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mucosal infections

• skin and various mucosae lining organs are in continuous contact with environmental microbes

• aberrant immune responses to harmless agents — celiac disease

  • most pathogens that cause death are either mucoasal surface pathogen or enter through mucosal surface

prevention of infection in the gut

• presence of thick mucus layer keeps most organisms in lumen away from intestinal epithelium

• antimicrobial peptides produced by intestinal epithelial cells

• IgA antibodies (produced via plasma cells in lamina propira) → transported into the lumen and neutralizes pathogens before entry into epithelium

distinctive features of mucosal immune system

• intimate interactions between mucosal polarized epithelia and lymphoid tissues

• discrete compartmants of diffuse lymphoid tissue and organized stuctures — peyer’s patch, isolated lymphoid follicales and tonsils

• specialized antigen uptake mechanisms

• broad surface area in contact with environmental agents/microbes

epithelium types

• simple columnar

• pseudostratified columnar epithelium

• non keratinized stratified squamous epithelium

• keratinized stratified squamous epithelium

effector mechanisms of the mucosal immune system

• activated/memory T cells predominate even in the absense of infection

• multiple activated ‘natural’ effector/regulatory T cells present

• production of mucins and mucus

• secretory IgA antibodies

• production of antimicrobial peptides (AMPs)

• presence of distinctive microbiota

Discrete compartments of the mucosal immune system

• cells of mucosal IS located both in anatomically defined compartments + scattered through mucosal tissue

• lymphocytes are in organised tissue (peyer’s patch), isolated lymphoid follicles forming GALT

• mesenteric lymph nodes (gut draining LN) — connected to peyer’s patch and intestinal mucosa via afferent lymphatic vessels (largest LN in body)

immunoregulatory environment of MIS

• active down regulation of immune response predominates at homeostasis (to food and other innocuous antigens)

• inhibitory macrophages and tolerance-inducing dendritic cells

• high number of FoxP3+ Treg cells and FoxP3-Tr1 cells

goblet cells

• simple columar epithelial cells that develop from stems cells in bass of crypt

• contain mucopolysaccharide in secretory granules — expand up to 500-fold after release

• act as a physical barrier to the motility and feeding of bacteria and other pathogens (intestinal parasites)

• releases Th2 cytokines including IL-5 and IL-13

gastrointestinal cells and host defence

• intestinal epithelial cells produce antimicrobial peptides that kill pathogens or reduce their entry into the epithelium

• M cells transport antigens to underlying DC

• goblet cells secrete mucus in response to microbes + metabolites (Th2 cytokines)

• Paneth cells produce antimicrobial peptides

• tuft cells detect pathogen molecules → produce IL-25 → induce ILC2 → produce IL4 and IL13

Microfold (M) cells

transport antigens from the lumen of the intestine to underlying dendritic cells for antigen delivery

Paneth cells

• in the small intestine

• produce antimicrobial peptides such as cathelicidins and defensins

tuft cells

detect pathogen molecules (chemoreceptions), and produce IL-25, which induced ILC2s to produce IL-4 and IL-13 and help initiate a Th2 type response

M cell structure

• thin glycocalyx, short microvilli and large fenestrations in htier membranes

• able to transport antigens in endocytic vesicles across the cytosol and deliver them via endocytic vesicles across the cytosol → deliver them by exocytosis at the basolateral membrane

• convoluted basal membranes in M cells form “pockets” → allows close contact with DC for antigen presentation

mucosal macrophages

• lamina propria contains largest population of macrophages in the body → highest population in small intestine

• located immediately under the epithelium, rep 75% of all mononuclear phagocytes

• highly phagocytic but with a non-inflammatory profile

major function of gastrointestinal tract humoral immunity

neutralise luminal microbes and mediated mainly by IgA produced in the lamina propria and transported across the mucosal epithelium into the lumen

MIS antibody production

• IgA is produced in higher amounts than any other antibody isotype, smaller quantaties of IgG and M are also secreted into the gut lumen

• IgA-secreting plasma cells are widely dispersed in the lamina propria of the gastrointestinal tract not just in lymphoid follicles

intrepithelial lymphocytes

long-lived resident effector cells that are interspersed between epithelial cells

instestinal conventional and unconventional T cells

• IEL

• most T cells are CD8+ in small intestine

• Mostly CD4+ in Lamina propria

• most T cells in healthy lamina propria have been activated by DC and express markers of effector or memory T cells

