Infectious Diseases PDF

Infectious Diseases Background, classification and principles What is a virus? 'A piece of bad news wrapped in a protein' 'A package of misinformation' Definition: Protein code that has misinformation (RNA or DNA) in the virus particle. This RNA or DNA instructs the cell to do something different than it was doing before. - Small, obligatory intracellular, infectious agents. - Virus ONLY replicates in HOST and is fully dependent on this HOST for energy metabolism, lipid biogenesis and protein synthesis. - Virus particles do not grow or divide. They are built de novo from assembly of pre- formed components. Formal definition: Obligate intracellular parasites or symbionts that possess their own genomes encoding information required for virus reproduction and, hence, a degree of autonomy from the host genetic system, but do not encode a complete translation system or a complete membrane apparatus. Tabacco plants were dying -> Martinus Beijerink wanted to know if a bacterium was causing this. He used a filter (only things smaller than bacteria could go through) -> ‘contagious living fluid’ (first virus described). Terms: RNA or DNA in virus particle (virion) encapsulated by a capsid. The viral nucleic acids together with the capsid are called the nucleocapsid. This is surrounded (not always) by an envelope. This is always derived from the host when the virus buds through the membrane. Replication cycle: 1. Virus enters the cell by attachment mediated by a molecule on the cell (receptor). This triggers the entry of the nucleocapsid into the cell. 2. The nucleocapsid releases the nucleic acids resulting in replication of the RNA/DNA. 3. RNA synthesis, new nucleic acids are made, new capsid proteins are made. 4. They come together and are released through a process called budding or the virus lyses the cell to release itself (and then it does not acquire an envelope). Viruses are extremely diverse, but ALL viruses must make mRNA that can be read by host ribosomes -> 1000s of viruses but only 7 genome types (Baltimore classification) dsDNA: DNA needs to be transcribed into mRNA. +ssRNA: can be directly translated by the ribosome. -ssRNA: transcribed into mRNA strand. Retrovirus: reverse transcribed into DNA, first single then double stranded. The DNA is then transcribed into mRNA. +RNA: polymerase (RDRP) encoded on the viral genome (host does not have RDRP). RDRP uses + strand to make – strand. Virus encodes proteins to create structural components (capsid, etc.) -RNA: enters the cell and first needs to make +strand, with use of RDRP. This RDRP is already in the virus particle sitting on the -strand. Then same as in +RNA (polymerase + structural components). RNA virus classification: Viruses within family/genus are NOT transmitted the same way, NOR do they cause the same diseases. Conversely, viruses from different families can cause similar symptoms. General principles of diagnostics Patient arrives: generally start with anamnesis (history), then do clinical examination and samples, laboratory analysis of collected samples and review of laboratory data. Diagnostics support the differential diagnosis (weighing the probability of one disease versus that of other diseases). CRP: C reactive protein. Based on several GP tests you determine the severity. Hospital then determines the type of infection for targeted treatment. Sensitivity = the probability that a truly infected individual will test positive. Specificity = the probability that a truly uninfected individual will test negative. PPV (positive predicted value) = probability that those testing positive are truly infected. NPV (negative predicted value) = probability that those testing negative are truly uninfected. Prognostics (predict progress of disease) Example: HPV can cause cervical cancer; some serotypes are associated with a high-risk progression. The presence of certain serotypes predicts development of disease. Example: RSV leads to disease in about 1-2% of children. The course of disease is very difficult to predict. It is important to predict which children need hospitalization (it is therefore important to develop a prognostic tool -> how do we classify?). Pathogen- and host-based diagnostics: Detection methods: - microscopy-based: Viruses can only be seen with EM. Staining procedures are used on bacteria (gram positive/negative). - culture-based (viruses, bacteria, fungi): Take cell layer and add clinical specimen and look whether it is causing cytopathic effects (CPE). Based on these effects we can distinguish the type of virus. CPE can be used to quantify viruses (plaque assay) -> count the number of locally lysed areas -> quantify number of infectious viruses. Bacterial cultures -> can also determine sensitivity against antibiotics (determine minimal inhibitory concentrations based on the halo). We now also use mass- spectrometry (MALDI-TOF MS) -> determine spectrum (different for every species). - antibody-based: fluorescently labelled antibodies. - Molecular: PCR (also quantitative: SYBR Green, TaqMan) to detect viruses/pathogens and Whole Genome Sequencing (if more information is needed). Quantification (amount of RNA in the plasma) is used to stage disease activity, predict disease progression and to monitor the efficacy of treatment and the transmission. Determination of which test to use depends on the condition of the patient. Metagenomics: Unbiased: sequence everything in your sample to determine which types of microbes live together in a community. (But you look at the data in a biased manner) - Very expensive: only used when there is no idea what pathogen is present (PCR primers are designed based on the suspicions). - This also results in false positives, since you detect ‘innocent bystanders’ that are present, but not the cause of disease. Can also help with reconstruction of viral genomes and is also used to analyse the microbiome. Host-based biomarkers (looking at the host response): - ‘Serology’ = antibody response to infection - Measure IgG, but this is still present long after you’ve already recovered from an infection. - IgM is more interesting, since this is the initial response and declines sooner compared to IgG. - CRP (C Reactive Protein) and PCT (Pro-Calcitonin): CRP is an acute phase protein (complement system (always sense for damaged cells)), when there is infection -> activates complement system -> CRP synthesis (can be measured very shortly after onset of infection). PCT is produced by the thyroid gland, the exact mechanism is unknown. It is needed to quickly distinguish between viral and acute bacterial infections. The information on susceptibility to antimicrobial agents needs improvement. Disease outbreaks need to be tracked in early phases, allowing for identification of new pathogens and track their outbreaks. Challenges: wide variety of specimen and diagnostics need to be applied to different patient populations in different settings. Point-of-care: increase the turn-around time of sampling to diagnosis and treatment. è increase turn-around time, focus on host transcripts and proteins (biomarkers of infection). Biased and unbiased identification of protein biomarkers: Biased: antibody-based Unbiased: proteomic profiling, mass spectrometry Interactive Lecture Virus replication Polio -> poliomyelitis (inflammation of the spinal cord) - Oral fecal transmission - Virus shedding weeks - Replication in the intestine - protects the 5’ end (otherwise nucleases destroy the RNA). Poly-A tail at the 3’ end. Looks like cellular mRNA on the 3’ end, not on the 5’ end. Cap-independent translation of viral RNA Cellular mRNAs have a 7-methyl guanosine (m7G) cap (also referred to as 5’ cap) and poly A tail at the 5’ and 3’ end. What are their functions? Translation initiation, RNA stability. How does poliovirus ensure translation of its viral RNA by the host ribosome? IRES -> Internal Ribosomal Entry Site How would the virus benefit from this approach? It can translate under conditions where normal cap-dependent translation cannot occur. Stress response phosphorylates eIF2alpha -> all cap-dependent translation stops. Protease of the virus cleaves the eIF4G. Cellular mRNAs usually encode a single protein; the (single) viral RNA of poliovirus encodes 11 proteins. What strategy does the virus use to achieve this? Polyprotein processing. One large ORF with start and stopcodon -> translation. Protease cleaves the large protein into smaller chunks. Virus diagnostics making use of assessing the cytopathic effect (CPE) in cell culture is not routinely used in laboratory diagnostics. Why is this the case? Which of the following answers about cell culture-based virus detection in NOT true. 1. Waiting time until CPE can be observed is often very long 2. CPE is often not specific, making identification of the infecting virus difficult 3. CPE can also develop after the virus infection in the patient has been cleared What is the purpose of adding a semisolid agarose matrix on top of the cell monolayer during a plaque assay? 1. It prevents the cells from drying out 2. It prevents possible cell-culture contaminations with bacteria and fungi. 3. It prevents virus particles from freely floating through the cell culture dish 4. It is used to stain the cell monolayer and visualize plaques 5. It provides nutrients and growth hormones to the cells. Which statement about a quantitative PCR is true? 1. The more DNA in the sample, the lower the Ct value 2. The more DNA in the sample, the higher the Ct value 3. The more DNA in the sample, the lower the maximum fluorescence 4. The more DNA in the sample, the higher the maximum fluorescence Which of the following methods can be used to detection infectious virus? 