Immune response to viruses

Question 1

What are the main cells in the innate response?

Answer 1

• Neutrophils (phagocytose bacteria and other pathogens)
• Antigen Presenting Cells (APCs) (phagocytose the pathogen)
  −Macrophages and dendritic cells
• Natural Killer (NK) Cells (non specifically kills virus infected cells)
• Eosinophils (parasites)

Question 2

What is the function of NK cells?

Answer 2

*Rapidly seek out and destroy
virus-infected cells (not specific)
* Identify markers of stress on
the infected cells
*Has two type of receptor
−Activating receptor
−Inhibiting receptor (prevents NK cells from killing healthy cells)
* no memory or MHC restriction

Question 3

How are NK cells activated?

Answer 3

Transformed or infected cells sometimes increase expression of molecules that activate NK cell receptors (activating ligands). This activating signal may override the inhibitory MHC class I molecule signal and allow NK cells to attack.

Question 4

How do NK cells kill?

Answer 4

−Bind to infected cells
*Release perforins
* Induce apoptosis
*Release interferon gamma

Question 5

What are APCs?

Answer 5

• Involved early in the host
  response
• Carry immunoglobulin Fc and
  C3b receptors (promote
  phagocytosis)
• Professional antigen presenting
  cells
  *MHC class II protein binds to antigen fragment and presents antigen
• Initiate the adaptive immune
  response

Question 6

What are PAMPs?

Answer 6

Pathogen-Associated Molecular Patterns recognised by innate immune system
* Un-methylated C-G dinucleotides (CpG motifs) on DNA viruses
* high frequencies in viruses
* low frequency in mammalian cells
* Double-stranded RNA (only RNA viruses produce this)
* Uracil-rich, single-stranded RNA

Question 7

What are toll-like receptors (TLRs)?

Answer 7

• A type of PRR found on the surface of cells (TLR-1,2, 4, 5, 6) and within endosomes of phagocytic
  cells (Neutrophils and APCs) (TLR-3, 7, 8, 9)
• Detect extracellular viruses
• Sense the presence of viral nucleic acid and other conserved molecular
  components of invading pathogens

Question 8

Examples of TLRs?

Answer 8

TLR-3 binds ds RNA
TLR-7 binds Uracil-rich ss RNA (e.g. HIV)
TLR-8 binds ss RNA
TLR-9 binds CpG motifs within viral DNA

Question 9

What are cellular PRRs?

Answer 9

• Found in the cytoplasm
• Retinoic acid inducible gene-1-like receptors (RIG-1 and MDA-5)
• RIG-1 recognises ss RNA
• Melanoma differentiation association gene-5 recognises ds RNA
• NOD-like receptors (NOD2)
• Cytosolic DNA sensors

Question 10

What are interferons?

Answer 10

• Protects adjacent cells from infection
• Inhibition of viral replication
• Helps activates T-cell mediated immunity
  −Activation of macrophages
  −Up-regulates MHC receptors on virus-infected cells

Question 11

What are INFalpha and INFbeta (type 1)?

Answer 11

• Produced by most cell types early in infection (Innate response)
• Activate genes that have antiviral activities
  −dsRNA dep protein kinase R
  −RNase L
• Helps stimulate MHC class I (enhance presentation of viral peptides
  to T cells)
• Activates NK cells
• Induces apoptosis

Question 12

What is INFgamma (type 2, proinflammatory)?

Answer 12

• Involved in the regulation of nearly all phases of immune and
  inflammatory responses (both innate and adaptive)
• Produced by NK cells and T lymphocytes
• Enhances MHC expression on APCs
• More important as an immunoregulator than as an antiviral agent
  −Enhances the cytotoxic activity of T cells, macrophages and NK
  cells

Question 13

What are the components of the adaptive immune response?

Answer 13

Virus-specific immune response, recognise viral proteins and carbohydrates (antigens); takes days
Humoral Immune response (B lymphocytes)
− Antibody production
* Cellular Immune Response (T lymphocytes)
− Cytotoxic T cells (CTLs) CD8+
* Kill virus-infected cells
* Cytokines that eliminates viral RNA
− T Helper cells (TH) CD4+
* Activate macrophages
* cytokines

Question 14

What is the humoral immune response?

Answer 14

B lymphocytes
* Resident in the lymphatic tissue
* Respond to antigenic stimulus by
producing and secreting antibodies
* Carry highly specific receptors that
recognise viral epitopes
* Recognise Ag in their native form (no
processing)

Question 15

How are B cells activated?

