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Molecular Mechanisms > Antibody development and function > Flashcards

Antibody development and function Flashcards

1
Q

what are antibodies?

A

a soluble protein secreted by plasma cells
highly specific - each Ab recognises a single antigen epitope

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2
Q

how do the structure of antibodies relate to their function?

A

their variable domains contain an antigen-recognition site - Fab arms

heavy chain has Fc region for FcR binding or complement binding
- directs innate immunity to fight specific infection

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3
Q

why are B cells important?

A
  • they secrete antibody
  • highly diverse: 10^10 unique B cells
  • each B cell expresses >10^5 copies of the BCR
  • when binding antigen, they can develop into plasma cells or memory cells
  • B cells are constantly turned over - tight regulation needed
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4
Q

where are B cells produced (foetus and adult)?

A

Foetal B cells are made in the liver
by birth B cells are made in the bone marrow

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5
Q

what is the key aim of vaccination?

A

Inducing Ab is a key aim of vaccination – target surface-exposed antigen

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6
Q

what is the consequence of failure to produce antibodies?

A

A failure to induce functional Ab responses increases risk of infection / childhood death – children lacking B cell compartment will die without treatment
- this disease is rare, as children don’t live long enough to pass on genes to next generation

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7
Q

what is the result of poor control of B cell responses?

A

autoimmunity and cancer

or lack of productive Ab response

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8
Q

how do individual naive B cells differ?

A

Individual naïve B cells express shared molecular signatures (eg CD19, CD20) but differ in their BCR diversity - one B cell recognizes one target

B cells look the same, but naïve B cell varies in its BCR – BCR confers its identity

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9
Q

what is the general life of a B cell?

A
  1. B cell precursor rearranges its Ig genes
  2. Immature B cell bound to self cell-surface antigen is removed fro repertoire - negative selection of autoreactive B cells
  3. mature B cell migrates to peripheral lymphoid organs and engage to foreign antigen - activation
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10
Q

what does B cell development require?

A

Development requires the successive acquisition of properties that are essential for function:
- Allows host to control the process – quality control in development

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11
Q

how is B cell development controlled?

A

To enable CONTROL - B cells develop in a series of sequential steps

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12
Q

what is the process of B cell development?

A
  1. stem cell progenitor with germline H and L chains, with no surface Ig
  2. VDJ rearrangement of H chain from early pro-B cell to large pre-B cell
    - functional H chain formed which is expressed at surface
    - surrogate non-functional L chain scaffolds H chain at surface
  3. ## VJ rearrangement of L chain in small-pre B cell
  4. surface expression of IgM in immature B cell
  5. immature B cell leaves bone marrow and differentiates into mature, naive B cell with surface expression of IgD and IgM, derived from differential H chain splicing
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13
Q

how efficient is B cell gene rearrangement?

A

Many joins are not productive - 70% fail for each chain

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14
Q

what drives somat?ic recombination?

A

RAG genes

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15
Q

what is allelic exclusion of light chains?

A

Rearrangements can occur from either inherited chromosome, but only one chromosome is
used at a time
- There are 2 light chains, kappa and lambda, to choose from
- The two kappa genes are used first, then the two lambda genes - SEQUENTIAL and highly controlled
- This is called allelic exclusion and ensures that one B cell produces antibody of one specificity!!

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16
Q

what is the ratio of kappa:lambda light chain in normal blood of humans?

A

2-3:1 - this is used clinically
- when ratio is perturbed, indicates uncontrolled antibody production

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17
Q

why is one chromosome used at a time during H chain rearrangment?

A

H chain, L chain, 2 chromosomes:
- If process is uncontrolled – potential to make B cell with multiple specificities
- avoids non-specificity

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18
Q

what is the advantage of controlled recombination?

A

Using all potential genes raises the chance of making a successful BCR - enhances diversity
- makes BCR of single specificity

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19
Q

how is somatic recombination terminated?

A

Further recombination is stopped by preventing RAG expression or function

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20
Q

what does somatic recombination produce?