• other unconventional T cells reside in intestinal epithelium — TCRγδ and TCRαβ

IEL function

• recognise and destroy intestinal epithelial cells that display properties of infection, damage or stress

• highly motile within the intestinal epithelium and present a fully mature effector status

microbiome

• community of microorganisms that naturally that exists in a particulr environment

• bacteriome — major component of human microbiota

• virome — primarily composed of bacteriophages

• archaeome — methanogens i.e Methanobrevibacter smithii

• mycobiome — Saccaromyces, Malassezia and Candida genera

• parasitome — Blastocystic spp.

gut microbiota

• one of the largest components of our body

• composed of mostly of bacteria, also arachaea, viruses, fungi and protozoa

• symbiotic relationship with human body — protect and support the structure of intestinal mucosa

• may be influenced by a complex combination of environmental, genetic and lifestyle factors

dysbiosis

observed in metabolic disorders i.e diabetes, obesity, in cancer, autoimmune disorders, and stress related neuropsychiatric disorders i.e depression and anxiety

probiotic organisms

non-pathogenic, living microbes that provide a benefit to the host

probiotics

specific nutrients (usually complex carbohydrates) that support and encourage the growth of beneficial commensal microbes

postbiotics

referes to the waster left behind after digestion of prebiotics and probiotics and may include nutrients ie vitamins B and K, amino acids, and antimicrobial peptides

immune privilege

• coined by Peter Medawar in 1948 to describe the absence of an immune response to allografts placed into the anterior chamer of the eye or brain

immune privilege sites

• brain, eye, testes, placenta, and foetus

• have mechanisms that suppress inflammation and promote immune tolerance

• lack of classical lymphatic drainage system for antigen-presenting cells that reside in the parenchyma and perivascular spaces

• lack of cell-mediated response to instilled antigens

• prolonged survival of tissues when grafted into the brain, eyes, testicles, compared to other sites

parenchymal microglial cell

• brain resident macrophages

• enters brain very early in embryogenic phase — derived from yolk-sac precursores

• functions as immune sentinels and contributes for maintenance of brain homeostasis — synaptic remodeling, neurogenesis, and the routine clearance of debris and dead cell

choroid plexus

• modified ependymal cells that produce CSF

• acts as diffusion barrier between blood and csf and it is home to various immune cells

• choroid plexus is key point of entry for peripheral immune cells into csf space

epiplexus cells

• innate immune cells located in the choroid plexus of brain ventricles

• express markers of macrophaes, dendritic cells and microglia

• function includes phagocytosis, antigen presentation, iron accumulation and NO production

meningeal, perivascular, and ventricular macrophages

• re-stimulation of lymphocytes with peptide-MHC complexes

• allow primed lymphocytes to enter the CNS parenchyma

• derived from blood-borne monocytes (population homeostasis)

• neutrophiles and other granulocytes are absent from the health CNS

Cerebrospinal fluid

• CSF surrounds the brain and spinal cord, both internally and externally

• mainly produced by the choroid plexuses — highly vascularised tissues located within each ventricle of the brain

• reabsorption into blood via arachnoid villi

immune cell content of CSF

• 90% T cells

• 5% B cells

• 5% monocytes

• <1% dendritic cell

• T cells constitutively monitor the CNS by trafficking through CSF

CSF cytology

• Colourless and clear

• antibodies and complement is normally absent

• up 5 WBCs per mm³ in adults and 20 WBCs per mm³ in newborns

blood brain barrier

• depends on stringent ionic homeostasis

• strictly controls the movement of solutes across the CNS vasculature

• direct membrane-membrane contact between endothelial cells — tight and adherens junctions seal capillary endothelium

• pericytes aid in regulation of transcellular barrier and maintenance of vessel function

glia limitans perivascularis

inner most layer of BBB formed by astroglial endfeet surrounding blood vessels

BBB disruption

• vasogenic agents — histamie, thrombin, proinflammatory cytokines

• infectious agents — bacteria, bacterial toxins, viruses, parasites, and fungal pathogens

T cell cross reactivity

reaction of T cells to more than one distinct peptide - MHC ligand

molecular mimicry

resemblance between epitopes contained in microbial and host proteins, leading to cross-reacitivity of T cells in the host

TCR binding degeneracy

refer to the promiscuity of T cell receptor engagement that allows a single TCR to bind to different peptide-MHC complexes

original antigenic sin

• A footprint of immne response is established during the first exposure to a pathogen.

