E) and membrane proteins (M) which are encoded by the 3 'end of the viral genome. Among the 4 structural proteins, the S glycoprotein, being a large multifunctional trans-membrane protein , plays the role of viral attachment, fusion and entry into the host cell. Protein S is made up of S1 and S2 subunits, which are further divided into several functional domains. The S1 subunit has 2 functional domains: N-terminal Domain (NTD) and Receptor Binding Domain (RBD) and the latter contains conserved receptor binding motif (RBM) SARS-Cov2 uses the human angiotensin-converting enzyme 2 (hACE2) receptor to seize the target cell through the spike glycoprotein (S-protein).

47. Antibody responses Covid -19 IgM and IgG antibodies to SARSCoV-2 are detectable within 1–2 weeks after the onset of symptoms in most infected individuals The major target of neutralizing antibodies to coronaviruses is the S protein ( S1 and S2 domains). S1 is membrane distal and contains the RBD that binds to the cellular receptor ACE2. S2 is membrane proximal and has a role in membrane fusion. The S proteins of SARS- CoV and SARS- CoV-2 are 88 % identical and both bind to ACE2 with high affinity . Certain monoclonal and polyclonal antibodies raised to the S protein of SARS- CoV can cross- neutralize SARS- CoV-2 . Antibodies that bind to the S1 RBD block its interaction with ACE2, whereas those that bind to other regions of S1 and S2 can inhibit conformational change of the S protein and block membrane fusion , respectively

48. The major types of vaccine for COVID-19 are: Inactivated viral vaccines: produced by culturing the SARS-CoV-2 virus in cell cultures and chemically inactivating it . Live attenuated vaccines: produced by generating a genetically weakened virus that replicates to a limited extent, not causing disease but inducing immune responses similar to those induced by natural infection . Recombinant protein vaccines: based on the spike protein, or on the receptor binding domain (RBD) or on virus-like particles (VLP ) Viral vector vaccines: typically based on an existing virus (usually an incompetent adenovirus for replication) carrying the sequence of the genetic code that encodes the spike protein . DNA vaccines: based on plasmids, modified to carry genes that generally code for the spike protein which is then produced in the vaccinated individual . RNA vaccines: based on messenger RNA (mRNA) or a self-replicating RNA that provides the genetic information for the spike protein. The global COVID-19 vaccine landscape

49. Major antigen candidate In case of SARS-CoV2, it has been shown that only antibodies directed to protein S can neutralize the virus and prevent infection. S protein consists of S1 and S2 subunits Consequently, all SARS-CoV-2 vaccines in use and under development include at least a portion of the S protein . These can only be limited to the S1 or domain the RBD. The primary goal of all COVID-19 vaccine programs is the formulation of a vaccine that stimulates the production of protein S neutralizing antibodies in vaccinated individuals. On NCBI is availability of a database of more than 5000 full-length SARS CoV2 genomes isolated from various countries available, which facilitates the delineation of polymorphisms in the S protein.

50. Live attenuated viral vaccine Live attenuated virus strains is obtained by mutation or deletion virulence genes . Coronaviruses have several genes that are not required for replication and that can be deleted, leading to attenuation in vivo. Deletion of the E protein leads to attenuation and generation of an efficacious vaccine strain, but reversion of the attenuated phenotype has been reported. Deletion of virulence factors may provide a preferred mechanism of attenuation . The use of an attenuated strain of a pathogen as a vaccine requires demonstration of its inability to genetically return to becoming pathogenic . This is particularly challenging in the case of coronaviruses as they are known to recombine in nature, and an attenuated vaccine strain could, in theory , recombine with wild coronaviruses to recreate a pathogenic strain.

51. Inactivated viral vaccines Viruses are physically or chemically inactivated ( vaccine against poliomyelitis, hepatitis A and flu) Inactivated viruses can be generated and scaled quickly in a pandemic situation using established infrastructures and methods Inactivated viral vaccines have few security concerns, unlike their attenuated live counterparts , and express a wide range of native viral antigens , including surface antigens with retained epitope conformations that can induce dependent conformation antibody responses Inactivated viral vaccines often require an adjuvant and repeated administration to be effective. The use of alum as an adjuvant makes them unsuitable for respiratory mucosal delivery Currently, there are various clinical and preclinical studies evaluating inactivated SARS-CoV-2 vaccines

52. Recombinant viral-vectored vaccines A vaccine based on viral vectors is a promising prophylactic solution against a pathogen. These vaccines are highly specific in delivering the genes to the target cells, highly efficient in the gene transduction, and efficiently induce the immune response. They offer a long term and high level of antigenic protein expression and therefore, have a great potential for prophylactic use as these vaccines trigger and prime the cytotoxic T cells (CTL) which ultimately leads to the elimination of the virus infected cells

53. Protein subunit vaccines Subunit vaccines primarily induce CD4 + T helper cells and antibody responses. Most of these vaccines contain the full-length SARS-CoV-2 S protein or parts there of with the aim of inducing neutralizing antibodies. Proteins or peptides alone are poorly immunogenic and generally require more than one adjuvant but also repeated administration, and are poor activators of the CD8 + T cell response. Furthermore, this platform is generally unsuitable for vaccination of the respiratory mucosa In this regard, there are clinical and preclinical studies of vaccines and that use COVID-19 protein subunits . It appears to have comparable efficacy to the two RNA-based vaccines, but with a technology that has been in use for 30 years.

