Viral Replication Cycle PDF

The main function of viral capsids is to protect nucleic acids from: ¨ Physical damages: Mechanical protection. ¨ Physical damages: UV radiation (from sunlight) leads to chemical alteration, causing mutations. ¨ Enzymatic damages: nucleases and esterase derived from dead cells or freely secreted as...

The main function of viral capsids is to protect nucleic acids from: ¨ Physical damages: Mechanical protection. ¨ Physical damages: UV radiation (from sunlight) leads to chemical alteration, causing mutations. ¨ Enzymatic damages: nucleases and esterase derived from dead cells or freely secreted as cellular defense against infections, ¨ Viral genome packaging ¨ , In naked viruses, iteraction with host cells: capsid proteins of naked viruses mediate the binding and penetration of the virus inside the host cell., ¨ Stimulation of the host immune system: the protein capsid of naked viruses are often the major antigens of the virus. Pericapsid : It is present only in some families of viruses (orthomyxovirus, herpesvirus, retrovirus, poxvirus, etc..). ¨ The pericapsid is composed by phospholipids derived from cytoplasmic or intracellular membranes. ¨ It also contains some viral glycoproteins, which can act as virus receptor to target cells, and can stimulate immune responses. ¨ The presence of pericapsid accounts for the sensitivity of certain viruses to organic disinfectants (ethers, alcohols) that can dissolve lipids. Viral Genomes Molecular Weight: from 1.5 to 240 x106 Codifying Capacity: from few proteins to more than 200 proteins RNA viruses with a negative polarity (-). Their genome can act as a template for the synthesis of messenger RNA. These viruses have the RNA-dependent RNA polymerase enzyme associated with the virion. A cell, to be infected, must be: - Susceptible: the cell has got the receptors necessary for viral entry and - Permissive: the cells has got all the machinery necessary to carry out the entire viral life cycle. • Productive infection: The infection of a permissive cell leads to the production of viral particles. • Abortive infection The virus enters the target cell but the life cycle cannot be completed. There is no production of viral particles. • Restrictive infection When cells are permissive only in a certain phase of their life-cycle. Acute non-persistent infections ¤ Most of viral infections are extinguished with the healing and the disappearance of the virus from the body. ¤ The virus is not detectable anymore, except in cases of reinfection. ¨ Persistent infections ¤ In other cases, the infection after the acute phase may become persistent in the form of: Latent infection, Chronic infection, Slow infection. Viral replication cycle It can be divided in 6 phases: -ADSORPTION PENETRATION -UNCOATING -REPLICATION -MATURATION RELEASE. The period between the penetration of virus into cells and the maturation of viral progeny is called period of eclipse, as the virus is no long available as a morphologic entity in the cell. Viral tropism is the ability of the virus to recognize specific receptors and thus to infect specific cell types. In influenza virus, M2 protein is present in the viral envelope. It acts as an ion channel and causes the acidification of endocytic vescicle containing the virus. This results in fusion between the pericapsid and the vescicle membrane, thus favoring the release of the nucleocapsid into the cytoplasm. DNA viruses follow 4 different replicative strategies for the synthesis of mRNAs: 1° group :herpesviruses adenoviruses papovaviruses. The expression of viral genes occurs in the nucleus through the cellular enzyme RNA polymerase II. mRNAs are trans-located into the cytoplasm where they are translated. For Papovaviruses, the expression of viral genes occurs in 2 steps. For Herpesviruses and Adenoviruses, the expression of viral genes occurs in 3 phases. 2° group Poxviruses :Despite having a DNA genome, the entire replication cycle is completed in the cytoplasm. Viral gene expression is carried out by a DNA-dependent RNA polymerase (encoded by viral genome) in the cytoplasm. 3° group Parvoviruses :They have a single-stranded DNA. Viral gene expression requires 2 steps: - Synthesis of the complementary DNA strand. This gives origin to a doublestranded DNA - Gene expression by the cellular RNA polymerase II These events occur in the cell nucleus. 4° group Hepadnaviruses: Among these, hepatitis B virus is the only pathogenic for humans. HBV has a partially double-stranded DNA. The positive strand is shorter and should be completed by cellular enzymes. Once completed the synthesis of the + strand, viral gene expression is carried out by the cellular RNA polymerase II. The mRNAs are then transported in the cytoplasm for translation. The fulllength mRNA is also used for the synthesis of viral genome by the Reverse Transcriptase enzyme. Replication of viral genome for DNA viruses: DNA viruses require a DNAdependent DNA polymerase to replicate their genome. • Herpes viruses, poxviruses and adenovirus have their own DNA polymerase Their genome encodes for a DNA polymerase • HBV uses a RNA-dependent DNA polymerse (the reverse transcriptase) to synthesize its genome. • Parvovirus, and Papovavirus do not have their own DNA polymerase, thus they use the DNA polymerase of the host cell. The cellular DNA polymerase is produced during the S-phase of the cell cycle, and thus it is present when the cell is actively replicating. • To use the cellular DNA polymerase, Parvovirus can replicate only in cells that are in active proliferation. These cells are the progenitors of the red blood cells. • To use the cellular DNA polymerase, Papovaviruses induce the progression of the cell cycle from the G1 to the S phase. This allows these viruses to infect notreplicating cells. RNA viruses follow 4 main strategies. Replication strategies vary according to the polarity of RNA genome: positive or negative. The first group, viruses with (+) sense RNA genomes, includes the Picornaviridae, Flaviviridae, Togaviridae, Caliciviridae, Coronaviridae. The complete linear RNA genomes of the Picornaviridae and Flaviviridae (including HCV) act as mRNA and is translated into a viral polyprotein which is subsequently cleaved into individual proteins by the viral protease. In togaviruses, coronaviruses and caliciviruses (hepatitis E virus), there is only the translation of the 5’ portion of viral genome. This translation determines the production of RNA dependent RNA polymerase enzyme. The RNA polymerase is necessary for: -the synthesis of viral genome -the synthesis of mRNAs for structural proteins. Scheme of viral replication of + RNA virus: The viral RNA dependent RNA polymerase is responsible for the synthesis of new molecules of RNA with positive polarity that will be used as new molecule of viral genome and as mRNAs. This newly transcribed (+) sense RNA has 3 possible functions: (i) mRNA (ii) a template for production of additional (-) strands or (iii) packaged as progeny virus. Riboviruses with negative RNA genome Replication scheme -Single-stranded RNA with negative polarity: (-) RNA -It cannot serve as mRNA. Thus the virus needs a RNA dependent RNA polymerase, already present in the virions to synthesize mRNAs. RNA-dependent RNA polymerase synthesizes mRNAs that will be used for translation. mRNAs are also used as template for the synthesis of viral genome by viral