A

This leads to lots of individual B cells that differ in their BCR
- One B cell recognizes one target
- B cells with IgM, IgD, different surface molecules
- Broad repertoire of B cells which can recognise diversity of antigens

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21
Q

what kind of antigen do BCRs/Antibodies recognise?

A

Abs recognise conformational, 3D epitopes

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22
Q

how is B cell autoreactivity limited?

A
  • B cells are removed or edited if they recognise self-antigens during development
  • First wave of control for autoreactivity
  • The self-antigens they can encounter/screened against are mostly extracellular and not intracellular – extracellular is easily accessed
  • Therefore, this process is not perfect and some self- reactive B cells to intracellular self-peptides “get through”
  • Hence why autoantibodies can target dsDNA or cytosolic antigens
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23
Q

what happens to an immature B cell that encounters multivalent self-antigen on the surface of cell?

A

multivalent antigen: same antigen copied next to each other on surface
- Lots of antigens to bind multiple surface BCRs – transmits signal of autoreactivity
Leads to clonal deletion or receptor editing
Easy to make Ab response to multivalent antigen than single antigen due to amplified BCR response

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24
Q

what happens to a naive B cell that encounters soluble self-antigen?

A

Could also encounter soluble self-molecule at lower conc on way to secondary lymphoid tissue:
- Strength of input signal is lower as they are spatially distanced
- B cell becomes anergic and can’t produce immune response, eventually dies

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25
Q

what happens to a B cell which has no reaction to self?

A

If no self reaction:
- Enters secondary lymphoid tissue and resides as mature, naïve B cell in follicles of spleen or lymph node

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26
Q

what are the stages of B cells which develop in bone marrow (summary)?

A

Pro-B cells: HC gene rearrangements

Pre-B cells: LC gene rearrangements

Immature B cells: express surface IgM; exits the BM; if the cell strongly binds self-antigen, then eliminated (negative selection)

Mature B cells: reside in the follicles of lymph nodes and the spleen and can circulate between different lymph nodes – maximise likelihood that it encounters target antigen in lymph nodes

Mature B cells: express surface IgM & IgD and can now enter immune response

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27
Q

what triggers a mature B cell to become a plasma cell?

A

when it engages target antigen

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28
Q

what are the two possible fates of mature, naive T cells?

A

B cells can become plasma cells and plasma cells secrete antibody

B cells can become memory B cells and memory B cells do not secrete antibody unless antigen is re-encountered

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29
Q

what influences the function of an antibody?

A

the Ig H chain
- H chain usage can chain for effector function
- H chains contribute in different ways
- B cells can change the type of antibody they express and secrete by differential H chain splicing

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30
Q

what are the main Ig classes and summarise their main function:

A

D and M are surface Ig

-M is in primary immune response – pentamer – high avidity

G: subtypes 1-4 which all have different properties e.g. complement activation, FcR usage
- mainly secondary response

E – found in serum at low levels and on surface of mast cells to encounter parasites (hypersensitivity reactions)

A – associated with mucosa, 2 subtypes, second most found in serum

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31
Q

what are the intrinsic features of a resting B cell?

A

Resting B cell has high levels of surface Ig – searching for antigen
High expression of surface MHCII to cross-talk with CD4 T cells

Doesn’t secrete Ig – avoid non-selective Ab entering circulation

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32
Q

what are the inducible features of resting B cells?

A

growth, somatic hypermutation, isotype switch

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33
Q

what are the intrinsic features of plasma cells?

A

PC – terminally differentiated B cell
- Gone through selection process
- low surface Ig
- no surface MHCII
- Producing tons of soluble Ab for decades - high secretion rate

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34
Q

what are the inducible features of plasma cells?

A

none

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35
Q

what are the two pathways for generating antibody responses?

A
  1. extrafollicular responses - T-independent
    - B cells differentiate to IgM and IgG plasma cells, producing antibody of modest affinity
  2. Germinal centre responses – T-dependent (requires processed, peptide epitopes)
    - selective process to generate plasma cells that produce high affinity, class-switched (e.g. IgG, IgA, IgE) antibody and memory B cells
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36
Q

what are T-independent responses? are they good vaccines?