1. Antibody ELISA against IgG 2. Electron Microscopy 3. Plaque assay 4. Quantitative PCR 5. Rapid antigen test Two related methods to quantify infectious particles rely on CPE: Plaque assay End point dilution (no agar) -> dilution, at some point it starts to become negative -> TCID50 means 50% is positive. Use susceptible cell (HELA, VERO, BHK-21) Detection of genetic material (quantitative PCR): qPCR (Taqman), measures fluorescence over time (Ct is number of cycles where the fluorescence is above a certain threshold). Starting number does not matter, the end number is always the same. Arboviruses Arboviruses = arthropod-borne viruses (transmitted by arthropod vectors) - mosquitos (mostly tropical, subtropical) - sandflies - gnats (mostly transmit to farm animals) - ticks (lyme, but also others) Arboviruses evolved in different virus lineages. Most arbovirus infections are asymptomatic or results in mild, mostly generic symptoms (flu-like symptoms). These symptoms are primarily caused by the activation of the immune system. There are also viruses that cause severe symptoms (such as Dengue virus). - Increase in capillary permeability -> hypovolemic shock (organ impairment, plasma leakage and internal bleeding. - Disease is not always due to direct infection. Vascular permeability in Dengue is mostly due to activation of endothelial cells by high levels of cytokines. Direct infection of these cells does NOT seem to be the major cause of the plasma leakage. Other viruses causing extreme symptoms are Zika, West Nile virus, yellow fever induced. è All part of the Flavivirus genus. Tropism Zika tropism: - Infection of the placenta -> affects unborn child. - Affect neuronal progenitor cells, in unborn children this leads to incomplete development of the brain (CZS -> microcephaly) (GBS -> degeneration of axonic myelin sheets caused by the immune response) - Uterus, vagina/testis. Zika can be transmitted from mosquito to a human host, it replicates in both human and mosquito host (also transmission through sex and blood transfusion) Viruses are obligate intracellular parasites and therefore need to enter a cell (too large to diffuse across the plasma membrane) Initial encounter between virion and cell is governed by chance (viruses have no power of locomotion) Attachment of a virion protein to specific receptor molecules on cell surface (in some cases, multiple receptors are required [e.g., HIV-1]) Virus usually enters via an endocytic pathway DC-SIGN is considered one of multiple possible receptors that is involved in the entry process for ZIKA and DENV. DC-SIGN is expressed on Dendritic Cells, explaining DENV tropism for these cells. Another pathway in DENV: Fc receptors on macrophages/monocytes that recognize the conserved tail of an antibody, leading to degradation of the pathogen. DENV exploits this, it escapes the degradation. 1. Virus binds to receptor -> endocytosis -> RNA into cytoplasm. 2. Antibodies cover the entire virus (neutralizing) -> binding to Fc receptor -> internalization of the virus. 3. Antibodies (non-neutralizing) because affinity is too low -> endocytosis -> change in pH -> virus fuses to membrane and can enter the cell. Low pH induces conformational change -> spiky pattern -> allows virus to insert part of the protein into the membrane of the endosome -> fusion of viral and endosomal envelope. Flavivirus structure: + strand RNA-virus Encode a single polyprotein. Individual proteins are maturated through cleavage. Viral mRNA is capped but not poly adenylated. RNA structures in the 5’ and 3’ UTRs are needed for replication/translation. 3’ UTR gives rise to a non-coding RNA called subgenomic flavivirus RNA (sfRNA) (this is thought to inhibit immune responses). Translation of the viral polyprotein occurs at the membrane of the ER, several proteins are transmembrane proteins. C, prM, E = structural proteins NS = non-structural proteins (NS5 = polymerase, RdRp) Cleaved by proteases (both viral (NS3) and from host). Dengue virus Exists as 4 serotypes ADE (antibody dependent enhancement) is a major problem for developing a dengue virus vaccine: ADE occurs when antibodies raised during infection with one serotype are non-neutralizing in a heterologous infection with a second serotype. - There was even ADE between DENV and ZIKV. A vaccine should produce neutralizing antibodies against all serotypes to prevent vaccine-induced antibodies from enhancing disease. è Dengue vaccines are generally tetravalent. Dengue virus replication: 1. Attachment of the virus particle: To host receptor To DC-SIGN (receptor on dendritic cell) To Fc receptor (bound by antibody) 2. Receptor-mediated endocytosis 3. Acidification of endosome results in conformational change of envelop protein (fusion with membrane) 4. Translation of polyprotein 5. Replication of genomic RNA 6. Formation of nucleocapsid 7. Budding into the lumen of the ER (membrane is ER-derived) + assembly of immature virions 8. Maturation of virion (Cleavage by the Golgi protease Furin results in maturation of the E protein from prME) + release of mature virus particles. Common routes of virus infections - Limited number of entry ports for viruses. - Skin is an effective barrier. Virus replication also takes place in mosquitos. Bloodmeal -> virus goes to midgut where the virus infects the epithelial cells. It replicates there and eventually escapes the midgut and replicates in other tissues to accumulate enough to infect the salivary glands. (also barriers in mo Entrinsic incubation period = The time between taking an infectious bloodmeal and being able to transmit the virus. Vector competence: intrinsic ability to transmit a specific virus, how well a virus can be transmitted by a mosquito. Vectorial capacity: rate at which a mosquito population generates new infections from a single infectious host. Most arboviruses are not transmitted human to human, but between mosquitos and wild animals (enzootic cycle). - Example: West Nile virus (Culex) (prefer to feed on birds). General symptoms like fever, swollen lymph glands, nausea, muscle aches, with the exception or few extreme cases. Major implication: a vaccine in humans can NOT stop the virus circulation (enzootic). Urban epidemic cycle (CHIKV, DENV, etc.) -> prefer to feed on humans. These mosquitos are adapted to an urban environment (adaptation to breed in urban environment) -> contribute to the vectorial capacity of Aedes mosquitos. - Aedes mosquitos (aegypti and albopictus) both in Southern hemisphere, albopictus also in US and parts of Europe. Climate change -> expansion of Ae. albopictus in Europe. Example: CHIKV outbreak on La Reunion (French island). Aedes aegypti is not present on the island (principal vector for CHIKV) and Aedes albopictus was present but is a poor vector for CHIKV. Scientists looked at amino acid sequence -> single amino acid change resulted in far less virus needed in the blood to lead to infection. The virus adapted to albopictus causing the large outbreak. Vector control: - Insecticides - Repellents - Eliminate breeding sites - Prevent bites However, this might result in resistance. Wolbachia - Gram-negative bacterium - Maternally transmitted - Many insects are already infected by this bacterium It induces cytoplasmic incompatibility -> when a Wolbachia infected male mates with an uninfected female -> offspring will not develop. This enhances the presence of Wolbachia in the population. Wolbachia infection can protect from virus infection in drosophila, it can be introduced in Aedes aegypti in the lab to replace the population with Wolbachia infected mosquitos. - It can also result in reduced Dengue incidence. Interactive Lecture Chikungunya 1. Describe the genome organization of CHIKV. a. How many different RNA molecules are present in the CHIKV virion? The CHIKV virion contains a single, positive-sense RNA molecule. 1 piece of RNA in the virus particles b. How many different viral RNA molecules are present in infected cells? In infected cells, there are multiple RNA molecules present, positive, negative, positive and sgRNA. 3 different molecules. c. Which structures are found at the 5’ end and 3’ end of the viral RNA? The CHIKV RNA has a cap structure at the 5’ end and a poly-A tail at the 3’ end. d. How are structural and non-structural proteins organized on the viral RNA, and how is their translation regulated? The viral genome is organized into two main open reading frames (ORFs). The 5' end of the genome encodes the non-structural proteins (nsP1–nsP4), which are involved in replication, while the 3' end encodes the structural proteins (C, E3, E2, 6K, and E1). The non-structural proteins are translated directly from the genomic RNA, while the structural proteins are translated from a subgenomic RNA that is transcribed during replication. Translation regulation is mediated by the use of host cell machinery and the production of subgenomic RNA for efficient synthesis of structural proteins. e. Which viral protein is the core of the RNA-dependent RNA polymerase? The viral protein nsP4 functions as the RNA-dependent RNA polymerase. Stopcodon in front of nsP4. This can also be ignored and will then be cleaved later on. Further processing of the fusion protein -> positive sense. Alphaviruses need much more of the structural proteins. 2. Describe the replication cycle of CHIKV. a. How does CHIKV enter the cell? CHIKV enters the cell through receptor-mediated endocytosis. The viral envelope proteins interact with specific cell surface receptors, triggering the endocytic pathway. b. How does the uncoating process work? Once inside the endosome, the acidic environment induces conformational changes in the viral envelope proteins, facilitating the fusion of the viral envelope with the endosomal membrane. This leads to the release of the viral RNA into the cytoplasm. Acidification of the endosomes induces conformational change. c. At which location in the cell are new nucleocapsid particles and mature virions assembled? New nucleocapsid particles are assembled in the cytoplasm. Mature virions are assembled at the plasma membrane, where the nucleocapsid interacts with the viral envelope proteins embedded in the host cell membrane, and the virions are then released from the cell through budding. (Budding at the surface of the cell at the plasma membrane.) 3. What type of diseases is associated with CHIKV? CHIKV is associated with an acute onset of fever, severe joint pain, muscle pain, headache, nausea, fatigue, and rash. In some cases, chronic joint pain can persist for months or even years. 4. What type of vaccine is Ixchiq? Live-attenuated vaccine. 