RNA polymerase. Riboviruses with double-stranded RNA: Reoviruses and Rotaviruses (Reoviridae) - These viruses have a segmented double-stranded RNA. - Despite the presence of a (+) RNA strand, these viruses behave as viruses with (-) RNA genome. - Thus, only the (-) RNA strand is used for the synthesis of (+) RNAs by the viral RNA-dependent RNA-polymerase. Viral RNA polymerase uses (–) strand to synthesize (+) RNAs (+) RNAs serve as: - mRNAs for translation Template for the synthesis of (-) RNAs that represent new molecules of viral genome. Retroviruses Riboviruses with diploid genome - Two linear single-stranded RNA molecules with a positive polarity, (+) RNA, linked at the extremity 5’ together forming a dimer. - It does not work like mRNA. -The only function of the genomic RNA is to be used for the doublestranded DNA synthesis. - The synthesis of DNA from an RNA template is carried out by the reverse transcriptase enzyme. - DNA molecules migrate in the nucleus where they are integrated in the host genome. This step is catalyzed by a viral enzyme defined as integrase. - The integrated DNA is then transcribed by the cellular RNA polymerase II. The (+) RNA that are produced can serve as - mRNAs - New molecules of viral genome to be included in the viral particles. VIRAL GENETICS. A genetic change can be stabilized in viral population: - due to random fluctuations - if it confers a selective advantage (immune pressure/drug pressure). For some viruses (HIV and HCV) the concept of quasi-species was developed. The term quasipecies indicates the presence, in the same infected individual, of a swarm of genetically different viral variants. It is a concept derived from the observation that, in an individual infected for a long time, there is a continued evolution of the virus, as a result of spontaneous mutations and selective pressures exerted by host’s immunity and drug therapy. Viruses can be genetically modified by : - acquiring mutations (point mutations, deletions, insertions) during the replication of viral genome - Reassortment/recombination. Reverse transcriptase of HBV and retroviruses is probably the most error-prone viral replicative enzyme. In fact, the frequency of spontaneous mutations reaches 104 per nucleotide per replication cycle. The viruses that use DNA polymerase have a relatively low mutation rate (1 mutation every 108 -1011 base-pair per replication cycle), similar to that observed in cellular organisms. DNA viruses are generally more genetically stable than RNA viruses Some RNA viruses (es. paramyxoviruses) are stable from a genetic point of view. It has been postulated that these viruses adapted to human millions of years ago, and thus have reached their mutational threshold. The continuous and gradual antigenic variation, both spontaneous or induced by the host immune system, is referred to as antigenic drift.  Influenza virus, HIV, HBV, and HCV are subject to antigenic drift. Influenza viruses are a classic example of viruses subjected to antigenic drift. In these viruses there is a continuous, slight seasonal variation of the external glycoproteins, hemagglutinin and neuraminidase, which allow the virus to remain in human populations, despite production of neutralizing antibody. Antigenic shift is due to extended changes in antigenic properties, following a genetic reassortment. The resulting virus can infect hosts already infected with the parental virus. Recombinant viruses may acquire the ability to infect different cells, thereby enlarging their host spectrum and conquering new replicative niches.  The antigenic shift assumes importance in the epidemiology of influenza viruses type A, that can infect both human and animal hosts.  When there is a mixed-virus or human-animal virus infection, the progeny derived from reassortment may have replication properties compatible with the human species and antigenic properties -dependent from haemagglutinin and neuraminidase - completely different from the parental human virus, leading to pandemic infections. Influenza virus is prone to antigenic shift due to the segmented nature of its genome. When more virions are found in the same cell, they can interact both involving their genomes (genetic interactions) or their end products (non-genetic interactions). Viral recombination: exchange of homologous stretches of genetic material between two virus strains (usually of the same species) co-infecting the same cell, with the generation of a viral progeny presenting stable phenotypic characteristics of both parent viruses. Viral recombination between virus and host cell: specific feature of recombination between the host cell genome and the genome of retroviruses, with acquisition of cellular genes by viral genomes. In DNA viruses, recombination processes are frequent and involve the break and rejoin of homologous nucleic acid strands already synthesized.  For RNA viruses this phenomenon occurs during replication, when the viral enzyme can jump from one template strand to another, dragging the chain of newly synthesized one and with the generation of an hybrid polynucleotide strand.  This phenomenon can occur during the synthesis of RNA (mixovirus, coronavirus, etc..) or DNA (retroviruses). The frequency of recombination reflects the features of replicative enzyme, and it is high in those viruses in which the exchange of the template strand is a normal event in the process of synthesis of the viral genome (retrovirus: >20% of HIV isolates are recombinants between different subtypes, CRFs). Why is HCV characterized by a degree of genetic variability? Its polymerase lacks the proofreading-function, High virion production: >10*12 virions per day. Daily generation of a huge number of mutations (>10*10 point mutations produced per day). Double passage by viral HCV polymerase to create a (+)RNA template for protein synthesis. The virus is very variable: At patient level: the high degree of variability gives rise to the concept of quasispecies. At population level: different genotypes and subgenotypes. Hepatitis C Virus is Classified Into 7 Genotypes and 67 Subtypes. Genotype 1 is by far the most frequent genotype in chronically infected patients worldwide as well as in Europe. Although both genotype 1 and 3 can induce intracellular lipid accumulation, genotype 3 seems to be the most efficient in driving steatosis. HCV genotype 1b is significantly more prevalent among patients with liver cirrhosis and those with decompensated liver disease requiring liver transplantation than among those with chronic active hepatitis C. HCV genotype is the most important baseline predictor for response to Peg-IFN + Ribavirin Combination Therapy. HIV shows considerable genetic and phenotypic variability This is due to the particular mechanism of viral genome replication, that contains a series of steps involving a large number of transcription errors.  Several factors are known to contribute to the formation and evolution of numerous viral variants:  The high error rate of "transcription" and lack of "proofreading" function by RT enzyme, Recombination during the process of viral DNA synthesis, The high daily rate of viral production, The rapid selection of species with different viral replicative capacity under immune and pharmacological pressure. Why HIV Develops this Great Genetic Diversity?  At every replication cycle, changes are made in viral genome "Mutations"  RT enzyme has an error rate during transcription of 1:2,000-10,000 bases.  