A

no T cell response due to a lack of protein antigen, e.g. purified bacterial capsular polysaccharide antigen
- these vaccines can produce effective antibody responses in younger age groups
- extrafollicular only

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37
Q

what are T-dependent responses?

A

T cell involvement and guidance to B cells, as antigen is processed peptide

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38
Q

what are the outcomes of T-independent responses?

A

low affinity IgM and some IgG

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39
Q

what are the outcomes of T-dependent responses?

A

IgM, IgG, IgA, IgE
- high affinity antibody
- induction of memory which is long-lived

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40
Q

how can the involvement of T cell help alter the B cell response?

A

Improves B cell selection:
- Antibody produced after a primary B cell response is detectable at low levels by a week after immunization
- Secondary responses occur much more rapidly because of B cell memory – already been through selection process – institutional memory
- Memory B cell responses are faster & more extensive than primary B cell responses
- Instead of 3 days to get small numbers of B cells to expand <24 hours to get lots of memory B cells to proliferate
- More antibody, sooner!!!!
- Induction of T cell immunity is a major aim of vaccination programmes

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41
Q

what are the benefits of vaccination and B cell memory to protein antigens?

A

e.g. diphtheria infection:
- Death before sufficient Ab response to the toxin – not enough Ab to neutralise toxin as bacterium increases
- Toxoid vaccine
- Primary response kinetics are the same – modest Ab levels
- during infection, this shoulder of antibody buys time
- Make more Ab to neutralise the toxin to clear the infection

42
Q

where do adaptive immune responses develop?

A

Adaptive immune responses develop in the spleen or one of the approx. 700
lymph nodes and can “postcode” the response locally
- Starts in white pulp/T zone in spleen/lymph node respectively
- strategic location of lymph nodes to restrict dissemination of antigen – traps in one place
- Sentinal sites to stop antigen spread – local response

43
Q

how do adaptive responses develop?

A

Antigen is internalised by DC:
- Processed and presented by MHCII – costimulation activates antigen-specific T cell
- T cell interacts with antigen-specific B cell had internalised antigen with BCR and presented it via MHCII

some B cells migrate to pulp/medulla of spleen/lymph node and forms extrafollicular responses – PCs are short lived

Other B and T cells migrate into follicles to form GCs
- Forms long-lived PCs in BM and memory B cells

44
Q

what B cell response does T-independent induce?

A

capsular polysaccharide antigen isn’t a protein epitope, so not T cell help:
- extrafollicular
- B cells enter T zone and form short-lived PCs of modest longevity

45
Q

what are the 2 signals which T-dependent antibody responses requre?

A
  1. Specific interaction between B and T cell – cognate physical interaction where they recognise same antigen
    - B cell uptake of antigen via BCR to present to T helper cell TCR, with co-stimulation
    - Physical contact necessary – increases specificity and enhances level of control of the process
  2. Soluble signal via cytokines to reinforce interaction
46
Q

what type of specialised CD4 T cell is involved in T-dependent responses?

A

TFH cell:
- Tfh recognise antigen (peptides) presented through MHC Class II
- To be primed CD4 T cells recognize peptide presented through MHCII by DC
- CD4 T cells positively select B cells they recognize peptide presented through MHCII by B cells – cognate interaction – MHCII used by DC and B cell to communicate to CD4 T cell
- This positive selection signal to B cells is therefore through cognate interaction

47
Q

what signalling molecules are important in T-dependent antibody responses?

A

CD40 (B cells) and CD40L (T cells)
- absence of this = hyper IgM syndrome (non-productive Ab responses)

Soluble messengers called cytokines help reinforce the signals from cognate interaction
- Important cytokines include IL-4 and IL-21

48
Q

what are the features of a T-independent 2 repsonse?

A

Purified capsular polysaccharide (CP) is a multivalent antigen
- Same repeating epitopes
- No protein = no T cell
- TI-2 antigen are clinically important – most bacterial vaccines target CPs
- response to TI-2 anitgens are fast but at lower level
- Limited class switch to IgG
- Lack memory and boosting effect (hyporresponsiveness – same TI-2 vaccine given twice, second response is weaker)
- Short-lived memory to TI-2 – only effective for couple years
- Can’t use in infants <5 years

49
Q

how can T-independent responses be improved?