• specific memory T populations are preferentially re-expanded when reexposed to the same antigen or one that is similar

• limits the clonal expansion of new antigen specific T cells

trained immunity

long term functional reprogramming of innate immune cells by exogenous or endogenous activation events → altered response to a secondary challenge after the return to a non-activated state

immune surveillance

physiologic function of the immune system is to recognize and destroy clones of transformed cells before they grow into tumours and to kill tumours after they are formed

tumour specific antigens

antigens that are expressed on tumours cells but not on normal cells

• some are unique to individual tumours, whereas others are shared among tumours of the same

tumour associated antigens

tumour antigens that are also expressed on normal cells.

• these antigens are normal cellular constituents whose expression is aberrant or dysregulated in tumours

Products of mutated genes

• oncegenes and mutated tumour suppressor genes produce proteins that differ from normal cellular proteins → can induce immune responses

• tumour antigens may be produced by randomly mutated genes whose products are not related to the malignant phenotype

antigens of oncegenic viruses

products of oncogenic viruses function as tumour antigens and elicit specific T cell responses that may serve to eradicate the tumours

Antigens of oncogenic viruses

• viral peptides are foreign antigens — virus induced tumours are among the most immunogenic tumours known

• virus encoded tumour antigens are not unique for each tumour but are shared by all tumours induced by same type of virus

oncofetal antigens

proteins that are expressed at a high level in cancer cells and in normal developing foetal but not adult tissues

• encoding genes are silenced during development — de-repressed with malignant transformation

tissue-specific differentiation antigens

tumours may express molecules that are normally expressed only on the cells of origin of the tumours and not on cells from other tissues

principal mechanism of adaptive immune protection against tumours

• Killing of tumour cells by CD8+ CTLs

• surveillance function by recognizing and killing potentially malignant cells that express peptides from tumour antigens

cross presentation of tumour antigens by dendritic cells

• most tumour cells not derived from APC → do not express co-stimulators needed to initiate T cell responses/class II MHC needed.

• cross presentation by DC needed sometimes

Escaping immune recognition by loss of antigen expression

• Immune responses to tumour cells impart selective pressures that result in survival and outgrowth of variant tumour cells with reduced immunogenicity

• process is called tumour immunoediting

• tumours developing in setting of normal immune system become less immunogenic over time

immune evasion by tumours

• Tumour immunoediting

• Class I MHC expression down regulation → not recognised by CTL

• secreted products of tumour cells may suppress anti-tumour immune responses → TGF-B

• Treg may supress T cell response to tumours

phases of tumour immunity

• elimation phase — number of immune cells can recognise and eliminate tumour cells

• equilibrium phase — variant tumour cells arise that are more resistant to elimination

• escape phase — one variant may escape killing mechanism/recruit regulatory cells to protect it → spreads unchallenged

Tumour vaccines

• id of peptides recognized by tumour-specific CTL and cloning of tumour-specific antigen genes provides candidate antigens for vaccines

• most are therapeutic vaccines — have to be given to host after encountering the tumour → has to overcome immune regulation that cancers establish

immune checkpoints/checkpoint inhibitors

• novel group of monoclonal antibodies with proven effectiveness in a wide range of malignancies

• promote anti-tumour immune response by blocking signalling via CTLA4 or PD1 pathway

CTLA 4 mechanisms of action

• blocking CTLA-4 with antibodies results in increased immune responses

• CTLA-4 expression is low on most T cells until the cells are activated via antigen → once expressed it terminates continuing activation of responding T cells

• expressed on Treg and mediates suppresive function of cell by inhibiting activation of naive T cells

PD-1 check point protein

• ligated PD-L1 expressed on APC and many other cells

• ligand leads to inactivaion of T cells or inhibiting signal transduction from TCR

• terminates peripheral response of effector T cells

• antibodies that block PD-1 and PD-L1 allows T cells to kill tumour cells

CAR T cells

• Chimeric antigen receptors in T cells

• patient peripheral blood T cells are isolated — stimulated with anti-CD3 or anti-CD28 and subjected to gene transduction with car encoding vectors