The genome of HIV is composed of 9749 nucleotides.  Therefore, each new virus has at least one mutation! The population of viral variants in the same individual is highly heterogeneous. SIVcpz (Simian Immunodeficiency Virus of chimpazees) is the Immediate Precursor of HIV-1. A current estimate indicates that > 20% of HIV isolates represent recombinants between different subtypes (CRF: Circulating Recombinant Form), and in some areas the CRF forms are becoming prevalent in the population. Today, >100 HIV-1 Circulating Recombinant Forms have been described. Different HIV-1 Subtypes Differ in Disease Progression. Y181C and Y181I mutations of HIV-1 RT, naturally occurring respectively in groups O and in all HIV-2 isolates, make these viruses resistant to all almost NNRTIs!.  Among HIV genome regions, env gene is the most prone to variation. Almost every step of HIV replication is target of at least one drug. 1 Attachment inhibitor (AIs): FTR, 2 Entry inhibitors (EIs): MVC Ibalizumab, 3 Fusion inhibitor (FIs): T20, 4 Nucleos(t)ide reverse transcriptase inhibitors (NRTIs): AZT, ddI, ddC, d4T, 3TC, ABC, TDF, FTC, TAF, 5 Integrase inhibitors (INIs) RAL, EVG, DTG, BIC, CAB, 6 Protease inhibitors (PIs) SQV, IDV, RTV, NFV, fAPV, LPV, ATV, TPV, DRV, 7 Capsid inhibitor LEN, 8 Non-nucleoside reverse transcriptase inhibitors (NNRTIs) EFV, NVP, DLV, ETR, RPV, DOR. …Today more than 100 mutations have been associated with HIV drugresistance. HBV is a DNA virus characterized by a high genetic variability that is lower only to that of RNA viruses such as HIV and HCV, that are the prototype of variable viruses. • HBV uses the Reverse Transcriptase for the replication of its genome. This strategy is unique among all DNA viruses. • The RT introduces mutations in HBV genome at a rate similar to that observed for the RT of HIV (1 mutation / 105 nucleotide). • HBV has the smallest genome of known animal DNA viruses (only 3200 bp). • This has forced the virus to optimize the genomic organization…Through an extensive overlapping of HBV open reading frames. Thanks to this unique genome organization, HBV genome contains all the information necessary for its life cycle!! • A mutation can be synonymous or have little effect on one viral protein, but may have severe consequences on an overlapping gene. • In this case, this mutation is not selected. y the same drug (such as tenofovir) may work as monotherapy against HBV, but not for instance against HIV. The pattern of lamivudine resistance mutations M204V+L180M+V173L in RT correspond to I195M+E164D in the HBsAg, that strongly reduce the binding affinity with neutralizing antibodies including those induced by the vaccine. Genetic variability in S gene can modulate HBV oncogenic potential. Deletions and stop codons in the S antigen can induce an oxidative stress thus favoring the neoplastic transformation of the hepatocytes. The detection of deletions in pre-S regions correlates with increased risk of liver cancer. GENETIC VARIABILITY - PRACTICAL IMPLICATIONS • Viral evolution can explain seasonal outbreaks, host change and pandemics. • The variability of viruses regulates the efficacy of natural and artificial immunity; in some cases is broad (i.e. against all viruses of that species), in other cases is strain- or typespecific (and therefore of limited clinical relevance). • The development of a vaccine can be relatively easy in some cases, and very difficult (nearly impossible) in others. • Viruses with high propensity to variability require drug combinations to be controlled – This is not the case whereby a structural constraint limits the fixation of mutations generated during replication. • HBV: High efficacy of antivirals, excellent vaccine. VIRAL PATHOGENESIS Pathogenesis of Viral Infections ¨ The pathogenesis is the process by which a viral infection causes a disease. ¨ The virion represents the inert form of the virus. In fact the virus activity takes place during virus-cell interaction. ¨ The penetration of the virus into the host cell creates a new entity: the infected cell. ¨ The viral pathogenesis is generally linked to: ¤ The pathogenic activity of the virus itself; ¤ The permissiveness of the host and its organs. ¨ Balance: double result of the spread of the virus and host survival. ¨ Virulence for each type of virus can be determined by small mutations in the genome. Important events in the viral pathogenesis are: ¨ Viral entry into the host. ¨ Viral Replication in the place of first implant, which may coincide or not with the site of entry. ¨ The overcoming of local defenses (eg. lymphocytes, macrophages, IFN). ¨ The diffusion from the place of implant. ¨ The dissemination to target organs. ¨ The elimination of the virus by the host organism. Viruses can penetrate mainly through: ¨ Respiratory route (very common); ¨ Gastrointestinal route (very common); ¨ Genital route (less common, but frequent); ¨ Skin lesions caused by insects, animals, medical and surgical instruments. ¨ Blood transfusion. Blood products are a potential source of infection (eg. hepatitis B, hepatitis C, HIV) (less common, but frequent). ¨ Organ transplants, particularly bone marrow and kidney (cytomegalovirus and Epstein-Barr virus). ¨ Maternal-fetal transmission. Vertical = From mother to child: Perinatal Transfer through the delivery channel, breast-feeding: HIV, HBV, HSV2 (very early treatment after birth of HIV newborn can reduce the risk of HIV transmission). Trans-placenta: CMV, HIV Rubella Virus, etc - asymptomatic - Fetal deformities - Abortion (search of the virus in the amniotic fluid by molecular biologies procedures). Gametic Cells: Endogenous Retroviruses. The route of entry determines the modality of transmission and the epidemiological characteristics of viral infection. Transmission by respiratory route (very common): it is poorly influenced by social and economic conditions, but it can suffer environmental and seasonal influences. ¨ Transmission through the gastrointestinal tract (very common): it is influenced by the social and economic conditions, as in case of contaminated food and poor environmental remediation. ¨ Transmission by genital route (less common, but frequently): it is influenced by the degree of sexual promiscuity and it interests adults only. ¨ Transmission through skin lesions caused by insects and animals: it may be subject to environmental and seasonal influences. ¨ Transmission through medical or surgical instruments, blood transfusions and blood products, organ transplantation (especially bone marrow and kidney): this kind of transmission especially interests particular individuals at risk: professionals, politransfused, drug addicts. Viruses Entering Through the Skin: They are capable of replication in cells residing in the superficial layers of the dermis. Routes of Dissemination ¨ After successful entry of the virus, a key factor for the establishment of infection is the availability of sensitive and permissive cells. ¨ When the virus is infecting a sensitive cell, at the site of entry it begins to multiply and to locally spread towards neighboring cells. ¨ Otherwise, the virus can be carried by body fluids and it can thus migrate up to find cells permissive for viral replication (less efficient mechanism). ¨ The virions produced in situ may: ¤ Continue to spread in the same area, extending