A

Use conjugate vaccine (physically link TI-2 antigen to protein) – allow T cell involvement – overcome limitations

protein Conjugate vaccines work in infants and save 100000s lives each year

50
Q

how does a conjugate vaccine work?

A

Conjugate capsular polysaccharide vaccine:
- B cell reocgnises CP antigen and internalises it, processes protein into peptide and present on MHCII to T cell
- T cell specific to peptide is activated – cross-talk with B cell
- Enables high affinity Ab, isotype switching, long-lived response, memory

51
Q

how are long-lived plasma cells and memory cells developed?

A

via germinal centre responses

52
Q

what are germinal centre responses?

A

GC are necessary for the production of long-lived plasma cells and nearly all B cell memory
- The antibody that is produced is of high affinity and class-switched to IgG, IgE or IgA
= To achieve this requires B cells to undergo further recombination of their Ig V genes – risk
- This has to be carefully regulated to prevent auto-immunity and cancer (B cell lymphoma)
- This has resulted in the GC being a complex and organized process

53
Q

where and when do germinal centres form?

A

Germinal centres form in the follicles of lymph nodes during T-dependent responses

54
Q

how is the germinal centre response activated?

A
  1. DCs and B cells internalise and process antigen
    - B cells move to T zone via CCR7
  2. naive T cells are primed by cognate interaction with DC
  3. cognate interaction of primed T cell with activated B cell - cross talk
  4. activated B and T cells move to follicle where GCs develop
55
Q

what cell types are involved in the germinal centre response?

A

Centroblasts (Cb)

Centrocytes (Cc)

Follicular dendritic cells (FDCs)

TFH cells

TFH and FDC help with selection

56
Q

what are centroblasts?

A

Centroblasts (Cb) – are proliferating GC B cells that undergo somatic hypermutation – dark
zone

57
Q

what are centrocytes?

A

Centrocytes (Cc) – are GC B cells that have undergone affinity maturation and are out of cell cycle – light zone

58
Q

what are follicular dendritic cells?

A

Follicular Dendritic Cells (FDC) – These are different to iDC, they reside in follicles.
- They have intact antigen in its native conformation (not processed) bound to their surface and Cc compete to bind this antigen – light zone

59
Q

what are follicular T helper cells?

A

Follicular T helper cells – These are GC T cells that give survival signals to Cc after they come out of the dark zone – light zone

60
Q

What important molecules are involved in the germinal centre response?

A

Activation-induced cytidine deaminase (AID) when expressed in B cells and is essential for class switch recombination and IgV hypermutation
– lack of AID means lack of GC response and IgG

B Cell Lymphoma-6 (BCL-6) is the master transcription factor for committment of GC B cells and is required for the generation of TfH cells

CD40 on B cells and CD40L on T cells

IL21 is important for the generation of TfH cells

Blimp-1 is a transcription factor required for the plasma cell programme

61
Q

what are somatic hypermutation and affinity maturation?

A

random point mutations in V region genes of Ig molecule to enhance the affinity of the antibody to its antigen target
- B cells with a stronger affinity are selected for and survive

62
Q

how do the first and second wave of antibodies differ in infection?

A

First wave of Ab – no selection process so IgM is produced – pentamer with 10 Fab arms – high avidity but low affinity, constantly binding lots of antigen

second wave is IgG which has 2 fab arms – higher affinity, lower avidity

63
Q

what is the process of the germinal centre response?

A
  1. In dark zone, dividing and mutating centroblasts (Cb)
    - random mutations in IgV genes
    - proliferation
  2. Selection is needed to test affinity of the Ab and see if it still targets antigen in the light zone. Cb become centrocytes (cc)
  3. Cc are selected by FDC which holds native antigen on surface – Cc compete for antigen binding on FDC
    – more tight binding promotes survival of Cc – positive selection
  4. Cc winners get survival signals from Tfh cells, but most die from neglect
  5. Survivors differentiate to PCs and memory B cells
64
Q

what is the germinal response critical for generating for vaccines?