54. Virus- like particles -VLP VLPs are forming spontaneously particles composed of different structural viral proteins that they are co-expressed or mixed. In case of envelope coronavirus , VLPs are formed when viral proteins S, M and E, with or without N, are co-expressed in eukaryotic producing cells . This results in an active budding from VLP-producing cells which are structurally identical to the infectious virus but lack the viral genome and therefore they are not infectious . The presence of protein S on the surface of VLPs allows them to bind and insert ACE2 + in the same way as the parent virus . Currently, there is only 1 VLP- based COVID-19 vaccine in clinical trials, with 12 more under preclinical development . Medicago , a Canadian company, produces its own SARS- CoV-2 VLP from genetically modified plants .

55. Nucleic acid- based vaccines mRNA vaccines are non-infectious and they are synthesized by in vitro transcription, free of microbes molecules . These beneficial features differentiate mRNA vaccines from live attenuated viral vaccines , inactivated viral vaccines, subunit and recombinant vaccines viral vector vaccines in terms of safety , efficacy and problems of anti-vector immunity, enabling their rapid and economical and repeated production vaccination it is relatively simple . Plasmid DNA vaccines share several characteristics with mRNA vaccines, including safety, ease of production and scalability. However, they are poorly immunogenic, requiring multiple doses and the addition of an adjuvant .

56. How works the anti Covid vaccines The messenger RNA vaccines Pfizer- BioNTech The BNT162b2 mRNA vaccine ( Comirnaty ) is based on messenger RNA technology: it is composed of modified mRNA contained in lipid nanoparticles . The mRNA encodes the full-length, membrane-anchored spike protein SARS-CoV-19 and contains mutations that stabilize the spike protein in an antigenically preferred prefusion conformation. Lipid nanoparticles protect non-replicating RNA from degradation and allow it to be released into host cells after intramuscular injection. Once inside the host cells, the mRNA is translated into the SARS-CoV-2 spike protein, which is expressed on the surface of the host cells. Transient expression of this spike antigen induces neutralizing antibodies and cellular immune responses against it, which may confer protection against COVID-19

57. The mRNA does not enter the cell nucleus and therefore does not interact or modify our DNA. Furthermore, mRNA naturally degrades after a few days once it has performed its function. Efficacy : 95%; the protection would appear about ten days after the first administration but completes it 7 days after the second administration. Storage : the fragility of mRNA requires temperatures between -90 ° C and -60 ° C; resists between 2 ° C and 8 ° C for up to 5 days . Regimen: 2 intramuscular injections at least 21 days apart.

58. Moderna Moderna's mRNA-1273 vaccine ( Spikevax ) is based on mRNA technology: mRNA encodes the spike protein of the SARS-CoV-2 virus. Once taken up by cells, the RNA is used to produce the protein, which then triggers an immune response. The RNA does not enter the nucleus where the cell’s genome resides, and is degraded by the cell within a day of the injection . The difference with the Pfizer vaccine is the composition of the lipid nanoparticle that encloses the RNA and the Moderna formulation allows the vaccine to be stored at a temperature of -20 ° C for 6 months and in the refrigerator (at about 4 ° C) for 30 days . This makes it possible to simplify the logistics of vaccine distribution, particularly in rural areas and in countries with limited health infrastructures. The messenger RNA vaccines Each vial contains 10 doses ready for use. Regimen: 2 intramuscular injections 28 days apart in subjects over 18. Efficacy: 94.1%; immunity is considered acquired 14 days after the second administration.

59. Vaccines that use virus vector Oxford-AstraZeneca The AZD1222 or Covishield vaccine, authorized for emergency use in the UK and other countries, uses a chimpanzee virus rendered harmless to humans, the vector ChAdOx1, to deliver the code needed to produce the spike to the cell. The DNA sequence will be read by the cells and transcribed into mRNA, the translation of which will lead to protein synthesis . Storage : DNA is more stable than RNA and the genetic material is well protected from adenovirus, so the low temperatures of mRNA vaccines are not required. Efficacy : 62% to 90% depending on the dosage; generates a response already after the first dose, the second should be administered at 28 days.