the infection in the same organ or system; ¤ Migrate. ¨ The most common route of dissemination is systemic blood (viremia). ¨ Some viruses can spread trough a nervous route, using the peripheral nerves to reach the central nervous system. Localized infections ¨ They represent infections that remain confined to the tissues of the site of entry, or to those adjacent to it. ¨ They are superficial epithelial infections: skin, conjunctiva and mucous membranes of the gastrointestinal, genital and respiratory tract. ¤ Eg. rhinoviruses, whose infection does not go beyond the nasal cavity. ¨ Although viral replication is restricted to certain areas, the effects on the body can be much more generalized (eg. flu). Distant Spread: It occurs in all systemic infections. ¨ It presupposes the lymphatic drainage of the virions and their release into the circulation. ¨ For some viruses, the blood dissemination can continue even after the production of neutralizing antibodies. In fact, the virions can be conveyed within cells (monocytes or lymphocytes). I Event: Possible replication in the mucous membranes of the first implant. II Event: Dissemination. First and transient viremia . III Event: Internal organs infection. Possible massive replication . IV Event: Dissemination. Second and longer phase of viremia. V Event: Infection of target organs. Diffusion Trough Nervous Route: • Rhabdoviruses • a herpes viruses • Polio viruses. Viral Elimination ¨ It is a phenomenon of considerable importance for both the spread of infection and to the setting up of prevention strategies. ¨ Main modalities of elimination: ¤ Airway ¤ Digestive tract ¤ Urogenital tract. The incubation period is the interval between the acquisition of infection and the onset of disease. The generation time is the interval between acquisition of infection and transmission of infection to another individual. Often, the possibility of transmitting the infection may precede the onset of the disease state. Latent Infection: The virus is detectable during the acute phase of replication, but it is no longer detectable as infectious virus if not in successive reactivation. ¨ The virus remains in the form of integrated nucleic acid into the host cell genome or present in the cell nucleus in the episomic form. ¨ Sometimes the virus can "wake up" and begin a new cycle of productive replication (relapse), which in turn ends with a phase of latency. ¨ Ex: herpes virus, papovavirus, adenovirus, hepatitis B and other viruses with DNA replication intermediates. ¨ Some forms of latency are suspected for RNA viruses: retroviruses (HIV), measles virus and some enteroviruses. Chronic Infections ¨ They are characterized by the continuous presence of the virus, that continues to be produced even after the acute phase but usually at much lower levels and for a long time, compatible with the survival of the host. ¨ Classics examples: hepatitis B and C, human retroviruses as HIV and HTLV and Papovavirus. ¨ The damages to the host are typically derived from accumulation (immunodepletion, autoimmunity, chronic inflammation, oncogenesis). Slow Infections ¨ They are characterized by a long incubation period that can last several years, followed by a progressive disease. The acute stage is completely absent. ¨ Belong to this kind of infections those caused by "nonconventional agents" such prions: eg. progressive spongiform degeneration of the CNS, with fatal outcome. Viral Effects on Host Cells ¨ The penetration of the virus into the cell creates a new entity: the infected cell. ¨ The multiplication of the virus alters the physiological state of the cell: changes in cell morphology, metabolic disorders, genetic and regulatory changes. ¨ The infection can lead to 2 distinct events (which depend on the type of the virus and the type of cell): ¤ Cell death (cytocide infections) through lyses; ¤ Transformation. Viral Effects on Host Cells ¨ Cytopathic effect (CPE): morphological alterations that occur both in the host and in cell cultures in vitro. The type of alteration is characteristic for different groups of viruses and is useful in laboratory diagnosis. ¨ The cytopathic effect may be represented by: ¤ rounding of the cells with subsequent lyses; ¤ formation of multinucleated giant cells (syncitia) due to the presence of specific fusion proteins of the virus (eg, paramyxovirus, herpesvirus and some retroviruses); ¤ formation of clusters of viral constituents and/or altered cellular structures in the nucleus or cytoplasm of the cell (inclusions). ¨ Many viruses also have the ability to inhibit the synthesis of cellular macromolecules (shut-off) and this prevents the cell to repair the damage caused by the virus. Virus-induced Apoptosis ¨ The cell infected by a virus may experience apoptosis. Apoptosis: programmed cell death. ¨ It is a phenomenon encoded by specific genes, often considered as a form of cell suicide. ¨ The elimination of virus-infected cells prevents very effectively the spread of the virus within the host organism. ¨ Viruses have evolved in parallel to the host and in some cases have developed mechanisms to overcome host processes specifically activated to fight viral infections. ¤ Eg. viruses can encode proteins which interfere with the antigen presentation, the action of specific cytokines, complement activation and they can inhibit or promote apoptosis. the major animal viruses capable of inhibit the expression of proteins directly involved in apoptosis and their related targets. Adenovirus p53, CMV p53, EBV p53, HBV p53, HPV p53, Adenovirus bcl-2 ,EBV bcl-2, Poxvirus caspases, Adenovirus TNF receptor. the major animal viruses capable of stimulate the expression of proteins directly involved in apoptosis and their related targets: HPV p53 HIV bcl-2, HBV, HCV, HIV TNF receptor, Fas Ligand. Apoptosis may represent a process facilitating the release of the virus, especially for naked viruses that uses the host cell lyses for their release. Transformation: the process by which a cell loses control of its proliferation and become cancerous. It consists in the induction of morphological, biochemical and biological changes at a genetic level, stably transmitted to cellular progeny. It is a multifactorial process (chemical, physical and viral), divided into 2 phases: ¤ Immortalization: indefinite multiplication of cells with the formation of lines or clones; ¤ Unordered stacked growth, with loss of contact inhibition that can cause the formation of a tumor when inoculated into an appropriate host. ¨ The tumor is the result of several cumulative effects that change the control of cell proliferation. In some cases the cells express tumor antigens (T) at their surface. Trasformating Infections ¨ The first exposure to an oncogenic virus or chemical carcinogen (initiator) induces heritable changes in some cells. ¨ The exposure to a second agent (promoter) results in the formation of the tumor. ¨ Oncogenic viruses, chemical and physical agents can be either initiators or promoters. ¤ Eg. EBV is able to immortalize B lymphocytes, T protein of polyoma virus can transform immortalized cells, etc. ¨ Some agents are defined as complete carcinogens, capable of initiatives and promotions. ¨ Approximately 15% of human cancers has a viral etiology, that was also associated with 2 widespread tumors: female cervical cancer (HPV) and cancer of the liver (HBV and HCV). Oncogens and Tumor Suppressor