A

The germinal centre response plays a critical role in vaccinology:
i) Ig class switching;
ii) high-affinity antibody;
iii) memory B cells;
iv) long-lived plasma cells

65
Q

what is key for the control of germinal centre responses?

A

Organisation is key for development and function and brings multiple cell-types together – selection and control

66
Q

what do vaccines target?

A

All licensed human vaccines target exposed antigens – legitimate debate between role of T and B cells in protection
- Virus – e.g. SARS-Cov-2 spike (vaccines target spike viral protein)
- Bacteria – Capsular polysaccharides or lipopolysaccharides (carbohydrate), tetanus toxin (protein)

67
Q

how successful have vaccines been?

A

narrow” spectrum of vaccines (EPI) has:
- averted 154 million deaths, including 146 million among under 5s, 101 million under 1s
- For every death averted, 66 years of full health gained (102 billion years of full health gained)
- Vaccination - 40% of the observed decline in global infant mortality, 52% in the African region
- Does not include all vaccines
- Does not include natural immunity
- No vaccines to many pathogens – 33 bacterial pathogens cause 8̴ million deaths / year (2nd leading killer globally)

68
Q

how is antibody selectivity achieved?

A
  1. Antigen internalised by DC via pinocytosis (random) – processes and presents via MHCII to T cell, generating antigen-specific T cell
  2. Uptake of antigen by B cell is more selective as it relies on BCR – internalises specific antigen and presents on MHCII as peptide to T cell
    - what the B cell recognises is different to what it presents to the T cell: native vs processed
  3. Extrafollicular response without T cells – short-lived PCs form
  4. Forms GCs in follicles – long-lived PCs in BM, different heavy chain usage, memory B cells
69
Q

why is it important to have different antibody effector functions for different infections?

A
  • IgG1 and 3 better at activating complement that IgA
  • Complement activation at mucosal surface via IgA would cause local inflammation, break in mucosal surface, so bacteria in gut can escape – hence why IgA is poor complement activator

Selection matters – antibody that is generated impacts immune response

70
Q

how do antibody regions relate to antigen-recognition and function?

A

IgM = pentamer – high avidity
IgA = dimer – forms immune complex with antigen target to remove from circulation
IgG3 = large hinge region – highly flexible compared to other IgGs, can get to hard-to-access sites, but more prone to degradation – shorter lived
IgG1 is longer lived - more stable

71
Q

what are the two types of salmonella?

A
  • typhoid
  • invasive non-typhoidal salmonella
72
Q

what are some features of typhoid salmonella?

A
  • mostly seen in North Africa
  • 3 vaccines available
  • induces normal gut infection with little risk
  • can also be treated with antibiotics
73
Q

what are some features of invasive non-typhoidal salmonella (iNTS)?

A

mostly in sub-saharan Africa
- lacks capsule
- uses different types of LPS components
- S. Typhimurium – USES 04 LPS O-ANTIGEN
- S. Enteritidis – USES 09 LPS O-ANTIGEN
- not gut restricted, found in blood stream
- tends to infect HIV+ adults
- tends to infect healthy, young infants who are HIV-
- no vaccines available
- 25% chance of death

74
Q

why does the location of iNTS increase mortality?

A

Bug enters gut
- Spreads to mesenteric lymph node, which acts to constrain spread
- If fails, bug enters blood and lymph to enter spleen, liver BM – systemic
- Low risk in gut
- Enhanced risk when disseminating to organs
- Ab constrains dissemination
- IgG response to salmonella decreases risk

75
Q

how can salmonella infections be modelled?

A

Salmonella infections can be modelled in the mouse to study the induction and functioning of immunity in multiple organs

76
Q

what can mouse models show about salmonella infections?

A

Give mouse bug:
- spreads to spleen, which provides model to see how B cell response develops
- Spreads to liver – assess effector functions of immune system
- Culture bacteria from organs to see how disseminated the bug is
- In spleen and liver, bacterial colonisation is over mil bugs – peak colonisation
- In kidney, lung and brain at low numbers
- In blood, numbers are modest

77
Q

how can secondary salmonella responses be modelled in mice?