5. What are putative problems with this type of vaccine? Immunocompromised Adverse reactions Reversion to virulence 6. What is a main limitation of the vaccine trial used to analyze Ixchiq’s efficiency? A major limitation of the vaccine trial could be the relatively small sample size or short duration of follow-up, which might not accurately capture long-term efficacy or rare adverse effects. Additionally, a limited geographical scope may not fully account for the genetic diversity of the virus in different regions. Sample size and population diversity. Difficult to assess efficacy against chronic infection. Respiratory Infections Virus transmission: Zoonosis: infection from animals to humans. SARS-CoV Transmission from bats to humans (market) followed by human-to-human transmission. - High mortality rate Interventions: Infection control (face masks) Check for fever Rapid quarantine upon detection of symptoms MERS-CoV Bats -> camels -> humans (camel markets) - Similar symptoms to SARS-CoV, fewer cases, but higher mortality rate. SARS-CoV2 (COVID-19) - Coronavirus family Limit in sizes of RNA viruses: RdRP is error prone (about 1 mutation per 10 kb) so viruses are usually not larger than 10 kb. Low proof-reading and no correction These mutations are both good and bad: Resistance and adaptability Could generate defective viruses This is the reason the genomes are this small. The genome of SARS-CoV2 is 29.9 kb (large) + ssRNA 13 ORFs Cap and poly-A-tail - Non-structural proteins - RdRp - Proteases (cleave itself off) - Spike proteins! - Proteins that are there to interfere with immune response of the host, especially effective since it has multiple of these proteins Viral tricks for viral gene expression: Ribosomal frameshift: in other ORF (not random) and occurs when there is a 'slippery' sequence (A & U) that slows down the ribosome causing the 'slipping', this is used to regulate the relative amount of the proteins Polyprotein processing Leaky scanning Subgenomic RNA: in addition to full length RNA you get a set of RNAs that correspond to the last bit of the sequence, seperate for all ORFs (all with cap and A tail) Subgenomic RNAs are created by discontinuous transcription during (-)RNA synthesis. When the polymerase encounters transcription-regulating sequences the RdRP skips to the TRS to create the short sequences. (in SARS-CoV2 this occurs in negative strand synthesis -> these negative strands are then used to make positive strands). Entry SARS-CoV2: 1. It uses the spike protein to attach to cell surface receptors (ACE2 (expressed on lung epithelial cells) 2. Enters at cell surface or endosomal compartment (release of RNA in the cytoplasm) -> see explanations below Cell surface entry (more relevant) The spike is a trimer (S1 (on top of S2, binds receptor) and S2) is expressed as protein during translation. During maturation on the way out, the virus is cleaved, so you get S1 S2 heterodimer on the surface of the virus. S1 S2 occurs 3 times -> trimer on the surface. RBD of S1 binds ACE2 receptor S2' is cleaved after binding to ACE2 receptor by the protease TMPRSS2 (it cleaves at the S2' site). After binding to ACE2 the virus is docked on the cell surface -> need other proteotic cleavage S2' to activate the spike protein (FP!!!! (very hydrophobic)) -> conformational change -> insertion in plasma membrane of host. (bridge between viral and host membrane) Endosomal entry If there is no TMPRSS2 the virus will be endocytosed. In the endosomes are proteases like Cathepsin L will mediate the cleavage -> entry and release in cytoplasm. After entry: - Replication of the viral RNA including nested set of sgRNAs. - Viral protein expression of all sgRNAs. - Genome encapsulated by nuclear protein made from sgRNAs. - It is bound by nucleocapsid protein. - Buds into Golgi apparatus. - Virus received its envelope including the spike proteins. - Follows cells excretory route to infect the lumen. It remodels the intracellular membrane to generate shielded compartments where they duplicate (for SARS-CoV2 = double membrane vesicles). High viral RNA concentration present in throat and nasopharynx early in infection. This was compared between SARS-CoV and SARS-CoV2. The viral load in CoV2 was about a thousand- fold higher than in CoV, it also peaked sooner and there was an early detection in all patients. There is thus a very efficient transmission of SARS-CoV2. - Many clinical symptoms, mainly in respiratory system, but also kidney, neuronal, etc. SARS-CoV2 causes direct virus-induced cell/tissue damage AND damage by hyperactivated immune response (IFNs and cytokines recruiting many cells). Drug development against COVID: - Drugs that inhibit viral proteins (RdRP, protease) - Host-targeting drugs (reducing inflammation) Dexamethasone: glucocorticoid steroid (anti-inflammatory) Safe and cheap drug RSV (Respiratory Syncytial virus) (mononegavirales (non-segmented, -RNA)) lower respiratory tract infection (epithelial cells) highly contagious virus and immune-induced damage problematic in young children RNA is already bound to proteins it needs -> L protein (RdRP), P protein (essential cofactor for L), N protein (shell) and on the surface F protein (entry) and G protein (binds host receptor). Replication RSV: - Entry by G proteins binding to molecules as initial attachment. - Fusion is then mediated by F protein interacting with the receptors. - Virus enters either through release of RNA at surface or via an endocytic pathway (macropinocytosis) - Replication -> transcription -> translation - Assembly and budding But also, cell-to-cell spread. - NS proteins modulate immune response, interfere with expression of immune proteins Influenza A Four types (A-D) Natural virus reservoir is wild aquatic birds 8 -strand ssRNA segments that encode 10 genes Replicates in the nucleus Lipid envelope with main proteins: - HA (haemagglutinin) binding: HA attaches by binding to glycans on the cell surface -> either alpha2,3 (preferred in birds) or alpha2,6 (preferred in human) - NA (neuraminidase) cleaves sialic acid from cell surface molecules -> can be trapped, helps it to be released and approaching the cell surface. Nucleocapsid proteins are directly interacting with viral RNA. The envelope is supported by the matrix protein (M1). - Influenza strains are classified based on antigenic properties of HA and NA. Entry process: - Binding to receptor -> insertion of fusion peptide into the membrane of the host cell -> conformational change -> fusion of viral membrane and host membrane. - Viral RNAs and polymerase released into cytoplasm and into the nucleus (protein complex sitting on the RNA has nuclear localization signal) - RNA replication in the nucleus, translation in cytoplasm (ribosomes), encapsulation in nucleus again (mRNA is capped and poly adenylated). - Assembly and budding. Antiviral drugs: Targeting of HA-NA activity to suppress IAV -> block virus production. Adamantanes blocks ion channels (when low pH -> release of RNA) RdRP inhibitors Prevention of maturation of HA. Inhibition of cap dependent endonuclease (cap snatching) Many different Influenza subtypes named after countries. Seasonality of epidemic flu: - Immune response -> antigen change -> immune response -> etc. - Evolutionary pressure to keep changing antigens (HA and NA). Antigenic drift: Point mutations -> new viral strains that are not/poorly recognized by host immune response Antigenic shift: Virus has different segments -> reassortment of the segments into a new virus. - Because there is a different preferred conformation for HA binding in humans and birds, direct bird-human or other way around infection does not occur. Pigs have two sialic acids, so pigs can be infected with both. Reassortment in pigs can then lead to new viruses for which there is no pre-existing immunity. Interactive Lecture Coronavirus Dexamethasone Infect susceptible cell -> cell death Medical need -> relevant mechanism -> lead compound -> drug candidate -> clinical testing (whole process takes about 5-10 years and millions of euros) Drug repurposing: test already approved drugs for other disease for antiviral effects and/or clinical benefit. 1. The first papers discusses remdesivir. What is this drug? What is its mechanism of action? If the information cannot be found in these papers, refer to other (reliable) sources. Remdesivir (GS-5734), an inhibitor of the viral RNA-dependent, RNA polymerase with in vitro inhibitory activity against SARS-CoV-1 and the Middle East respiratory syndrome (MERS-CoV),5-8 was identified early as a promising therapeutic candidate for Covid-19 because of its ability to inhibit SARS-CoV-2 in vitro.9 In addition, in non- human primate studies, remdesivir initiated 12 hours after inoculation with MERS-CoV10,11 re- duced lung virus levels and lung damage. It pauses the RdRP or induces chain termination. Remdesivir needs several enzymatic steps before it works. Cellular enzymes convert it into its active nucleoside triphosphate. Triphosphate -> highly charged. 2. Different setups exist for clinical trials. What do the following terms mean, when referring to clinical trials: Randomized: In a randomized clinical trial, participants are randomly assigned to different groups (such as a treatment group or control group). Randomization helps reduce bias by ensuring that each participant has an equal chance of being assigned to any group, making the results more reliable and allowing for a fair comparison between treatments. Double-blind: A double-blind clinical trial means that neither the participants nor the researchers know who is receiving the active treatment and who is receiving the placebo (or an alternative treatment). This setup helps prevent bias in reporting and assessing outcomes, as neither party is influenced by knowing which treatment was given. Placebo-controlled: In a placebo-controlled trial, one group receives the experimental treatment, while another group (the control group) receives a placebo—a substance with no therapeutic effect. The placebo group serves as a benchmark to measure the treatment’s actual effect, helping to determine whether the benefits observed are due to the treatment itself or other factors. Multi-centre: A multi-centre clinical trial is conducted across multiple locations or centers, rather than at a single site. This setup allows for a larger, more diverse population sample and can increase the generalizability of the findings, as the treatment's effects are tested in various settings and among different patient demographics. BEST SETUP FOR CLINICAL TRIAL -> COMBINATION (randomized, double blind, placebo-controlled, (multi-centre)) 3. With reference to question 2, what was the setup of the clinical trials in the paper? How many patients were included? Randomized, double-blind, multi-center and placebo controlled Why is the number of patients important? -> statistics, statistical power, the more people, the more reliable your results 4. The papers refer to primary and secondary outcomes (or: endpoints). What do these terms mean? Primary Outcome (Primary Endpoint): This is the main result that the study is designed to measure and assess. The primary outcome is the most important question the trial seeks to answer, often directly tied to the main benefit or effect of the treatment. For example, in a trial for a new drug to treat hypertension, the primary outcome might be the reduction in blood pressure over a set period. Secondary Outcome (Secondary Endpoint): These are additional effects or measurements that the study is designed to observe but are not as central as the primary outcome. Secondary outcomes often provide supplemental information on the intervention’s effectiveness or potential side effects. For example, in the same hypertension trial, secondary outcomes might include changes in heart rate, cholesterol levels, or quality of life. 5. What is the main conclusion of the studies by Weigel? A 10-day course of remdesivir was superior to placebo in the treatment of hospitalized patients with Covid-19. Patients who received remdesivir had a shorter time to recovery (the primary end point) than those who received placebo. 6. Clinical journals usually request authors to openly discuss the limitations of the study (sometimes under a separate heading in the Discussion). Did the author report limitations to the study? If so, which? Travel restriction. Limited supplies. Hospital restrictions. 