Lecture 2 - Immune Evasion by Antigenic Variance

Question 1

T. brucei infection in man

Answer 1

• Lives extra-cellularly in mammalian bloodstream
• Elicits host immune response
• Peaks and troughs of parasitaemia represent repeated cycles of immune destruction and regrowth of parasite population

Question 2

Variant surface glycoproteins form protective surface coat on trypanosome cells

Answer 2

• Abundant surface protein 7-10% of total cellular protein

-Electron dense, homogenous ‘surface coat’ - covers the entire cell surface

No enzymatic or receptor function known - but parasite protective function

> 2000 VSG genes/pseudogenes - antigenically diverse - immunologically distinct VSGs typically have <25% amino acid identity

Question 3

Variant Surface Glycoprotein (VSG)

Answer 3

Homodimer of two 50–60kDa subunits - held on extracellular face of plasma membrane by glycosylphosphatidylinositol (GPI) anchor

VSG molecules free to diffuse in the plane of plasma membrane and there is no evidence for intermolecular affinity between VSG dimers

Large N-terminal domain (NTD) 350–400 residues and 1 or 2 small C-terminal domains (CTD) 20–40 residues - domains connected by flexible linkers

Comparisons of VSG primary sequences have categorised N-terminal domains into three types - A, B and C - and C-terminal domains into 6 types 1 – 6, based on sequence similarities and position of cysteine residues

Packing density - 5.7 x 10^6VSG dimers- access to plasma membrane physically obstructed by VSG

Question 4

Crystal structure of VSG N-terminal domain

Answer 4

NTD forms a rigid dimer (one monomer is dark blue - the other light blue)

VSG shields surface from antibodies and prevents complement proteins forming ‘membrane attack complex’

Question 5

Modelling suggests VSG can adopt two conformations

Answer 5

1. Tightly packed VSGs elevate VSG above transmembrane proteins
2. Relaxed conformation maintains protective coat at reduced protein density

Many plasma membrane proteins e.g. hexose transporter have small extracellular domains (<10 kDa) - VSG coat prevents antibody access to these proteins

However, other proteins have extracellular domains of similar size or even larger than VSG - how these proteins avoid immune recognition is unknown

Question 6

VSG coat elicits a robust antibody response by the host

Answer 6

Low levels of anti-VSG antibodies – trypanosomes internalise VSG-Ig complex, degrade antibodies and return VSG to surface

Turnover of VSG coat occurs solely via flagellar pocket – this occupies ~5% of cell surface but entire pool of VSG recycled in ~12 minutes - VSG bound to antibody is turned over even quicker

Question 7

How are antibodies bound to cell surface VSG removed from the surface?

Answer 7

Movement of immune complex (towards posterior end) depends upon forward cellular motility

Hydrodynamic flow acting on ‘swimming’ trypanosomes causes directional movement of Ig-VSG complex within the plane of the plasma membrane (GPI anchored)

Antibody bound to VSG acts as a
‘molecular sail’ to sweep Ab-VSG
complex back to flagellar pocket

Uptake via flagellar pocket is
dependent on endocytosis

Question 8

Removal of antibodies from cell surface renders trypanosomes resistant to complement mediated lysis (time at 37C before addition of complement)

Answer 8

Resistance to complement depends upon concentration of anti-VSG antibody

Question 9

Short stumpy trypanosomes remove VSG-antibody complexes faster than long slender forms

Answer 9

Stumpy forms show greater resistance to antibody-mediated killing –stumpy forms to predominate at peak parasitaemia and extend period during which infection is transmissible to tsetse flies

Stumpy forms not replicative, so need to be able to withstand immune system for longer

Pleomorphic stumpy cells have a lower half life of cell surface fluorescence

Question 10

VSG recycling can only protect the trypanosome for a limited time

Answer 10

At high levels of anti-VSG antibodies – complement fixation, agglutination and phagocytosis occur as VSG-Ab clearance can no longer protect the parasite and cells are lysed - except for trypanosomes that have switched VSG expression

Question 11

Transcription of VSG genes in bloodstream form cells

Answer 11

VSG genes ONLY expressed from dedicated sites in the genome called Expression Sites (ESs) - located at telomeres

Expression sites are transcribed by RNA Polymerase I which allows for high levels of gene expression – estimated 512-fold higher rate of VSG mRNA production compared to typical Pol II transcribed gene (VSG mRNA is also very stable)

Polycistronic transcription - individual mRNAs generated by processing of the polycistronic transcript (trans-splicing and polyadenylation)

Bloodstream ES contains Expression Site-Associated Genes (ESAGs) – encode various proteins e.g. transferrin receptor – as well as VSG gene

ESAG repertoire differs between different ESs