A

After infection, bacteria reaches peak and then drops over months for clearance
- Innate system controls magnitiude of response early on
- Can vaccinate first and look at secondary infection – see impact of pre-existing antibody and Th1 responses

78
Q

how effective is salmonella vaccine in mouse models?

A

In spleen and liver, within 4 hours, vaccination reduces bug number – pre-existing antibody provides protective barrier that is rapid
- To stay healthy, you don’t need vaccine-mediated protection to cause sterile immunity in every case
- Large no. bacteria during infection, but Ab acts as a barrier, for T cells to come and clear pathogen

79
Q

how do vaccines buy time for clearing infection?

A

Antibody-mediated protection induced to vaccines is rapid and work before infection establishes and when the total bacterial numbers are lower:
- T cell help induce antibody and help to clear – overlap of immune response
- Ab buys host time to make functional responses to prevent overwhelming infection

80
Q

what is antibody function related to?

A

its structure and type;

the capacity to bind its target antigen and

the immune consequences (effector functions) of binding its target or pathogen

81
Q

what is the structure of salmonella?

A

On surface of generic salmonella is the capsule - TI-2 antigen
– can be used as conjugate vaccine

Other salmonellas typhimurium and enteritidis lack capsule, so LPS is exposed –
- LPS O-antigen = structural identity
- easier for antibody to access

Cell wall-associated antigens – more buried

Secreted antigen

82
Q

how do antibodies sequester salmonella?

A

IgM and IgG can control infection by binding capsule
- vaccine needs to produce long-lived antibody

83
Q

how many antibodies can bind to covid vs salmonella?

A

IgG can surround sars-cov-2 – not many antibodies can bind to surface
- few surface antigens (25-100), low surface area
- only a few hundred Abs can bind

Bacteria is bigger with more surface antigens (100s) – thousands of Abs can bind

84
Q

how complex is the bacterial cell wall of salmonella?

A

LPS:
- Diverse surface on bacterial surface
- Multiple Abs can interact with antigens
– LPS-O antigen
- LPS is essential for bacterial integrity (millions of copies)
- More buried proteins e.g. OMPs – hundreds of thousands of copies

85
Q

what is LPS O-antigen?

A

Lipid A induces inflammation – toxic to humans – provides bacterial cell wall integrity
- O-antigen sugar stretches out – salmonella expresses O4
Core and lipid A is conserved, but O-antigen is diverse – can differ by one sugar – Ab cannot cross bind and protect
- O-Ag differs between different serovars/serotypes, occludes cell-surface Ag but is a target of protective Ab

86
Q

how does the LPS O-antigen limit antibody binding?

A

LPS O-Ag forms a “forest canopy” over the bacterium and a single sugar change in LPS limits
cross-protection between different Salmonella types despite high protein homology
- Bacteria introduce diversity to evade antibody
– specificity of Ab can’t overcome this
– one sugar means if infected by one bug, you aren’t cross-protected against another bug of same species

87
Q

how is the O-antigen an effective shield against antibody?

A

Membrane moves in real time:
- Antibodies bind O-antigen to dock on surface
- O-antigen moves – Ab needs to dock on surface, but vibration keeps antigen moving
- Big core leaves gaps in LPS layer for Ab access
- If small, there is no gap, so Ab cannot access

Many Abs produced in response to natural infection will be specific but cannot reach target – not protective

88
Q

how does the structure of LPS make antibody binding less energetically favourable?

A

If there is a big gap – Ab can access despite movement of O-antigen
- But Ab has to work harder as LPS is constantly moving – less energetically favourable to target underlying proteins compared to O-antigen
- Need to make productive antibody response

89
Q

how can we predict what what antigens will be easily bound?

A

Combining what we know about the bacterial cell-wall and antibody
structure can help us “predict” what antigens might be protective
- LPS layer restricts Ab access to few epitopes
- LPS can combine and stabilise antigens to minimise Ab activity
- Boosting can increase likelihood of cross-protective Ab

90
Q

what is the serum bactericidal assay (SBA)?