7. How is remdesivir given to a patient? Intravenously. 8. Would you recommend using remdesivir for COVID patients? It depends, only on hospitalized patients. 9. The Hammond et al paper assesses the clinical benefit of nirmatrelvir. What is this drug? What is its mechanism of action? Nirmatrelvir is an orally administered severe acute respiratory syndrome coronavirus 2 main protease (Mpro) inhibitor with potent pan–human-coronavirus activity in vitro. Replication -> protease Nirmatrelvir and ritonavir (anti HIV drug) 10. What is the setup of the trial of the by Hammond et al? Randomized, double-blind, multi-center and placebo controlled 11. What is the main conclusion of the study? Treatment of symptomatic Covid-19 with nirmatrelvir plus ritonavir resulted in a risk of progression to severe Covid-19 that was 89% lower than the risk with placebo, without evident safety concerns. 12. Do the authors report limitations of the study or the drug? If so, which? Restricted to unvaccinated people. 13. Would you recommend the use of dexamethasone for COVID patients? Yes, but only to patients with a high risk of developing severe disease. Respiratory Virus Infections Mucosa -> open connection to the outside world, the mucosa consists of one or more layers of epithelial cells. It is the barrier and first line of defense, you will find the immune cells right under this layer and together this is called the mucosal immune system. Airway mucosa function: - Conduction of air. - Exchange of oxygen and CO2. Goblet cells -> mucus production Ciliated cells -> ciliary movement -> remove particles out of respiratory tract Subepithelial layer -> where immune cells reside Club cells and basal cells -> important when there is damage, regeneration of the epithelial cell layer Example: SARS-CoV2: prior infection vs no previous infection. Cumulative incidence is way higher when there was no previous infection. They also looked at post=-vaccination responses -> vaccinated vs unvaccinated -> vaccinated people less vulnerable for infection. When a new variant occurs, people got infected despite vaccination. What can be concluded about mucosal immunity based on these data? - Upper respiratory tract infections lead to protective immunity. - Parental vaccination induces limited protection against URTIs. - Mucosal immunity is essential to protect against infection. Mucosal immune system: Production sites: bone marrow, thymus Induction sites: lymphoid tissues Components: Cytokines (innate) Immune cells (innate and adaptive) Antibodies (neutralisation) Migration from site of infection to the nearest lymph node. Waldeyers ring: tonsils (protection and primary response) - APC present to the T cells in Waldeyers defense ring leading to a memory response è Local immunity (resident B and T cells) -> first line of defense Adenovirus (DNA virus) Causes mild diseases but also severe (acute bronchitis/pneumonia) Seven serotypes (distinguished based on serum) Type 3 causes most acute infections in Asia It has a capsid containing the DNA and interacts with multiple receptors As soon as it in endocytosed the virosome, it fuses with the lysosome (enzymes to degrade). The virus is then able to escape into the cytosol as a free particle -> it injects the DNA into the nucleus -> DNA copied -> transcribed -> assembly -> lysis of the host cell -> infects other cells. RSV (RNA virus) Infects epithelial cells -> antiviral response Very young children or old people A small percentage of children get a very severe infection Enveloped virus Fuses with membrane -> releases content -> transcribed -> new particles are formed -> this leads to syncytia (fusion of cells causing larger cells) Is antiviral immunity determined by the different viral characteristics? -> YES Nucleic acid (RNA/DNA) determines the response. - Different types of receptors -> different response - Different mechanisms (interferons etc.) - Primary response -> epithelial cells -> cytokines and chemokines - Infection -> cell migration -> gradient of chemokines T cells arrive at site of infection: 1. viral proteins captures in the cytosol 2. processed and presented as peptides in MHC1 molecules (all cells) to the CD8+ T cells 3. cytotoxic response -> lysis of infected cell (granzyme) 4. phagocytosis of cell remnants by APC to CD4+ cells -> memory Tissue resident memory T cells: - CD8+ and CD4+ Tissue resident memory B cells: - Production of IgA and IgG Mucosal antibodies are important for opsonophagocytosis (antibody-mediated uptake) on mucosal surface. The antibodies also have an important function in blocking interaction with the epithelium. There are all sorts of receptors to get those Igs translocated to the top layer of the epithelial cells. - FcRn receptor for IgG - pIgR receptor for IgA and IgM They are locally produced (some are derived from the blood) they are then translocated all the way up (primary translocation: basolateral -> apical). They can also be transported back where there are APCs taking them up (for immune response). How are mucosal immune responses studied? In vitro models (study interactions with viruses) Measure virus neutralization: use a less dangerous virus to express the most important proteins and look at specific interactions (add antibodies to study neutralization) Cellular surface markers and intracellular markers (label antibodies for different cells and then use flow cytometry) ELISA/bead based (to measure levels of soluble factors (chemokines/cytokines)) Principles of Vaccination Vaccine = biological preparation that improves immunity to a particular disease - Vaccines against infectious diseases consist of antigens resembling the disease- causing microorganism (weakened) or components of it. - Most vaccines also contain adjuvants (improves immune response) Variolation (prevention of smallpox (Variola)) by inoculation with infected matter led to vaccination. - Inoculated a child with smallpox virus -> vaccination Dutch immunisation programme: But still: - Vaccines are not always available in developing countries. - There are no vaccines against HIV and many other infections. - Continuous improvement of most vaccines needed. Many different infection stages of Malaria (parasite looks different in every stage) –> very difficult to create a vaccine The balance vaccine efficacy and vaccine-associated risk is very important. Types of vaccines: Live attenuated vaccine: Wild virus -> culture it -> weakened form. Wild virus -> culture it -> inactivate it -> weakened. Subunit vaccine: Weaker response Safe and controllable - Adjuvants (usually aluminum salts (amplifies immune response -> activated immune cells and antigens stick to it (depot effect))) Other molecules can also be used as adjuvants - Virus like particles (repetitive surface) Example: HPV (single protein can form a VLP (virus like particle) - Outer membrane vesicle-based vaccine Virus Like Particle: The repetitive surface causes a strong immune response. Vector-based vaccines: - Example: adenovirus (you put a piece of genetic material of another virus in (in this case Influenza)) -> it will express Influenza molecules on the surface. - Example: adenovirus vector that contains DNA from the spike proteins of a virus, so you get antibodies against the spikes so the virus cannot enter. mRNA vaccines - RNA given in lipid vesicle. Inactivated vs mRNA vaccines: mRNA virus only against spike protein (in COVID) causes a stronger response than inactivated vaccines against all parts of the virus. Routes of administration: Intranasal vaccination Parental vaccination Oral vaccination Polio vaccine: OPV = oral (by drops -> live, weakened virus) or IPV = via injection (inactivated virus) - No mucosal immunity in IPV. Efficacy trials: - Vaccine group and control group -> have to wait long to draw conclusions Controlled human infection models - Control > reduction of incidence of disease, prevalence of infection, ongoing measures required - Elimination > interruption of transmission of infection (>absence of disease) in a large geographical region - Eradication > complete termination of transmission by extermination of infectious agent worldwide Challenges of vaccine development: Public demand for safe and effective vaccines Translating basic science into real vaccines Production at large scale (millions of doses/year) Stringent regulatory requirements > impacts every single step in the process Vaccine manufacturing process must be described in detail > dossier Well-characterized end products Interactive Lecture RSV 1. The burden of disease: Who are most vulnerable to a severe course of a RSV infection? Young children (under age of 6) are not the only RSV-vulnerable population: older adults, in particular those with medical comorbidities, are also at increased risk of severe RSV infection. 2. What’s does syncytial mean? When cells have merged together to form a single multinucleated cell. Fusion of different cells because of fusion proteins. 3. Is RSV infection seasonal? RSV epidemiology was characterized by a seasonal pattern in most places around the globe. Countries around the equator may have more perennial transmission. After onset of COVID19 there were out of season cases. 4. What is the mortality and for which groups are hospitalizations rates high? RSV mortality rates are highest among children with known risk factors. However, these children repre- sent a minority of severely affected infants. Most children hospitalized for RSV infection are previously healthy term infants. Not sure about the mortality -> the numbers are not clear. Netherlands: Almost all children Pre-fusion proteins 8. What has been the current strategy in prevention of a severe RSV infection in children, and for which risk groups is this therapy indicated? The primary strategy for preventing severe RSV infection in children has been monoclonal antibody therapy with Palivizumab (and more recently Nirsevimab). Monoclonal antibody (half life is 28 days igG) Palivizumab for high risk 9. Multiple vaccine candidates and monoclonal antibodies are in development (see Figure 6 of the review article), what are the advantages and disadvantages of the following strategies: 1. Monoclonal Antibodies: Nirsevimab Advantages: Provides immediate protection; effective for high-risk infants and immunocompromised individuals; bypasses need for immune system to generate antibodies. Disadvantages: Temporary protection; requires periodic dosing; can be costly. Nirsevimab has a longer half life. 2. Subunit Vaccines Advantages: Safer with minimal risk of causing disease; focuses immune response on specific antigens, reducing side effects. Disadvantages: Often requires adjuvants to enhance response; may need booster doses for long-term immunity. 3. Vector Vaccines Advantages: Strong immune response; can be modified for various pathogens; single dose can provide lasting immunity. Disadvantages: Risk of pre-existing immunity to vector; potential safety concerns in immunocompromised individuals. 