Genes: Normal cell proliferation is under the control of positive (oncogenes) and negative (tumor suppressor genes) regulatory genes, which may lead to neoplastic transformation in response to various alterations. ¨ Oncogenes are absolutely normal regulating genes, and are necessary for cellular homeostasis. ¨ They were discovered in the genome of retroviruses (v-onc). Subsequently, very similar genes have been identified in normal cells too (c-onc). ¨ The proteins encoded by oncogenes may be: ¤ Growth factors. ¤ Membrane receptors for growth factors. ¤ Tyrosine proteinchinases. ¤ GTP-binding proteins. ¤ Nuclear proteins. ¨ The proteins encoded by tumor suppressor genes such as Rb and p53, are able to negatively control cell growth. ¨ Oncogenes may determine a neoplastic transformation due to: ¤ Gene amplification (c-myc); ¤ Point-mutations (ras), gene-breaks (erb-B), rearrangements (myc, dbl). ¨ Some DNA viruses, or RNA viruses which synthesize DNA during replication (retroviruses), are able to induce transformation and/or tumor in vitro and in vivo. They possess oncogenes in their genome. ¨ RNA viruses, with the exception of retroviruses (and HCV), do not have oncogenic properties. RNA viruses that can cause transformation during permissive infections. ¨ Retrovirus as HTLV-1, causing adult T-cell leukemia (ATL) : ¤ Introduce voncogenes in normal cells under the control of viral promoters (transduction). ¤ Integrate near c-onc and thus alter their expression (active in cis). ¤ Encode misregulatory proteins against c-onc and v-onc (active in trans, tax in HTLv1). ¨ Hepatitis C virus (HCV) is associated with (HCC) DNA viruses usually cause lytic infections and induce transformation in the nonpermissive ones. ¨ Hepatitis B virus (HBV) is associated with primary hepatocellular carcinoma (HCC). ¨ Epstein-Barr virus (EBV) is the causative agent of Burkitt lymphoma, with the translocation of c-myc oncogene. ¨ Herpes simplex virus type 2 (HSV-2) is associated with cervical cancer. ¨ Human herpesvirus 8 (HHV8) is the causative agent or a cofactor of Kaposi's sarcoma, and of primary effusion lymphoma (BCLB). ¨ Cytomegalovirus (CMV) is associated with cervical cancer. ¨ Adenovirus: malignancy associated with genes encoding E1A, E1B, able to transform human cells. ¨ Polyomavirus generally do not induce tumors in permissive hosts. They encode antigens, important for initiation and maintenance of transformation (eg SV40 large T antigen). Merkel cell polyomavirus (MCV), new virus linked to a rare skin tumor. ¨ Papillomavirus (HPV) is associated to anogenital cancers and Epidermodysplasia verruciformis. HBV and HCV-related hepatocarcinogenesis: The repeated cycles of cellular degeneration and regeneration can favor genome instability thus promoting neoplastic transformation of the hepatocytes. Approximately 20-30% of HBVrelated HCCs develop without cirrhosis, thus highlighting a direct role of HBV in hepatocarcinogenesis. HBV is not cythopatic : the hepatic damage that is observed in the hepatitis is host mediated. The accumulation of damage produced by the cytolytic action of CTL and subsequent fibrotic repair are responsible of the chronic hepatopathy and its outcomes in liver cirrhosis and hepatocellular carcinoma. The damage is evidenced by rising plasma levels of liver enzymes (transaminases). HBV Carcinogenesis: •High protein X expression •Integration of viral DNA in a particular position of the host genome •Active cell division in relation to repair processes •Inflammatory alterations. Integration of HBV genome in cell genome can favor: Genome instability involving genes encoding Such as Cyclin E1 and Tert p53, Wnt/b-catenin, cyclins A/D TGFb, and Ras. Loss of oncosuppressor genes such as p53 and prb. Up-regulation of oncogenes such as Cyclin E1 and Tert. HPV is recognized as the etiological agent of cervix cancer. It can also cause cancer mainly in anus, penis, oro-pharinx. E6 & E7 alter the activity of proteins p53 and Rb , respectively. These proteins arrest the cell cycle at G1 phase During normal replicative cycle of the virus, the production of E6 and E7 controlled by the product of E2 gene. • In warts and condylomas (benign neoplasia) viral genome is in episomial form. • In cervix carcinoma, viral genome is integrated in DNA of infected cell. However, E2 gene is lost during integration of genome of HPV in genome cell. The functional loss of E2 determines an increased expression of oncogenic proteins E6 and E7. Epstein-Barr virus (EBV/HHV4) The presence of viral genome in B lymphocytes immortalizes these cells. EBV is associated with Burkitt’s Limphoma (children in central Africa and New Guinea) and nasopharyngeal carcinoma. In AIDS patients it is supposed to be associated to oral leukoplakia. 90% CASES of TRANSLOCATON of c-myc from chromosome 8 to 14. ANTIVIRALS So far, there are drugs for the treatment of the following human infectious diseases:  Influenza virus infections  Respiratory syncytial virus infections  SARS-CoV-2 infection  Herpes simplex infections  Human citomegalovirus infections  Varicella-Zoster Virus  Hepatitis B infections  Hepatitis C infections  HIV infection  Human papillomavirus infections. In all pandemic phases, therapy and prophylaxis with antiviral drugs aim at the following: − reduce the number of individuals at risk of getting a disease (morbidity) − decrease the number of deaths (mortality) − prevention of the spreading of new virus subtypes (e.g. influenza) during pandemic alert period − protect people who play key roles in managing the response to pandemic (prophylaxis). An ideal antiviral must be, Water-soluble, Chemically and metabolically stable, Easily absorbed (apolar). must NOT be, Toxic, Carcinogenic, Allergenic, Mutagenic, Teratogenic. Antiviral drugs interfere with a specific function, e.g. an enzyme, essential for virus replication. In case they interfere with cell function, they must: Be crucial for the virus but not for the cell or kill the infected cell exclusively While there is a wide highly-selective range of drugs against bacteria, their employment against virus is more complex due to the viral ability to replicate exclusively in host cells of the organism. Anti-influenza antivirals • The 3 targets of anti-influenza virus (A and B) are: the neuraminidase enzyme - the M2 protein - the RNA polymerase. 8 drugs have been approved to treat influenza infections. • Neuraminidase is crucial for influenza virus life cycle. Indeed, it cleaves sialic acid residues on glycoprotein thus allowing: - the entry of virus into target cell during the first steps of influenza virus life cycle - the release of viral particles from infected cells at the end of maturation process . neuraminidase inhibitors Zanamivir, Oseltamivir, Laninamivir, peramivir.  