A

SBA is the workhorse of functional antibody testing against many bacterial pathogens including Salmonella
- Take bug, add serum antibody, add source of complement – combine in absence of cells
- Bactericidal Abs bind surface and punch hole and promote complement to kill the bug

91
Q

what is needed to produce a functional immune response against salmonella?

A

Serum and use source of complement and mix with salmonella:
- If no immune sera but complement present – no killing – LPS layer blocks complement
- Immune sera but no complement – Ab alone is not sufficient to kill – need both Ab and complement
- Immune sera and complement – bacteria die

92
Q

how do antibodies and complement function together in vitro compared to in vivo?

A

In vitro:
- Ab binds bug
- Recruits complement binding – punches hole – cell death within 10 min

In reality:
- neutrophils, macrophages also contribute – competition of immunity – important timing
- Can take 5 min to kill – opsonised bacteria within neutrophils and macrophages
- Multiple effector mechanisms that work in combination to control infection

93
Q

Which classical complement pathway components contribute to antibody protection in vaccinated mice?

A

WT and complement deficient mice vaccinated with OMV:
- vaccine of WT, C1q-/-, C4-/- and C5-/- lead to reduction in bug load compared to non-vaccinated
- vaccination of C3-/- mice does not reduce bug load
- C3 is the exception
- C3 sticks antigens on FDC surface – C3 is important for initial Ab responses

94
Q

is C3 redundant in antibody-mediated protection in mice?

A

C3 K/O mouse
- immune sera from WT source
- Transfer serum into mouse
- infect with bug

If Ab and immune cells are present, C3 is not essential

Redundancy in responses:
- C3 is important, but there are multiple mechanisms are available
- C3 is important for making Ab responses, rather than helping Ab function

95
Q

how can we track vaccination and infection in vivo?

A

Imaging can track vaccination and infection in vivo
- Label organ sections to see bacteria localisation

Spleen section from unvaccinated, infected mouse:
- Salmonella in red pulp and marginal zone
- In absence of vaccination, bacteria enter red pulp and marginal zone

In vaccinated infected mouse
- there is more IgG
- but, bacteria occupy same niches in red pulp and marginal zone – vaccination helps control infection, but not where bacteria localise

96
Q

how can macrophages outcompete neutrophils to capture salmonella?

A

Spleen and liver: bacteria found in macrophages whether vaccinated or not
- vaccination does not alter cell tropism

Intravital – image macrophage uptake of bug
- Non immunised animal:
- Introduce bacteria in real time bacteria – some bugs stick to macrophages, some don’t

In OMV immunised animal:
- Bug stick to macrophages and are taken up efficiently by macrophages
- Ab isn’t changing what cell type takes up the bug, it just improves efficiency

97
Q

why are macrophages important in infection? how can this be tested?

A

Macrophages are not essential for protection but are needed to restrict bacterial spread within organs like the spleen
Test macrophage using depletion:
- Lack vaccine but have macrophage – many bacteria in marginal zone
- Vaccine and macrophages: white pulp is free of bacteria – stopped spread of bug
- Remove macrophage with vaccine – bug can spread into white pulp

Macrophage doesn’t kill bug immediately, but stops spread of bacteria

98
Q

how can SBA experiments be reflected in human systems?

A

Bacteria with antibody and complement in THP1 macrophage cell line
- Lack antibody, but have complement – bacterial uptake by macrophage
- Ab with no complement – more uprake
- Both present = highest bacterial number uptake – complement and Ab contribute most to bacterial uptake
- No C3 – less efficient uptake

99
Q

how can long-lived persistor bacteria be observed?

A

THP1 macrophage infected with red bacteria or green bacteria or both kinds of bacteria:
- Persistor cell in yellow – long-lived bacteria

100
Q

does antigen recognition lead to protection?

A

Recognition of antigen doesn’t always equal protection – need effector function and Ab access

Multiple systems need to combine – redundancy in immune response for efficient killing

Molecular Mechanisms (6 decks)

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