Answer 15

• Activates on binding to virus/Ag; requires a signal from T helper cells
• Clonal expansion of B cells producing a single antibody
• Antibodies early on are low affinity (IgM); B cells evolve by hyper-mutation in the V regions to produce high affinity binding antibodies (IgG) later on

Question 16

What are IgG, IgM and IgA?

Answer 16

IgG: main circulating
antibody in secondary response; important for long term immunity

IgM: Circulating antibody
produced early in
infection; diagnostic marker

IgA: Secretory antibody; primary defence at mucosal surface

Question 17

What is the role of an antibody?

Answer 17

• Binds to circulating virus (outside the cell)
  −May prevent attachment to susceptible cells (Neutralises virus)
• Opsonisation
  −Virus-coated with antibody activates complement
  −Induces inflammatory response (attracts neutrophils and macrophages)
• Plays a major role in recovery following
  infection with viruses that cause viraemia

Question 18

What is the cellular immune response?

Answer 18

• T lymphocytes co-ordinate the activities of the cells involved in generating the immune response
  −T helper cells (CD4+ T cells)
  −Cytotoxic T cells (CD8+ T cells)
• Carry highly specific receptors that recognise specific regions on a pathogen (single T cell recognises a single T cell epitope) in association with MHC proteins
• On activation, T cells will divide and proliferate and secrete cytokines that regulate the immune response
• Some T cells become long-lived memory T cells

Question 19

What are cytotoxic T cells (CTLs)?

Answer 19

• Recognises viral peptides on the
  surface of infected cells in associated
  with MHC class I
• Kill virus-infected cells
• Granules within the CTL polarise
  towards the target cell
• Perforin is released and creates
  pores in the cell membrane
  causing lysis

Question 20

What are helper T cells?

Answer 20

• Recognises viral peptides on the
  surface of APCs in associated with
  MHC class II
• Plays essential role in initiation of B
  cell responses
• Secrete cytokines that regulate the
  immune response
• Main types are Th1 and Th2

Question 21

What is Th1?

Answer 21

Secretes IL-2
Inflammatory response
Augments immune response by
attracting macrophages to site of
infection
Activates IFN γ and TNF β
Promotes IgG2a production

Question 22

What is Th2?

Answer 22

Secrete IL-4, IL-5 and IL-6
Provide help for antibody production
Promotes switching of B cells (IgG2a
to IgG1)

Question 23

What are the targets of the humoral response?

Answer 23

• Surface exposed protein on virus particle
• Envelope proteins
• Capsid proteins (of naked viruses)
• Often spikes or loops that protrude from viral surface
• Epitopes can be linear or “conformational” (critical residues brought together by folding of
  the polypeptide chain)

Question 24

What is western blotting?

Answer 24

• Allows detection of antibodies to some or all the viral proteins of a particular virus
• Can be used to monitor present of antibodies to different antigens at different stages of infection
• Not used routinely in diagnostic setting (not easily standardised or scaled up for automation)
• Generally used as a confirmatory test

Question 25

How is a western blot performed?

Answer 25

1. Run proteins on a SDS polyacrylamide gel
2. Transfer separated proteins to a
   nitrocellulose/nylon membrane
3. Incubate membrane with antibody (from sample i.e. diluted serum sample). Wash, incubate with secondary antibody labelled with enzyme/tag that can be visually detected
4. Detect/visualise proteins on addition of
   substrate

Question 26

What is a virus neutralisation test?

Answer 26

• Loss of infectivity through reaction of the virus with specific antibody
• Virus is incubated with serum sample (serial dilutions)
  − Using constant concentration of virus
• Concentration where plaques are visible and beginning to decrease in number
  − Virus and antisera are then used to infect cell monolayers in a microtitre plate
  − Determine the highest dilution of antibody which when mixed with a standard virus concentration will prevent the formation of plaques

Question 27

Pros and cons of neutralisation assay

Answer 27

• Used for detection and quantification of virus specific antibodies
• Gold standard in virology
• Neutralising antibody correlates with protective antibody in vivo
• When new virus emerges – such tests can be operational within weeks (take up to a year for an ELISA to be fully validated)
• Cons:
  −Slow can takes several days to get a result
  −Require infectious virus
  −Cost to maintain cell culture facilities

Question 28

What are uses of serological tests?

Answer 28

Used when it is difficult to detect virus directly
* Widely used when animals are imported and exported from countries
−Governments want certification to ensure animals are not bringing
pathogens with them
* Used to assess vaccine efficacy
−Is vaccination being implemented correctly
−Eradication programs