Neuraminidase inhibitors block the activity of neuraminidase. They mime the structure of sialic acid residues and thus they act as competitive inhibitors. Zanamivir and Oseltamivir are structurally related neuraminidase inhibitors. They block the intracellular penetration of virus and viral release by infected cells. Zanamivir (Relenza) administered via inhalation, Oseltamivir (Tamiflu) administered per oral suspension. Oseltamivir and Zanamivir are inhibitors of Neuraminidase, which is found on the outer cell membrane of viral particles The drugs act directly on this enzyme, specifically, interfering with its ability to release and spread the infection throughout the body. • Another target of anti-influenza drug is the M2 protein. • The M2 protein act as a protonic pump. It determine an acidification of the endocytic vescile, thus allowing the release of viral genome into the cytoplasm. AMANTADINE AND RIMANTADINE inhibit influenza virus penetration Blocking the M2 viral proton pump; Matrix M1 protein does not separate from nucleocapsid and genome cannot migrate to nucleus. The anti-herpes virus drugs mainly target the viral DNA polymerase, an enzyme that is critical for viral life cyle. These drugs are: ACYCLOVIR, PENCICLOVIR,, GANCICLOVIR,CIDOFOVIR,LETERMOVIR, FOSCARNET. Acyclovir is used for the treatment of: - Herpes simplex virus Chicken-pox virus, Epstein-Barr virus, and herpes encephalitis in immunocompromised patients. - Acyclovir is an analogue of guanosine. This means that it is structurally similar to the guanosine. It differs from the guanosine since it lacks the 3’ hydroxyl group. Mechanism of action of acyclovir • The affinity of acyclovir for viral thymidine kinase is 200 times higher than that for cellular kinases. This means that acyclovir is specifically activated only in herpesviruses infected cells. • The concentration necessary to inhibit viral DNA polymerase is lower than that necessary to inhibit cellular DNA polymerase. This reduces the toxicity of acyclovir. Gancyclovir: It has an antiviral structurally similar to acyclovir It has high activity against cytomegalovirus (especially in case of retinitis in patients with AIDS). The mechanism of action is similar to that of acyclovir: activation due to phosphorylation by cell kinase transforms it in active triphosphate form. The latter acts as viral DNApolymerase inhibitor. The first round of phosphorylation is not carried out by a thymidine kinase (absent in CMV), but by a viral kinase encoded by the gene UL97 It has an intracellular half-life higher then acyclovir. HIV TREATMENT GOAL. Once initiated, ART should be continued for decades (lifetime?), with the following key treatment goals: • Maximally and durably suppress plasma HIV RNA • Restore and preserve immunologic function • Reduce HIV-associated morbidity and prolong the duration and quality of survival • Prevent HIV transmission. ART should be initiated in all individuals living with HIV, regardless of WHO clinical stage and at any CD4 cell count! .Even if HAART (higly active antiretroviral therapy) is effective in suppressing plasma HIV-1 RNA levels and restoring CD4+ T lymphocytes to levels where opportunistic infections are rare, it cannot eliminate HIV-1 from the infected patients. If therapy is stopped, HIV returns rapidly. What about the viral life cycle makes retroviruses so difficult to eradicate? Retroviruses synthesize a DNA copy of their genome after entry into the host cell. Integration of this DNA into the host cell's genome is an essential step in the viral replication cycle. The presence of proviral DNA and cellular reservoirs does not allow HIV eradication Although combination therapy for HIV infection represents a triumph for modern medicine, chronic suppressive therapy is required to contain persistent infection in reservoirs. • The availability of drugs more potent and with high genetic barrier (e.g. the integrase inhibitor dolutegravir) allowed to the possibility of changing the dogma of triple therapy!!!! Drug potency and genetic barrier are two important factors in the achievement of a good virologic response and to contain emergence of resistance. • Genetic barrier: The type and the number of mutations required by HIV to develop a fully resistant virus. If a single mutation is sufficient to confer high level of resistance >Drug has LOW genetic barrier> HIGH likelihood of clinically meaningful resistance. If a substantial number of mutations is required to confer high level of resistance >Drug has HIGH genetic barrier> LOW likelihood of clinically meaningful resistance. An ARV's intrinsic antiviral potency combined with its genetic barrier to resistance influences its ability to protect from virological failure. • The Env comprises three gp120-envelope glycoproteins and gp41transmembrane subunits bound non-covalently in a trimer of heterodimers. This facilitates HIV-1 entry through a multi-step process involving sequential structural rearrangements of both gp120 and gp41. HIV-1 Entry Process and Inhibition: Fostemsavir binds gp 120, Ibalizumab binds CD4, Maraviroc binds CCR5, Enfuvirtide binds GP41. Mechanism of action of Nucleos(t)ide reverse transcriptase inhibitors • Nucleoside analogues and nucleotide analogues arrest the synthesis of viral DNA by reverse transcriptase. •After phosphorylation by cellular kinases, these compounds are incorporated by reverse transcriptase into the nascent chain of viral DNA. Because these drugs lack a 3' hydroxyl group, no additional nucleotides can be attached to them, and the synthesis of viral DNA is arrested. NTRIs- tenofovir. • Lamivudine • Emtricitabine. • Nonnucleoside reverse-transcriptase inhibitors are small molecules that have a strong affinity for a hydrophobic pocket located close to the catalytic domain of the reverse transcriptase. • The binding of the inhibitors affects the flexibility of the enzyme, thereby blocking its ability to synthesize DNA. Integrase Inhibitors: Raltegarvir, Elvitegravir, Dolutegravir, Bictegravir, Cabotegravir. Integrase mediates the transfer of the proviral DNA into the host genome in two steps: 1) the 3’-end processing that excises a dinucleotide at the 3′- end and 2) the strand transfer step that integrates HIV DNA into the host chromosome. A third reaction which is the reverse of the strand transfer reaction called the disintegration terminates the process. Once integrated, the stable provirus is established after the DNA repair by the host enzymes. Integrase inhibitors interfere with the strand transfer process of the viral DNA by competitively binding to the active site of integrase. Protease inhibitors • The HIV protease cleaves large polyprotein precursors at specific sites, releasing the structural proteins and enzymes necessary for the assembly of infectious viral particles. • Protease inhibitors display a strong affinity for the active site of the HIV protease and inhibit the catalytic activity of the enzyme in a highly selective manner. Protease inhibitors impede the maturation of the viral particle. •Resistance to protease inhibitors is the consequence of amino acid substitutions that emerge either inside the substrate-binding domain of the enzyme or at distant sites. • Directly or indirectly, these amino acid changes modify the number and the nature of the points of contact between the inhibitors and the protease, thereby reducing their affinity for the enzyme. • Due to its high degree of genetic variability, HIV can escape the pressure imposed by the drugs, by acquiring mutations that confer drug resistance. • Drug resistance denotes the ability of the virus to replicate despite the presence of the drug. Thousands of people with HIV will now be offered a new long-acting injection to manage their condition if they would prefer to stop taking daily pills. ……..An estimated 13,000 people in England could make the switch. Cabotegravir and rilpivirine are given as two separate injections every two months, after an initial phasing-in period. The treatment is only suitable for those who have