• expanded in vitro and injected into patient

• undergo further robust proliferation in patient → tumour killing achieve via cytotoxic and cytokkine mediated mechanisms

CD19 CAR

single chimeric protein that is the recognition domain of a specific antibody against a tumour associated antigen and intracellular signalling domain capable of activating T cells

anti-tumour antibodies

• may eradicate tumours by the same effector mechanisms that are used to eliminate microbes — opsonization and phagocytosis, complement system etc

• outgrowth of antigen loss variants no longer express antigens that the antibodies recognize

use of FM

• measures and counts every single cell in a fluid stream and the

  • relative size

  • relative granularity or internal complexity

  • amount of fluorescence

• rapid and sensitive

advantages of flow cytometry

• multi-parameter analysis (1-8) — mulitple fluorescent colours to differentiate

• rapid analysis (2-10, 000 cells)

• significant number of cells sampled

• sensitive identification of rare events

disadvantages of flow cytometry

• single cell suspensions only

• no tissue context i.e location in a tissue

• unable to detect intracellular location

• cells cannot be recovered from intruments (unless using a cell sorter)

components of flow cytometer

• fluidics — introduce and focus cells in front of the lasers

• optics — generate and collect the fluorescent light signals

• electronics — convert light signals to proportional digital signals

fluidics

• introduce cells into strument and focus them in front of the laser

• cells must flow in single file to allow accurate analysis of each cell

• sample injected → unpressed into the sheath fluid → most instruments undergoes a process known as “hydrodynamic focusing”

• laminar flow is achieved → sample fluid flows in central core and does not mix with outer sheath fluid

excitation optics

• a laser is used to provide a single wavelength of light

• mulitple lasers can be installed to provide coincident light of different wavelenghts

• laser light beam is shaped by excitation lenses to focus on cells in fluid stream

collection optics

• emitted light is directed to appropriate detectors by lenses and filters

• light from forward scatter, side scatter and emitted fluorescence are detected

forward scatter

• related to cell size and surface area but NOT direct measure of cell diameter or circumference

• amount of light diffracted or scattered at narrow angles to the axis of the laser beam

• light reaches the FSC detecter — generates a voltage pulse signal which is proportional to the amount of light received

• larger the cell → wider the scatter

side scatter

• SCC related to cell granularity and complexity

• amount of light reflected or diffracted at 90 degrees to the axis of the laser beam

• light is focused through a lens and is collected by a detector usually located 90 degrees from the laser’s path

• anything with a greater density → more side scatter

fluorophore

mocule that:

• absorbs energy of a specific wavelength

• emits unused energy at a different (longer) wavelength of light

fundamental principle of vaccination

administer a killed or attenuated form of an infectious agene or a component of it that does not cause disease but elicits an immune response that provides protection against infection by the live, pathogenic microbe

lady Mary Wortley Montagu

observed in 1718 incoculation against small pox in the East and she was determined to promote it upon her return to England

herd immunity

indirect protection from an infectious disease that happens when a population is immune either through vaccination or immunity developed thorugh previous infection

who does herd immunity protect

protects individuals from highly contagious disease when some individuals cannot be vaccinated or initial vaccine-induced immunity has waned, and those who refuse immunisation

immunological basis for vaccination

• prevention of diseases by inducing the immune response

• generation of long-lasting and protective immunity

• formation of neutralising antibodies and cell-mediated immunity

T cell inducing vaccines

• CD8+ T cells controls viral replication by killing infected cells — i.e SARS-CoV-2, HIV, HCV, CMV

• CD4+ cell contribute to both humoral and cellular immune responses — stimulate proliferation and differentiation of B-cells and CD8+ T cells

• integration of cell mediate immunity effector functions are required to control intracellular microbes — Mycobacteria, Plasmodium spp., Salmonella, Listeria etc

antigen delivery

combination of different platforms, routes of delivery and regimes has the potential to induce superior protection i.e intranasal and intramuscular influenza vaccines

vaccine delivery

• most vaccines are administered via intramuscular injection which need administration by trained professionals and often causes pain at injection site