4. Nucleic Acid Vaccines Advantages: Rapidly developed and easily modified; strong cellular and antibody responses; no live virus needed. Disadvantages: Limited long-term data; may require cold storage; potential for lower efficacy in certain populations. 5. Live-Attenuated Vaccines Advantages: Mimics natural infection, providing robust and long-lasting immunity; often requires fewer doses. Strong immune response, you need less. Can be administered orally. Disadvantages: Risk of mild disease in immunocompromised individuals; complex storage and handling requirements. 6. Maternal Vaccination Advantages: Provides early protection to newborns through passive immunity; safe for both mother and infant. Disadvantages: Limited duration of protection; timing of vaccination is crucial for effective transfer of antibodies. 10. Which prevention strategies would you advice to reduce the burden of severe RSV infections? Maternal & monoclonal antibodies Vaccinating children after age of 6? -> nucleic acid vaccination since it is long-term or live-attenuated vaccine. Low & middle income countries -> maternal immunization (cheaper option) Retroviruses and HIV HIV: - Two types (most important one is HIV1, causative agent of AIDS) AIDS: severe immunodeficiency: Less than 200 CD4 T cells/µL Infections that do not cause disease in healthy people Sexual transmission is a major transmission route for HIV. HIV is a retrovirus: - + RNA -> - DNA -> dsDNA -> + RNA - Reverse transcriptase: enzyme that uses RNA to make DNA (counters central dogma) HIV contains two identical RNA pieces, lipid bilayer, spikes Core genes: GAG (group antigens) -> core POL (polymerase) -> enzymes ENV (envelope) gp120 and gp41 are important for binding the receptor and co-receptor. Enzymes in the capsid (POL): - Reverse transcriptase - Integrase - mRNA - Protease HIV infects T cells, so it binds to two receptors -> CD4 and coreceptor CCr5 or CXCR4 1. Gp41 with gp120 trimer binds CD4. 2. Induces conformational change, so it can bind with the T cell’s coreceptor. 3. Another conformational change that allows gp41 to extend into an elongated structure -> fusion peptide inserts into the host membrane. 4. Delivery of capsid into the cytoplasm. It always binds CD4, and it can 'choose' between CCR5 (R5 virus) or CXCR4 (X4 virus). Early in infection most HIV1 strains use CCR5 (R5). Over time strains that use CXCR4 (X4) emerge due to a coreceptor switch caused by mutations. - X4 viruses can infect naïve T cells and thymocytes, resulting in faster disease progression R5 is generally the variant that is transmitted. X4 results in faster disease progression, but just R5 can cause immunodeficiency. Heterozygous for deletion (+/32): less CCR5 -> HIV can enter less effectively. Homozygotes (32/32): almost completely resistance to infection. Replication cycle: 1. Entry 2. Uncoating 3. Reverse transcription (reverse transcription -> dsDNA in cytoplasm) 4. Nuclear import 5. Integration (virus becomes part of cells genome) 6. Transcription (5’ LTR is used as promoter) 7. Export of unspliced RNA 8. Translation (full length or gag or gag-pol) 9. Export unspliced genomic RNA 10. Spliced RNA (env) 11. Nuclear export and translation of spliced RNA 12. Post-translational modification gp120 13. Transport of gp41/120 to cell membrane 14. Assembly 15. Budding (gag binds RNA) 16. Maturation (protease -> conversion) Decision point: 1. Virus will be cleared 2. Virus establishes chronic infection (with you for life) Viral escape 1.Immune cell dysfunction. - Lower functionality of immune cells (exhaustion of CD4, CD8, B cells, monocytes and NK cells) Although HIV only infects CD4 cells, you can see immune dysfunction in almost all immune cell types. 2. Gp120 is highly glycosylated (glycan shield) -> escape immune response since it can change so easily (escapes antibodies). 3. MHC 1 is downregulated (mediated by nef (accessory proteins of HIV). HIV infection Geographical origin of HIV is central Africa. SIV (simian immunodeficiency virus) mutated in humans to HIV1. From GRID (gay-related immune deficiency) to AIDS. Worldwide there are 39 million people with HIV. Of which 24.000 people in the Netherlands. HIV is a single strand RNA-RT virus. It attaches to CD4. - So, the destruction of which cell will lead to AIDS? -> T helper cells - Replicates fast and therefore many mutations. Most people will have symptoms after infection -> acute HIV (high viral load, lower CD4 count, but after that our body is capable of suppressing HIV for many years (viral setpoint)). When do we call it AIDS? - Low CD4 cell count - Getting sick (presence of one of the 20 opportunistic infections) Common opportunistic infections: Cases: What do these patients have in common? - HIV infection - AIDS Medication: - Reverse transcriptase inhibitors - HAART (high active antiretroviral therapy) - cART (combination antiretroviral therapy) - combination of 3 drugs How many pills are needed daily to control HIV? -> 1 pill (containing 3 different drugs, it makes it statistically impossible to have 3 mutations at the same time) Antiretroviral drugs (HIV meds) prevent HIV transmission. Treatment goals: - HIV-RNA below 20/40 copies/ml (recovery of CD4 counts, delay progression to AIDS, decline of morbidity/mortality) - Improve quality of life - Prevent transmission Risk factors: Interactive Lecture HIV CCR5 is a chemokine receptor. 1) Why is it impossible to cure HIV-1 infection with cART (combination antiretroviral therapy)? HIV-1 cannot be cured with cART because the virus establishes latent reservoirs in long-lived cells and sanctuary sites, where it remains dormant and inaccessible to both drugs and the immune system. Latent reservoir (integrates but not active) Mutations (related to resistance of the drug Sanctuary sites Drug resistance Integration 2) How were the Berlin, London and City of Hope patients ‘cured’ from HIV-1 infection? Allogenic stem cell transplantation with cell lacking CCR5. 3) In Gupta et al Nature 2019 paper, study Figure 1. At the time point -27 days viral RNA load (Fig 1a) is undetectable, but HIV DNA load is very high (Fig 1b). How can this be explained? The immune system suppresses and gets rid of viral RNA, while DNA stays dormant. Many cells carry non-effective virus. Rt inhibitor and protease inhibitor, so integrated virus that is defective will not be affected. 4) Figure 1C shows an agarose gel. What does this experiment tell us? The patient acquired the two alleles of CCR5 32 genotype after the transplant. Pre-transplant only wt/wt and post-transplant only 32/32. 5) In Figure 2a and 2b, susceptibility to different virus strains is tested in cell culture. What is the aim of this experiment? Confirming the resistance of donor-derived immune cells to CCR5 dependent strains. The study confirms that CCR5 32 mutation blocks CCR5. tropic HIV strains but not CXCR4- tropic strains, demonstrating specific mechanism of protection conferred by the mutation. Flow cytometry (FACS Fluorescent Assisted Cell Sorting) 6) Why did the same approach used for the London patient not lead to cure of the ‘Essen patient’? The Essen patient had pre-exosting CXCR4 variants circulating before the stem cell transplant. 7) What are the main drawbacks with current cART regimens? Refer to Ndung'u T et al (doi: 10.1038/s41586-019-1841-8) and https://viralzone.expasy.org/5180 to answer this question. Expensive Resistance against treatment Higher risk of kidney, cardiovascular and bone disease Inability to clear reservoirs Medication taken for entire life 8) What are drawbacks of the approach used to cure the London patient? Only works for CCR5 strains Expensive High risk (very toxic) Not scalable Antiviral immunity Virus needs to cross intrinsic barrier (skin, stomach pH, cilia, etc.), then needs to escape the innate response and then adaptive response. Innate: - Does not require prior virus exposure - Response is within minutes to hours (initial response) Adaptive: - Highly specific - Memory: rapid and specific response to re-infection - Antibodies can prevent new infections - Cytotoxic T cells kill infected cells Interferon response (cytokines) Type 1, 2 and 3 -> type 1 alpha and beta (can be produced by all nucleated cells) Phases of the type 1 IFN response: 1. Sensing of the infection, leading to secretion of IFN alpha/beta. 2. Binding of IFN alpha/beta to IFN receptor, followed by JAK/STAT signalling. 3. Expression and effector functions of interferon-stimulated genes (ISGd -> many different ISGs, they collectively contribute to antiviral state). PKR (protein kinase R) (ISG) Translation initiation eIF2 in a GTP bound state brings in initiator tRNA. eIF2 is then reused and recycled (3 subunits, alpha is important for regulation of translation initiation) PKR phosphorylates eIF2-alpha to an inactive form. PKR is only activated in the presence of dsRNA (produced by replicating viruses). This inhibits all canonical cap-dependent translation (polio for example can translate independently of cap and thus evades this mechanism). OAS/RNaseL (ISG) Becomes active when it binds viral dsDNA -> uses ATP and OAS (oligoadenylate) makes a stretch of A’s 2’5’ (instead of 3’5’). This makes it resistant to RNases. It activates RNaseL which then degrades RNA. è Viruses do NOT express typical PAMPs (peptidoglycan, lipopolysaccharides, chitin, O/N mannan, beta-glycan) How are viruses recognized as non self? - dsRNA is a viral PAMP - dsRNA, more generally 'non-self nucleic acids' prime innate immunity - Another non-self-recognition factor -> uncapped RNA (some viruses) RIG-I-like receptors (RIG I, MDA5, LGP2, MAVS) RIG-I recognize non-self -> signaling cascade -> phosphorylation of transcription factors -> interferon expression. - RIG-I recognizes 5’ triphosphate (most viruses have their own capping enzymes or a different protein to avoid detection by RIG-I). Why are viruses then still successful? - They co-evolve with their host. - Inhibition of the IFN pathway. - Inhibition of ubiquitination of RIG-I. - Inhibition of sensing, signalling, etc. - And many other different ways. Herpesviruses: antiviral immunity, latency and reactivation General patterns of infection: Acute infection - efficient replication - virus eliminated by the immune system Chronic/persistent infection: 1. continuous productive replication (HIV, HepC) 2. latent infection (herpes) 3. integration into the genome (HIV, retroviruses) DNA viruses are well adapted to infect a broad range of individuals. - dsDNA viruses are transcribed to generate RNA for viral protein synthesis. - DNA viruses replicate their DNA to generate progeny DNA, replication can be by host DNA polymerase (polio/papilloma) or by viral DNA polymerase (adeno, herpes, pox) There are 8 different human herpesviruses. They have co-evolved with the human host. Human herpesvirus 1-8. - HHV1 causes cold sores - HHV3 genital herpes - HHV4 kissing disease (mononucleosis) -> Pfeiffer/B cell lymphoma - HHV6 and 7 are transmitted by saliva (toys children) -> chickenpox - HHV8 Kaposi Sarcoma Herpes replication: - Nucleocapsid and enveloped - Tegument present (viral matrix) = large number of proteins between capsid and envelope that the virus brings along when it infects a cell. These proteins have a function in DNA replication and initial immune response evasion. - Encode many proteins required for DNA replication. 