already achieved undetectable levels of virus in blood while taking tablets. Drugs available for the treatment of chronic hepatitis B virus. RT inhibitors Nucleoside RT inhibitors: • Lamivudine • Entecavir • Telbivudine. Acyclic nucleoside phosphonate analogues RT inhibitors: • Adefovir • Tenofovir. Immune-modulant drugs • Interferon α 2A • Interferon α 2B. Response to INFbased therapy depends on HBV genotype. HBV genome (cccDNA) persists and is archived inside the hepatocytes This makes HBV infection not curable. Due to the overlapping between the RT and HBsAg gene, some drug resistance mutations in the RT correspond to stop codons in the HBsAg. The issue of stop codons and deletions in S antigen is clinically relevant. The currently available anti-HBV drugs can suppress the synthesis of viral genome and viral particles production, but cannot suppress at all HBsAg production. •In presence of HBsAg stop codons or deletions, the production of truncated HBsAg can still go on even if the patient receives a fully suppressive therapy •Thus, in presence of HBsAg stop codons or deletions, the risk of liver cancer can persist even if the patient has undetectable viremia. HIV, HBV and HCV share several biological similarities, but … ORFs, open reading frames; cDNA, complementary DNA; cccDNA, covalently closed circular DNA. Differently from HIV and HBV: •HCV replication occurs only in cytoplasm •Viral genome is not archived into the genome of infected cells … This makes HCV curable!!!!. Inhibitors against hepatitis C virus (HCV) infections :Ribavirin, Interferons, NS3/4A protease inhibitors, NS5A inhibitors, NS5B polymerase inhibitors. Ribavirin It is a nucleoside analogue of guanosine that inhibits replication of several RNA and DNA viruses. The exact mechanism of action of the drug is not well defined; it is surely transformed into phosphorilated derivatives and inhibits the inosine monophosphate deidrogenase enzyme, with reduced synthesis of guanine nucleotides and inhibits the production of viral nucleic acids. Non competitive inhibitor of RNA polymerase. Low effect in vitro, often good in animals but poor in humans. Aerosol employment: syncytial respiratory virus. HCV:interferon. Interferon is a system of defense of organisms. Once it is excreted in the cell, interferon comes into contact with new cells and promotes in them virus resistance by induction.  Interferon has the following advantages: broaden action spectrum; low toxicity and modest antigenic properties. It is elaborated by cell genome when a cell matabolic response follows viral action. Interferon + Ribavirin: effects  This combination allows to improve the duration of virologic response (non-detectability of viral load, HCV RNA) compared to the monotherapy with ribavirin or with interferon.  This therapy also allows to achieve a sustained virological response (defined as nondetectability of HCV RNA for at least 6 consecutive months) in 50% of the cases.  Nevertheless there is a certain variability in the response depending on the viral infective genotype. VACCINE Body inoculum, able to stimulate an active immune response against a specific factor (microorganisms, viruses, toxins). Jonas Salk developed the first vaccine against poliomyelitis (inactivated vaccine). Albert Sabin developed the first vaccine against poliomyelitis which was orally givable (weakened live vaccine). All the viruses have a tropism exclusively human…have the possibility to be eradicated by a vaccine. Measles, rubella, HBV, smallpox and polio have not animal reservoir THEY ARE ONLY HUMAN!!!!!!!!!! Vaccines determine: - Reduction / absence of disease in individuals - Reduction / absence of circulation of a pathogen in unvaccinated and / or at risk people They are useful to both the health of every single individual and the protection of public health. Herd immunity (also called herd effect, community immunity, population immunity, or social immunity) is a form of indirect protection from infectious disease that occurs when a large percentage of a population has become immune to an infection, thereby providing a measure of protection for individuals who are not immune. The following vaccinations are so far obbligatory in Italy • Anti Tetanus • Anti Diphtheria • Anti Polio • Anti Hepatitis B • Anti Pertussis • Anti Haemophilus influenza b • Anti Measles • Anti Mumps • Anti Rubella • Anti Varicella. Two Types of Immunization • Passive Immunization – Methods of acquisition include natural maternal antibodies, antitoxins, and immune globulins – Protection transferred from another person or animal • Active Immunization – Methods of acquisition include natural infection, vaccines (many types), and toxoids – Relatively permanent. Passive immunization is a short-term immunization achieved by the transfer of antibodies, which can be administered in several forms; as human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous or intramuscular use, as high-titer human, from immunized donors or from donors recovering from the disease, and as monoclonal antibodies. Immunity derived from passive immunization lasts for a few weeks or months. It provides immediate protection, but the body does not develop memory, therefore the patient is at risk of being infected by the same pathogen later. Passive immunization is used when there is a high risk of infection and insufficient time for the body to develop its own immune response, or to reduce the symptoms of ongoing or immunosuppressive diseases. Passive immunization with immune sera depends from: • Halving rate of antibodies (generally 3 weeks) • Antibody titer The commercial sera are enriched with specific antibodies against specific antigens. Limitations: protection time, administration time, ok only if at the beginning of the acute infection. There is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin. Ideal characteristics of a good vaccine • Efficacy • Once-a-day administration • No side effects • Stability • Easy administration • Wide availability • Low prices. Vaccine constituents • Active ingredient • Preserving liquid (water o sterile solution saline) • Preservatives • Stabilizers (albumin) • Antibiotics • Adjuvants. The route of administration is chosen for each type of vaccine to optimize the immune response and can be: • Oral • Parental • Intradermal • Subcutaneous • Intramuscular. • Vaccines based on whole inactivated organisms • Vaccines based on attenuated living organisms • Vaccines based on toxoids • Vaccines based on specific subunits. Vaccines based on whole inactivated organisms: This is the first method by which vaccines were produced. The proper system of cultivation of the microorganism is treated with specific reagents (heat, formaldehyde, glutaraldehyde, UV rays) to inactivate the virulence. Little risk of being infected. Possibility of preservation by Lyophilization. The disadvantages of this method are that the vaccine produced in this way elicits a little (or completely absent) cell-mediated immunity. It needs booster doses. (Examples) • Whooping cough (pertussis) • Typhoid fever • Cholera • Plague (yersina pestis) • Rickettsiosis • Flu • Hepatitis A • Polio (Salk type) • Rabies • Anti-Respiratory Syncytial Virus (Dangerous!). Vaccines based on attenuated living organisms I They are produced by isolating a microorganism of low virulence from a case of mild disease. There is also the possibility of producing