• some are administered orally i.e rotavirus

• intranasal vaccines can activate immunes cells localised on mucosal tissue of upper and lower respiratory tract — i.e FluMist nasal spray

effect with intranasal vaccination

• Mucosal IgG and IgA

• CD4+ and CD8+ T_em cells

• T_rm cells in lungs and respiratory tract

effect with epidermal vaccination

• systemic IgG

• mucosal IgG, IgA

• CD4+ and CD8+ T_em cells

• T_rm cells (in skin, lungs, nasal mucosa, liver)

effect with intramascular vaccination

• systemic IgG

• Weak cellular response

• Poor T cell responses

live vaccines

• attenuated pathogens replicate sufficiently to produce a strong immune response but not so much as to cause significant disease manifestation

• aimed at immunocompetent individuals

• BCG, Measles, Mumps, varicella zoster

non-live vaccines

• contain only components of a pathogen or killed whole organism

• whole organisms, purified proteins from the organism, recombinant proteins or polysaccharidees, toxid, via vectors, nucleic acid bases RNA and DNA, and virus-like particles

correlates of protection

• an immune function that correlates with and may be biologically responsible for vaccine-induced efficacy

passive immunisation

normal human immunoglobulin or immunoglobulin with a high concentration of antibody specific to a particular disease

live/live attenuated vaccines

• contain a weakened form of the pathogen that replicates more slowly and is less virulent than original pathogen

• induce strong immune response — mimics natural infection

• theoretical risk that live attenuated pathogen reverts to wild-type virulent pathogen and cause disease

• should not be received by immunocompromised

examples of live vaccines

• measles

• mumps

• rubella

• varicella

• polio

• influenza

• BCG

• poxvirus

• rotavirus

rotavirus vaccine

• recommended — infants <6 months of age in a 2-3 dose schedule at 2 and 4 months (or 2,4,6 months)

• formulation — ≥ 10⁶ infectious units of rotavirus reassortants in cell culture media

• administration route — oral, vaccine virus replicates in intestinal mucosa, virus can be shed in the stool after 1st dose

contraindication of rotavirus

• history of intussuception

• congenital abnormality that may predispose to intussuception

• severe combined immunodeficiency in infants

beneficial non-specific effects of live vaccines against unrelated infections

• stimulation of PRRs leading to production of type I interferons and inflammatory mediators

• heterologous lymphocyte responses — cross reactive T cells or antibodies

• trained immunity — epigenetic alteration in innate immune cells and long term functional reprogramming

killed/subunit/toxoid vaccines

• killed vaccines contain an inactivated version of the pathogen, subunit and toxoid vaccines contain only select components of the pathogen

• primarily induce humoral immunity

• antibody levels against these vaccines generally decrease over time — requires booster

• no risk of reverting to wild-type form

• generally, can be safely used for immunocompromised

Hepatitis B

• recommendation — infants and children in a 4-dose schedule at birth and 2, 4, 6 months

• formulation: recombinant hepatitis B antigen protein, adsorbed into aluminium

• administration route: intramascular injection — may be offered in monovalent formulation or combination vaccines

VLP and particulate subunit vaccines

• virus-like particles (VLP) are a type of subunit vaccine based on virus-derived proteins, assembled to form a particle

• VLP self-assemble into particles that resemble or mimic the structure, size and symmetry of original viruses

• nonpathogenic (no intact virus genome) and incapable of infection/replication

Human papillomavirus (HPV)

• recommendation: adolescent and young adults aged 9-5 years, people with severely immunocompromising conditions, men who have intercourse with men

• formulation: 9vHPV recombinant protein particulate (VLP) contain major capsid (L1) of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 or 2vHPV (16 and 18)

• administration route: intramuscular injection

polysaccharide and conjugate subunit vaccines

• infection with encapsulated bacteria can be prevented by vaccination with capsular polysaccharides, either plain or conjugated to a protein carrier

• conjugate produce higher quality and longer term immunity (T cell dependent immunity) — bacterial capsular polysaccharide to a protein carrier

• Haemophilus influenze type B, Neisseria meningitis, Streptococcus pneumoniae

mRNA vaccines

• mRNA encoding the protein of interest is carried by lipid nanoparticles. Once transported inside cells, mRNA induces cell to produce the antigen encoded by the mRNA

• Comirnaty (Pfizer) and Spikevax (moderna)