1. Viral DNA enters the cell nucleus. 2. Circularization. 3. Early expression of genes giving rise to immediate early proteins. 4. These proteins then activate expression of delayed early proteins. 5. These proteins help in replicating the DNA and production of late proteins. 6. Late proteins are used to build the virus particle. è Rolling circle replication to form concatemers Coordinated viral gene expression is mediated by one of the proteins that is brought along in the virus particle (VP16). VP16 activates initiation of transcription of immediate early proteins. Replication cycle: 1. Virus enters through fusion of the viral membrane and host membrane. 2. Tegument proteins are released in the cytoplasm (some remain there, and some go to the nucleus). 3. Viral nucleocapsid docks at nuclear pore -> DNA enters nucleus. 4. VP16 activates transcription of IE (see above). 5. Early proteins -> late proteins (see above). 6. Nucleocapsids bud into the pernuclear lumen and then fuse with the outer nuclear membrane (the virus particle is too large to go through the nuclear pore. 7. Nucleocapsid buds into the golgi and acquire envelope proteins. 8. Via vesicles, the virion are released by exocytosis. Example: Varicella Zoster Virus (causes chickenpox in children) Child infected (chickenpox) -> latency -> reactivation later in life (shingles) - Throughout your life you most likely have reactivation Mechanisms of latency and persistence: Latency program: Virus reverts to a latent state. During latency viral DNA is copied by cellular DNA polymerases (i.e., not viral) probably during mitosis. Genome may be silenced using host epigenetic mechanisms (e.g., histone modifications). Latently infected cells express few viral proteins, and some herpesviruses encode noncoding RNAs. Latently infected cells are therefore not “seen” by the immune system, therefore not killed by cytotoxic (CD8+) T cells. Infection of long-lived cells virus can persist for a long time. Extensive modulation of MHC class 1 presentation. - Herpesvirus can prevent antigen presentation by MHC1 molecules by interfering with steps in this process. Interactive Lecture HPV HPV is sexually transmitted, but it can also go via skin-to-skin contact. Signs of HPV infection: No signs Infection at basal cell layer -> viral replication -> transmission Head, neck and throat cancers caused by HPV is increasing. Including L2 -> broader response L2 needs to be cleaved before virion can infect the cell. Influenza Haemophilus influenzae Gram negative coccobacillus (outer and inner membrane) Haemophilus = blood-loving (add blood to grow bacteria) Why influenzae? -> it was thought it caused influenza (it does not) First living organism fully sequenced Rough (unencapsulated) -> non typable vs Smooth (capsulated) -> type a-f Antibodies recognizing sugars -> if it agglutinates = type A/B If it doesn't agglutinate -> non typable - Capsulated mainly causes disease, but NTHi (non typable) is increasing in the elderly. Resident of our nasopharyngeal microbiome Spreads through airborne droplets Serotype b (Hib) strains associated with invasive diseases (bacteremia and meningitis) Serotype a (Hia) associated with invasive disease in indigenous populations (Alaska/Australia), because of genetics/environment Heamophilus meningitis caused mostly by type b (sore throat, headache, fever, neck stiffness) -> fast onset Very high prevalence but does not cause disease often since vaccine is very good. What makes it a pathogenic bacterium? Protective polysaccharide capsule (especially type b) Genetically heterogeneous (genome differs): Non typables have no capsule -> different genetically -> different phenotypes Hetergeneous lipooligosaccharide (lack the O-antigen) Haemophilus influenzae has many genes that can be switched on/off (phase variation) due to the presence of tandem repeats (usually 4 nucleotides (tandem repeats)) -> out-of-frame. One of the sugars that can be incorporated into LOS is sialic acid. Haemophilus influenzae can take up sialic acid from its surroundings. By doing this the bacteria becomes more resistant to complement-mediated killing. Resistance to complement-mediated killing (presence of sialic acid in LOS decreases iGM binding to bacterial surface -> survive better in human serum) Bacterial proteins can bind human complement regulatory proteins (C4 binding protein, factor H and vitronectin), outer membrane protein P5 was able to bind factor H. Strains with lower factor H show lower survival. Biofilm formation (trap nutrients for microbial growth) Self defense - Biofilms resist physical forces that sweep away unattached cells - Prevents phagocytosis by immune system cells - Increases resistance to penetration of toxins (e.g., antibiotics) Favorable niche formation - Nutrient availability Close association with each other - Facilitates cell-cell communication - Nutrient and genetic exchange Biofilm formation can be determined in a static or continuous flow system. Growth of mutant ampG was slightly lower, but biofilm formation was much higher. Several proteins are required for biofilm formation (protein K). DNA was essential for increased biofilm formation by the ampG mutant, more genomic DNA was detected in the mutant biofilms. Are there antibiotics that interfere with peptideoglycan biosynthesis? Yes, cefuroxime - Suboptimal concentration of antibiotics increases biofilm formation. - Fairly sensitive to antibiotics Haemophilus influenzae vaccines: - Hib capsule-based vaccine (very effective) - No vaccine for non typable (outer membrane vesicle-based vaccines are being tested) Tuberculosis Worldwide many cases of TB (because of HIV) 1 species Lipid cell wall Slow growing (divides every 24 hours) Stable genome, no plasmids, no horizontal gene transfer It spends a lot of energy on its cell wall. The cell wall is impermeable and plays a role in recognition, phagocytosis, etc. Transmitted by aerosols 1/4 of the world infected 90-95% will never fall ill after infection 5-10% ever progress from infection to active TB Immune system: Physical barrier Cellular (T cells, macrophages) and humoral response Inferon gamma binds -> activation of macrophage (mutation in interferon gamma makes you very susceptible to mycobacterial infection) - Crosstalk macrophage and T cell. - Neutrophils - Dendritic cells It is not sure if antibodies play a role. No neutralizing antibodies like in haemophilus (comes down to immune cells). - TNF-alpha (important cytokine). TNF-alpha is crucial in control. Immune response: 1. initial infection 2. establish chronic infection 3. adaptive immune system and granuloma formation 4. dissemination/transmission Tissue -> granuloma (tissue reponse, characteristic of TB) Necrotic central area Ring of macrophages and lymphocytes around it Giant cells TNF drugs -> interaction macrophage lymphocytes is off (no ring of macrophages etc.) TB examples: Classical pulmonary TB TB lymphadenitis TB pleuritis TB meningitis Risk factors for TB: High prevalence of HIV GDP Undernourishment (nutrition, certain vitamins are crucial) Diabetes (leads to more TB, underlying mechanism not known (dyslipidemia, hyperglycemia, drugs, insulin resistance???) Diagnosis and treatment: Look at sputum that is couched up Culturing (but this takes weeks) Ziehl Neelsen staining/fluorescence PCR Whole genome sequencing (read full genome, look at transmission (SNPs), and look at specific drug resistant genes) There is a range of drugs that act on different pathways in the cell. TB preventive therapy for latent TB infections: Diagnosis: - Intracutaneous tuberculin test: Injects mycobacterial antigen (Ag) under the skin to measure immune response (induration size). - Blood test (IGRA): Measures interferon-gamma (IFN-γ) production by T-cells when exposed to TB antigens, indicating T-cell memory. Target Groups: Preventive therapy is recommended for high-risk groups, including HIV- infected individuals, children, TB contacts, and those starting immunosuppressive treatments. Interactive Lecture Tuberculosis What distinguishes Mycobacterium tuberculosis from any other pathogen? Exists for 70.000 years already. It has co-evolved with humans for over 70,000 years, developing mechanisms to manipulate the immune system. Infects 1/4 of the human population. M. tuberculosis entered the human population 70,000 years ago—what does that mean? Became completely adapted to humans as a host species. Is M. tuberculosis an intra- or extracellular pathogen? M. tuberculosis is primarily an intracellular pathogen, residing within macrophages and other phagocytic cells. I Benefits: Protected. As soon as it is in the cell antibodies cannot reach it (small window for antibodies). But to get into the cell there is also an extracellular phase. What is the primary target cell for infection? The primary target cells for M. tuberculosis infection are alveolar macrophages. These are immune cells in the lungs (alveoli) that engulf the bacteria as part of the innate immune response. Why ‘alveolar macrophages’ (AM)? In direct contact with the air (ready to infect) Reach bottom of the lungs (far distance) if small enough. (exchange oxygen-CO2) Continuously exposed -> increased change of infection and prepared to not immediately respond to particles. Can survive is epithelial cells as well, but conditions are not favourable. AM is. Alveolar macrophages (AMs) are the primary target for Mycobacterium tuberculosis (M. tuberculosis) for several reasons: 1. First Line of Defense: o AMs are the first immune cells encountered by M. tuberculosis upon its entry into the respiratory tract via inhaled aerosols. o They reside in the alveolar spaces, making them readily accessible to the pathogen. 2. Phagocytic Role: o AMs are specialized in engulfing pathogens, but M. tuberculosis has evolved mechanisms to subvert this process to establish infection. 3. Permissive Niche: o AMs provide a relatively permissive environment where M. tuberculosis can survive, replicate, and avoid immune detection. o Early in infection, AMs exhibit anti-inflammatory characteristics to prevent excessive lung damage, which M. tuberculosis exploits to replicate intracellularly. 4. Immune Modulation: o AMs suppress inflammatory responses under normal conditions to maintain lung tissue homeostasis. This anti-inflammatory environment is advantageous for M. tuberculosis, allowing it to evade host defenses. What factors could play a role in the success of AM infections? 