attenuated microorganisms by performing multiple cultures on different culture systems, in order to decrease its virulence but not antigenicity. A third option is by chemical mutagenesis (eg. Salmonella typhi nitrosoguanidine). This category includes many viral vaccines. This system allows the in vivo replication of the attenuated microorganism. This exposes the body to a high dose of antigen and allows the use of low starting doses and fewer boosters. Their effectiveness is generally higher than that of vaccines based on inactivated microorganisms. In the case of intracellular microorganisms, a strong cell-mediated response is also induced. The disadvantages of this system include the possibility of a reactivation of the virulent microorganism (event quite unlikely to happens in the case of microorganisms with multiple mutations). In the case of viral vaccines, it is well known that some viruses can integrate their genome into the host cell (such event is not prevented by vaccines produced in this way). It cannot be used in immunocompromised persons. Difficulties of storage (at least -4°C, preferably 80 °C). Vaccines based on attenuated living organisms IV • Measles • Mumps • Rubella • Varicella-Zoster Virus • Poliomyelitis (Sabin type) • Yellow fever • Smallpox • Typhoid fever (Salmonella typhi var. Ty 21) • Tuberculosis (bacillus Calmette-Guérin). Vaccines Based on Toxoids Anatoxin or toxoid: toxin whose toxicity but not immunogenicity has been suppressed by chemical treatment (formaldehyde, iodide, diazonium salt). These vaccines are usually administered with an adjuvant. These vaccines are innocuous and easy to keep (at room temperature). Detossification process is not reversible. An adjuvant is a pharmacological or immunological agent that modifies the effect of other agents. Adjuvants may be added to vaccine to modify the immune response by boosting it such as to give a higher amount of antibodies and a longer-lasting protection, thus minimizing the amount of injected foreign material. Adjuvants may also be used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells; for example, by activating T cells instead of antibody-secreting B cells depending on the purpose of the vaccine. Adjuvants are also used in the production of antibodies from immunized animals. There are different classes of adjuvants that can push immune response in different directions, but the most commonly used adjuvants include aluminum hydroxide and paraffin oil. Vaccines based on anatoxins II • Diphtheria • Tetanus • Botulism • Poisonous snakes. Tetanus • Tetanus is an acute, often fatal, disease caused by an exotoxin produced by the bacterium Clostridium tetani. • It is characterized by generalized rigidity and convulsive spasms of skeletal muscles. • The muscle stiffness usually involves the jaw (lockjaw) and neck and then becomes generalized. Clostridium tetani • C. tetani is a • slender, • gram-positive, • anaerobic rod, • that may develop a terminal spore, giving it a drumstick appearance. • The organism is sensitive to heat and cannot survive in the presence of oxygen. The spores, in contrast, are very resistant to heat and the usual antiseptics. They can survive autoclaving at 249.8°F (121°C) for 10– 15 minutes. The spores are also relatively resistant to phenol and other chemical agents. The spores are widely distributed in soil and in the intestines and feces of horses, sheep, cattle, dogs, cats, rats, guinea pigs, and chickens. Manure-treated soil may contain large numbers of spores. In agricultural areas, a significant number of human adults may harbor the organism. The spores can also be found on skin surfaces and in contaminated heroin. • C. tetani produces two exotoxins:  tetanolysin  tetanospasmin. The function of tetanolysin is not known with certainty. Tetanospasmin is a neurotoxin and causes the clinical manifestations of tetanus. • On the basis of weight, tetanospasmin is one of the most potent toxins known. The estimated minimum human lethal dose is 2.5 nanograms per kilogram of body weight (a nanogram is one billionth of a gram), or 175 nanograms for a 70-kg (154lb) human. The basic cycle of the tetanus vaccination consists of three doses of vaccine, to be administered to the child within the first year of life, in particular in the third, fifth and twelfth months, simultaneously with the other childhood vaccinations. Currently, for the immunization of infants, the socalled hexavalent vaccine tends to be used, which, in addition to protecting against tetanus, also prevents diphtheria, poliomyelitis, hepatitis B, pertussis and Haemophilus influenzae type infections B (responsible among other things for meningitis). Two booster doses (always associated with the diphtheria and pertussis vaccine) are given at a later time: the first at 6 years of age, the second at around 15 years of age. To maintain an adequate level of immunity, further boosters can be planned every 10 years, using the vaccine for adults. The vaccine is given via intramuscular. Subunit vaccines: These vaccines consist of purified bacterial or viral components that elicit a protective immune response. These vaccines have better safety profiles than the others and are associated with a less risk of adverse reactions. These vaccines can be prepared directly from microorganism or through genetic engineering techniques. Subunit vaccines • Hepatitis B • Influenza • Human Papillomavirus (HPV) • Haemophilus influenzae • Neisseria meningitidis • Streptococcus pneumoniae. Vaccine against Hepatitis B It is composed by purified HBsAg, attained from transformed cells of Saccharomyces cerevisiae. Vaccine against human papillomavirus (HPV) –1991: Zhou et al. demostrated that the major capsid protein L1 can assemble to form Virus Like Particles without pathogen properties but with high immunogenicity. – Protection is type-specific. – Only epidemiologic relevant genotypes are considered. – There are 3 commercially available vaccines: - Bivalent (Cervarix): genotypes 16 & 18 -Tetravalent (Gardasil): genotypes 16, 18, 6, 11 - Nonavalent (Gardasil 9): genotype 6, 11, 16, 18, 31, 33, 45, 52, 58. Vaccines based on vectors: These vaccines are based on genome modification of a innocuous and weakened microorganism introducing the genic sequence of selected antigen in order to make it express. An upside of using vectored vaccines is that they are easy and relatively cheap to make. The adenovirus vector, for example, can be grown up in cells and used for various vaccines. “Once you make a viral vector, it is the same for all vaccines. It is just the genetic information in it that is different”. mRNA vaccines take advantage of the process that cells use to make proteins in order to trigger an immune response and build immunity to SARS-CoV-2, the virus that causes COVID19. Mechanism for Action mRNA vaccines have strands of genetic material called mRNA inside a special coating. That coating protects the mRNA from enzymes in the body that would otherwise break it down. It also helps the mRNA enter the dendritic cells and macrophages in the lymph node near the vaccination site. mRNA can most easily be described as instructions for the cell on how to make a piece of the “spike protein” that is unique to SARS-CoV-2. Since only part of the protein is made, it does not do any harm to the person vaccinated but it is antigenic. After the piece of the spike protein is made, the cell