60. Johnson & JohnsonIl vaccin Ad26.COV2.S, also known as JNJ-78436735, it is based on an adenovirus of serotype 26 (Ad26), a weakened version of the cold virus, which does not replicate in human cells but induces the production of the spike. With the same AdVac technology used here, the company has already developed the Ebola vaccine and is working on vaccines for Zika and H.I.V. Storage: at 2 ° C - 8 ° C for three months. Regimen : single intramuscular administration, but a two-dose regimen is also being studied. Johnson & JohnsonIl Vaccine virus vector

61. Sputnik V vaccine, or Gam- Covid - Vac , is based on two types of inactivated human adenovirus, unable to replicate and cause disease : Ad26 for the first dose and Ad25 for the second, 21 days after the first. Using two different types serves to avoid the danger that, after the first dose, the body produces antibodies with a consequent reduction in the effectiveness of the vaccination. As with other viral vector vaccines, it does not require very low temperatures for storage. The announced effectiveness is 91.4%; the virus is administered in Russia but also in other countries, such as Argentina and Belarus. Sputnik V Russian National Center for Epidemiological and Microbiological Research Vaccine virus vector

62. Protein-based vaccine NovavaxIl , the NVX-CoV2373 vaccine is composed of SARS-CoV-2 recombinant spike protein nanoparticles with an adjuvant (Matrix-M1 ™, based on saponin ) to enhance the immune response . It does not contain genetic sequences, but directly the spike proteins produced by insect cells infected by an engineered baculovirus with the genetic material necessary for their production. Regimen: consist in two doses 21 days apart. NovavaxIl Maryland biotechnology company

63. Inactivated virus vaccine Sinopharm The Chinese state-owned company has produced two vaccines based on a chemically inactivated version of the coronavirus that does not cause disease but stimulates the production of antibodies by the immune system: one made by the Wuhan Institute of Biological Products, tested on subjects for six years up, and one from Beijing Institute of Biological Products. The latter, approved in China for limited use and in use in the Emirates, Barhain and Egypt, is called BBIBP- CorV . The effectiveness declared by the producers is 79.34%; the regimen is two doses 21 days apart.

64. Sinovach Biotech The CoronaVac vaccine from Beijing-based Sinovac is based on an inactivated version of the coronavirus. It was grown in chimpanzee cells and chemically inactivated, i.e. made unable to replicate. The effectiveness declared by the Butantan Institute of Sao Paulo, relating to the Brazilian branch of the experiment, is 78%. The administration regimen is two doses 21 days apart and can be stored at refrigerator temperatures. Inactivated virus vaccine

65. Covaxin Bharat Biotech the Indian company's Covaxin vaccine is developed in collaboration with the Indian Council of Medical Research (ICMR) - National Institute of Virology (NIV); it is an inactivated and purified virus currently in phase 3 of experimentation . Authorized in India, the regimen involves two doses injected 4 weeks apart. Inactivated virus vaccine

66. Exploratory stage → this stage involves the laboratory research. During these studies different types of antigens were identified. These antigens could include, for example, VLP or weakened viruses and bacteria. Pre-clinical stage → this stage involves pre-clinical studies using tissue-culture, cell-culture or animal testing. The animal testing is an important process during which the safety, the immunogenicity and the ability of the vaccine to provoke an immune response are studied and verified. Clinical stage → this stage is subdivided in four phases. During all these four phases the vaccine is tested on human subjects. Stages of vaccine development 1 2 3

67. Clinical trial phases of a vaccine After obtaining an optimal vaccine product, it is tested in a series of 3/4 phase trials. Phase I study - 10-50 subjects - healthy adult volunteers. The first is to determine the safety, toxic, metabolic and pharmacological effects in a small number of individuals. Phase II study - A few hundred subjects. Any side effects, associated risks and immunogenicity. Phase III study – A few thousands of subjects (1000-3000). Duration is about 1 year. Phase IV study- Several hundred to thousands of subjects - Population targeted by the vaccine. In phase IV, on a very large number of vaccinated people, the emergence of the desired clinical effect, ie protection, is investigated in relation to the exposure to the risk of acquiring infection . The vaccine tested in a series of clinical studies on subjects who after vaccination were found to be protected, did not show phenomena of toxicity and side effects of little importance can be considered effective.

68.   « Herd immunity » or group immunity is a form of indirect protection that occurs when the vaccination of a significant part of a population ends up providing protection even to individuals who have not developed directly. This theory is the foundation for mass vaccination campaigns around the world. It currently provides that, for a population to be immune to an infectious disease, such as measles, at least 95% of the population must be vaccinated. In theory, therefore, with a 95% vaccination rate, the disease should be eradicated