1. Innate Immune Evasion: o M. tuberculosis inhibits phagosome-lysosome fusion, allowing the bacterium to survive intracellularly. o It detoxifies reactive oxygen and nitrogen species produced by AMs, which are normally bactericidal. 2. Access to Nutrients: o AMs provide access to essential nutrients like iron and fatty acids, which the bacterium uses for growth and persistence. o Lipid accumulation in infected AMs can further support bacterial metabolism. 3. Delayed Adaptive Response: o AMs fail to efficiently signal to adaptive immune cells early in infection, delaying the recruitment of T cells that could help control the infection. 4. Host Factors: o Environmental factors like smoking or age-related changes (inflammaging) can impair AM function, increasing susceptibility to M. tuberculosis infection. o Genetic variations in hosts may affect AM susceptibility to bacterial survival and replication. 5. Virulence Factors of M. tuberculosis: o Proteins like ESAT-6 (EsxA) and PDIM lipids facilitate bacterial survival by damaging phagosomal membranes and modulating AM immune signaling. o Bacterial effectors, such as SapM and PknG, inhibit phagosome maturation and autophagy. 6. Lung Environment: o Alveolar lining fluid, which includes surfactant proteins, antibodies, and hydrolases, can vary in its effectiveness in neutralizing M. tuberculosis. o Deficiencies in pulmonary surfactants or prior lung damage enhance AM infection success. Does infection by M. tuberculosis always lead to disease? No, infection by Mycobacterium tuberculosis does not always lead to disease (latent, dormant state). The majority of individuals (~90–95%) who are infected develop a latent tuberculosis infection (LTBI), where the bacteria are contained by the immune system and do not cause symptoms. Only 5–10% of infected individuals progress to active tuberculosis (TB), which involves symptomatic disease, often due to immune suppression or other risk factors. In some cases cleared. What is latent infection? Latent infection occurs when the immune system successfully contains M. tuberculosis without completely eliminating it. Key characteristics include: Absence of Symptoms: Individuals with latent TB do not exhibit clinical signs of the disease. Immune Control: The bacteria are confined within granulomas—structures formed by immune cells to restrict bacterial spread. Non-Transmissibility: People with latent TB do not spread the infection to others. Latent TB can exist as a spectrum, ranging from complete bacterial elimination to the persistence of viable but dormant bacteria. Can latent infection lead to clearance? Yes, in some cases, latent infection can lead to clearance, where the immune system completely eradicates the bacteria. However: Currently, there are no definitive tests to distinguish individuals who have cleared the infection from those who harbor dormant M. tuberculosis bacilli. For most, latent infection persists without progression to active TB unless immune function is compromised (e.g., HIV, malnutrition, or aging). What is reactivation of tuberculosis? Reactivation occurs when dormant M. tuberculosis in a person with latent TB begins to replicate, leading to active TB disease. This can happen years after the initial infection and is typically triggered by: Immune Suppression: Caused by factors like HIV, diabetes, corticosteroids, or aging. Other Risk Factors: Malnutrition, smoking, or other chronic illnesses. Reactivation often involves the breakdown of granulomas, leading to bacterial spread and symptoms such as coughing, weight loss, fever, and night sweats. After infection it can be cleared immediately, but often it recruits. Mycobacteria remain in a dormant stage. It can also be activated again. Why is the poised suppression of inflammatory responses in alveolar macrophages beneficial for M. tuberculosis (Mtb)? Alveolar macrophages suppress inflammatory responses under normal conditions to prevent lung damage from continuous exposure to inhaled particles. M. tuberculosis exploits this anti-inflammatory state to avoid immune detection and killing, creating a favorable environment for its replication and persistence. After 2 weeks, Mtb-infected AMs migrate to the lung interstitium. What happens? Once alveolar macrophages infected with M. tuberculosis migrate to the lung interstitium: Active Inflammation is Induced: o The host’s immune response activates, involving cytokines such as IL-1β and TNF-α. o Additional immune cells (e.g., neutrophils, monocytes, and T cells) are recruited to the site. Granuloma Formation: o Immune cells form granulomas around the bacteria to contain the infection and prevent dissemination. o This marks the transition from an early innate response to an adaptive immune response. What is the effect of active inflammation? 1. Immune Cell Recruitment: o The inflammatory response draws macrophages, neutrophils, and T cells to the infection site. o Dendritic cells migrate to lymph nodes to prime T cells, initiating adaptive immunity. 2. Granuloma Formation: o Granulomas help contain the bacteria but also provide a niche for its survival. o The inflammatory environment can limit bacterial replication but rarely achieves sterilization. 3. Tissue Damage: o Excessive or prolonged inflammation can cause lung damage, contributing to symptoms in active TB. 4. Potential Transmission: o In cases of active TB, inflammation leads to necrosis and cavitation in the lungs, facilitating bacterial transmission through aerosolized droplets. Foamy macrophage -> lipid droplets Neutrophils (when cell is damaged) -> NETs (almost no effect in tb) + ROS (induce damage to DNA, neutralization -> no damage can be caused) Recognition: When still outside the cell, it is recognized by different receptors (C type lectin receptors) -> pro- and anti-inflammatory cytokines. Detection -> intracellular signalling and intracellular trafficking Phagosomes and lysosomes fuse. Lysosomes contain enzymes and low pH and degrades microorganism taken up. PtpA prevents acidification. Blocking production of oxygen radicles. Influence and suppress adaptive immune system: Antigen presentation is blocked (before presentation of MHC2 and 1 blocked by degradation (MHC molecule affected)) or antigen is expprted before it is being presented. Decoy antigen: distract the immune system in wrong direction. Suppress DC migration to the draining lymph node -> no T cell presentation. Influences type of cytokines produced (IL10 and TGFB) No proper presentation of MHC1 is a sign for a cell to be destroyed by NK cells ‘Its ability to complete the infection cycle depends on both evading and taking advantage of the host immune responses. Examples? : Getting rid of the complement system. Avoid lysosomal degradation. Granuloma formation No oxygen (when aerobic pathogen does not have oxygen it dies, but in mycobacterium tb it can be reactivated. TGFB to prevent the response against mycobacterium tb. Design solutions: Accelerate the system, t cells should be readily available. (it usually takes 2 weeks for a colony to grow, because of the delay) Only 3 bacilli needed to infect someone. Strategies to improve protection by vaccination: IgM can bind more bacteria at a time (pentamer) High number of antibodies readily available Trained macroohages Poised protective T cells (to overcome delayed priming) Pneumonia and Vaccination S. Pneumoniae Gram positive: single membrane, thick peptidoglycan layer Microaerophilic coccoid bacteria (prefers less oxygen) Extracellular pathogen as opposed to mycobacterium Many children and also some adults asymptomatically carry the pathogen Case-fatality rates increased in elderly (children are vaccinated) è Commensal or pathogen?? (produces toxins so pathogen) Life cycle: Colonization (most important stage) Transmission by shedding Invasion (aspiration (from upper to lower respiratory tract) -> pneumonia, entry into blood stream -> bacteraemia, entry into brain -> meningitis, and it can also spread locally). Has developed immune evasive mechanisms: - Production of thick capsule. - Has a protein on the surface that binds factor H, so it prevents complement activation (Factor H -> complement regulator (prevents collateral damage)). - Make use of receptors on epithelial cells of BBB; to enable translocation over BBB (so it can cause meningitis (it causes neuronal cell death)). Co-infection with viruses (especially influenza) Relation: influenza causes epithelial cell damage, this causes decreased mucociliary velocity and allows the bacteria to pass more easily + there is an increase in type 1 interferons and reduced CCL2 (important for recruitment of macrophages and monocytes). How do we know whether the infection is caused by S. pneumonia? - If there is suspicion of meningitis (high fever, neck pain), they take cerebral spinal fluid by a lumbar puncture. This material is cultivated. Halos can be seen on the blood agar plates. Another test that can be done is the bile solubility test and more recently also PCR is used. Serotyping can be done based on capsule -> 95 different serotypes Capsule -> virulence factor (prevents uptake by phagocytosis) Genomic analysis of the core genome is studied and gives insight in how the pathogen interacts with the host. Antibiotic resistance based on MIC (minimal inhibitory concentration) values -> halo means susceptible. - Penicilllin (beta-lactam) is used to treat it. Since there is emerging resistance, there are also multiple antibiotics that can be used to treat it, but beta lactams are most often used for gram positive bacteria (they target the peptidoglycan synthesis). Horizontal gene transfer -> naturally competent (S. pneumonia actively transports DNA into the cell). This competence is induced by environmental signals (stress/high cell density). This involves killing of neighboring cells (fratricide) to release the DNA to then be taken up. This allows new variants to be introduced into the population. Penumococcal cell wall -> thick wall allows for the connection of all kinds of molecules, such as peptidoglycan binding proteins, lipoproteins, etc. ABC transporters It can take up amino acids (peptide importers) Metal ions!!!! Carbohydrates are taken up Transport dependent on ATP Shuttles back to translocon (opened by dephosphorylation of ATP) Enters the cell Single toxin: pneumolysin Forms pores -> content in released -> availability of nutrients for pneumococcus Balance between inducing inflammation and availability of nutrients Nasopharyngeal colonisation Carriage -> as soon as the density is high enough and release of pneumolysin -> inflammation -> shedding -> inhibition of mucin entrapment and agglutination -> pneumococcus degrades the IgA -> Age dependent decline (immunizing effect) Evolutionary point of view -> invasive disease is dead end road (either patient dies, or the pneumococcus is cleared by antibiotics) -> so NO evolutionary benefit. By accident it becomes invasive Why is S. pneumoniae an important cause of disease: - High carriage rates - Genetic adaptability - Ability to shift from commensal to pathogenic interaction with host. From colonization to invasive: Metabolically very adaptable -> increased growth as soon as it is in your bloodstream (different carbon sources). Pneumococcal vaccines: Conjugate vaccine (10 or 13 different purified capsular polysaccharides conjugated to carrier proteins) Purified polysaccharide (not conjugated) -> recommended for elderly (Select most prevalent serotypes) For manufacturing this vaccine, a diphtheria/tetanus toxoid is used (a toxoid is a detoxified toxin (done genetically or chemically)) and Haemophilus influenzae protein D (strongly immunogenic protein) - Adjuvanted with aluminium phosphate. This supports the T helper cell 2 (this is important for activation of B cells -> plasma cells -> antibodies) Why do we switch from PPV23 to a PCV (conjugate vaccine)? This has to do with the memory response. In a conjugate vaccine the B cell is more active in the uptake of the antigen -> presented to T cells -> T cell help -> memory response. How do we predict protection: Antibody levels (0.35 µg/ml) Opsonophagocytosis (antibody-complement-mediated phagocytosis) (primary defense mechanism) The vaccines work very well, but only against certain serotypes. Introduction of vaccines against certain serotypes results in an increase in the number of overall cases (of the serotypes without vaccine) à serotype replacement - Serotype-dependent protection Vaccines need to be affordable, target groups should not only be children (different adjuvant systems, etc.), reduction of carriage. Mucosal (intranasal) vaccines: Mucosal immunization -> local and systemic immunity Preventing disease and infection Asymptomatic colonization of pneumococci -> natural immunization What mechanisms are underlying protection? Antibodies play an important role - IgA on mucosal surface - Agglutination (IgG) might be important -> natural clearance (How do we measure this? -> opsonophagocytosis on mucosal surface (IgA and IgG)) T cells (Th1) (Th2) (Th17) are also important, they produce cytokine IL17A, this induces AMPs, neutrophils and IgA resulting in killing/clearance of pneumococci. Trm (T memory cells), Brm (B memory cells) cells are also important. The vaccine either has to penetrate the epithelial barrier or it needs to attract immune cells to take up the vaccine and induce a response. Outer membrane vesicles van be used as delivery ‘vehicles’. OMVs contain many PAMPs (Pathogen Associated Molecular Patterns) and you are able to put antigens on the surface to make it easily recognizable. With use of (E coli autotransporter protein) Hemoglobin protease (Hbp). - After detoxification of LPS Create new vaccine: - Culturing under different conditions -> proteomics analysis. - Focused mostly on the transporters involved in transport of metals. - Use of ICP-MS to detect how much of metal ions is present in the respiratory tract (in mimicked conditions). - Looked at proteins present in all conditions -> vaccine candidates. - Test vaccine candidates in mice. (Also looked at influenza co-infection) Interactive Lecture Whooping couch 1. What is the reproductive number (R0) of Bordetella pertussis? Between 12-17. How many people one infected person can infect around them. 2. Which age group is most severely affected by pertussis? Babies: -weaker immune system -narrow airways (bronchi and lungs are smaller) 3. How long does the disease last, and what are the three stages of the disease and their characteristics? About 100 days 1. Catarrhal stage 2. Paroxysmal stage 3. Canvalescent stage 4. How is pertussis diagnosed and what are the preferred techniques for this? PCR (early in infection), culturing, serology Clinical diagnosis in paroxysmal phase 5. Would you consider antibiotic treatment for pertussis infected individuals and when do you consider the treatment to be effective? Usually not really effective, doesn't shorten the course of disease. Only works when you are very early and sure its pertussis. But usually not. 6. Which cells are affected by the bacterium, and can you link the symptoms of the infection to these cells? Ciliated cells (transport mucus out of lungs), takes time to heal. Mainly affected by toxins of the bacteria, also influence immune response (inhibit macrophages and neutrophil influx in your lungs, and DCs and T regulatory lymphocytes) 7. What type of molecules are the main virulence factors of the bacterium, and what are their functions in general? Adhesins, pertussin toxin, 5 important toxins and a few virulence factors involved in adhesions, dampening immune system. 8. First whole cell vaccines were introduced (late 1940s). What does the whole cell vaccine consist of and what are the advantages and disadvantages of this vaccine? More antigens involved Strong efficacy record Adverse effects (local pain, redness, swelling, fever) 9. Acellular pertussis vaccines were developed since the mid 1970s. What do these vaccines consist of and what are the advantages of the acellular vaccines? Pertactin, fimbriae, FHA, weakened pertussis toxin Advantages and disadvantages of only choosing just a few proteins Higher adaptive immune response, can be more specific, higher amount of protein can be given, less adverse effects Expensive, waning immunity (not as strong) 10. Are both whole cell and acellular vaccines still being used? Yes -> wP is cheaper + in combination vaccines 11. Pertussis vaccination has brought a tremendous effect on the incidence of pertussis, although after an initial dramatic decline, infections are seen more often and case counts continue to rise. What are possible explanations for the rise in cases that is seen? Reduced immunity New strains have developed Improved diagnostics Increased awareness 12. Figure 1 (from the nonhuman primate article). What is an important conclusion from this figure? Acelullar -> spread it Longest time to clear infection with acellular vaccine 13. What do we know about asymptomatic carriage of Bordetella pertussis? Asymptomatic Bordetella pertussis carriage occurs when individuals harbor the bacteria without symptoms, acting as reservoirs for transmission, particularly in partially immune or vaccinated individuals. 14. When is a mucosal immune response initiated? A mucosal immune response begins when antigens encounter mucosal surfaces, triggering secretory IgA production and local immune activation. 15. Since a few years maternal vaccination with pertussis vaccines was introduced to limit severe infections. Why was this strategy chosen? Maternal vaccination protects newborns via transplacental antibody transfer, reducing severe infections in infants too young for vaccination. 16. This vaccine is preferably given at 22 weeks. Why do you think this is week 22? Vaccine given at 22 weeks, for when baby is born prematurely, but not earlier otherwise amount of antibodies is getting lower in the mother as well. 17. Does maternal vaccination influence the immune response after childhood vaccination? -> vaccine at 3 months Skin and Soft Tissue Infections SSTI = Skin and Soft Tissue Infections History Cause of SSTIs is divine entity Corpus Hippocraticum -> hypotheses about causes Robert Willam -> dermatoses into different orders (terms are still used) No antibiotics -> honey etc. were used in the past as treatment. - Types of honey have antibacterial properties. Skin: stratified organ Epidermis (keratinocytes) -> in contact with environment so protects Dermis (also fat and many immune cells) -> nerve endings Hypodermis (connects skin and muscle tissue) Fascia (important in infections) Skin: not completely sterile (skin microbiome -> bacteria, fungi, archaea, viruses Skin microbiome -> different roles in different niches (skin remains in homeostasis) Produce antimicrobial peptides Senses density of cells around them -> control them Makes sure health is maintained all over cell surface Dysbiosis is one of the causes of skin infection Other risk factors: Surgical site infection Hospital acquired infections Injury/wound Nosocomial infection -> transmission by hands The more superficial it is, the more benign the infection is. Pyogenic Impetigo (epidermis) Folliculitis (dermis) Erysipelas (dermis) Cellulitis Furuncles/carbuncles Surgical wound infections. Non-pyogenic (other infection) - Tetanus - Gas gangrene - Necrotizing fasciitis - Staphylococcal skin syndrome Caused by Staphylococcus aureus Cluster (looks like grapes) Gram-positive pathogen Partly hemolyses the blood (alpha hemolysis) Catalyses the breakdown of hydrogen peroxide into water 1/3 of people carries it Many strategies to ensure its virulence Alpha hemolysis: blood is not completely broken down Beta hemolysis: blood is completely broken down (yellow area around colonies) Gamma hemolysis: no hemolysis occurs S.aureus is in many part of the human flora, yet it can also opportunistically pathogenize sites of the human body. It can cause a wide variety of symptoms. Nasal carriers: Transmitted from hand to nose -> virus spreads all over body. Inappropriate = inappropriately treated (many people are diagnosed as polymicrobial when this is not always the case and this also happens with other pathogens (difficult to diagnose)). Antibiotics (non-penicillins) -> S. aureus is resistant to many antibiotics. Impetigo Most common bacterial infection in children Epidermis infected Highly contagious Without treatment it may resolve within 2-3 weeks Non-bullous (honey-coloured crust) (caused by S. pyogenes or S. aureus) vs bullous (blisters -> brown crust) (caused by S. aureus) SSSS (staphylococcal scalded skin syndrome) -> superficial blistering on the skin (caused by S. aureus) Cannot take a skin swab to diagnose it (because there are too many bacteria on the skin), so take culture of exudate or pus. Treatment: hygiene, topical/oral antibiotics For SSSS: IV something PAMPs (pathogen associated molecules) PRRs (pathogen recognition receptors) Interaction of PAMPs and PRRs -> cascade of immune responses. Keratinocytes are joined by tight junctions (desmosomes) 1. S.aureus invades and releases exotoxins, they then cleave off the protein component of the desmosome (desmoglein) 2. Extracellular connection is lost 3. Intracellular connection is lost (because of proteases) 4. Junction is open (crust on skin) Cutaneous abscesses - Caused by S. aureus Categorised based on site on infection: Hair follicle: folliculitis Dermis: furuncle/carbuncle Deep: abscess S.aureus invades the skin -> keratinocytes are activated -> release cytokines (IL-36alpha) (IL- 1beta released by intradermal infection immune cells) -> stimulate T cells -> release IL-17 -> recruit neutrophils and monocytes to the skin -> inflammation of the skin/ (or lead to abscess formatiom

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