Answers:Malaria Vaccine

Answers to case study exercise about malaria vaccines (NB: numbers etc. found in the databases 06-10-2026):

1 - What exactly is malaria?

1a) If you search for "malaria" on NCBIs Taxonomy page, you find some mosquitoes and some protozoans with the Genus name Plasmodium. Clicking the name of one of these gets you to a page where you can see the lineage:

• Genus: Plasmodium
• Phylum: Apicomplexa
• (Super)Kingdom: Eukaryota

1b) On NCBI's Taxonomy page is a function named ”Taxonomy common tree” which gives a nice overview. Alternatively you can open taxonomy pages for the two organisms to compare, and see on their lineages how much they have in common.

• Homo sapiens and Plasmodium: Eukaryota
• Babesia microti and Plasmodium: Aconoidasida

Here is the picture you can get from the "Taxonomy common tree" function:

1c) On CDC's page about malaria or on Tree of Life's page about Plasmodium you find:

• P. malariae, P. ovale, P. falciparum and P. vivax.

By looking up these four species in NCBI Taxonomy and looking at the table to the right, you see that all four species have a full genome in the databases (see the link named Genome under Entrez records).

 

2 - Identification of membrane proteins (potential vaccine targets)

2a)

5479 protein-coding genes.

2b)

The correct search strings

(taxonomy_id:5833)

or

(organism_name:"Plasmodium falciparum")

both give 23,379 hits in total, 509 from Swiss-Prot and 22,870 from TrEMBL.

If you only found 11,896 hits, it was because you used

(organism_id:5833)

which only gives those Pf proteins that do not have a specified strain or isolate — cf. question 3.4+3.5 in the UniProt exercise.

If, on the other hand, you found 24,313 hits, it was because you searched in All instead of specifying the search field:

Plasmodium falciparum

In that case, you will include some proteins that originate from e.g. humans but play a role in Plasmodium falciparum infection, which may be mentioned in some comment field or reference title.

2c)

This can be solved in several ways:

• (taxonomy_id:36329) (either selecting the right isolate from the drop-down menu or using the TaxID you found in the Taxonomy database)
• (taxonomy_id:5833) AND (organism_name:3d7)
• (organism_name:"Plasmodium falciparum") AND (organism_name:3d7)

They all give: 5,394 in total, 321 from Swiss-Prot and 5,073 from TrEMBL.

That corresponds approximately to the number of protein-coding genes found in 2a).

2d)

(taxonomy_id:5833) AND (cc_scl_term:*)

5,175 (410 from Swiss-Prot and 4,765 from TrEMBL).

2e)

32 hits, 27 from Swiss-Prot.

(taxonomy_id:5833) AND (cc_scl_term:"host cell membrane")
or
(taxonomy_id:5833) AND (cc_scl_term:SL-0375)

2f)

(taxonomy_id:5833) AND (protein_name:erythrocyte)

3,856, among these only 4 from Swiss-Prot.

2g)

(taxonomy_id:5833) AND (protein_name:erythrocyte) AND (protein_name:membrane)

3,610 hits, all from TrEMBL.

or

(taxonomy_id:5833) AND (protein_name:"erythrocyte membrane")

3,569 hits, all from TrEMBL.

2h)

(taxonomy_id:5833) AND (protein_name:erythrocyte) AND (protein_name:membrane) AND (fragment:false)

174 (or 172 if the words "erythrocyte membrane" are combined)

2i)

(taxonomy_id:5833) AND (protein_name:erythrocyte) AND (protein_name:membrane) AND (fragment:false) AND (database:pdb)

8 hits, called "Erythrocyte membrane protein 1" or "Erythrocyte membrane protein 2": Q6UDW7, Q8I098, Q8I639, Q8IHM0, W7K270, A3R6S4, A0A024V5I6, and I1X0L2.

 

3 - Analysis of membrane protein domain structure

3a)

InterPro identifier: IPR008602
Pfam identifier: PF05424
It is found 4 times in Q8IHM0, 5 times in Q8I098 and 6 times in Q8I639.

3b)

The transmembrane segments are in the following positions:

• Q8I098: 3124-3146
• Q8I639: 2650-2667
• Q8IHM0: 2695-2717

The extracellular parts are the N-terminal parts (all the positions before the transmembrane segments), and the intracellular (cytoplasmic) parts are C-terminal (positions after the transmembrane segments). All the Duffy binding domains are in the extracellular part.

3c)

The following positions are structurally determined by X-ray in the three proteins:

• Q8I098: No X-Ray, only EM (Electron Microscopy).
• Q8I639: 2333-2634, covering Duffy_binding domain 6
• Q8IHM0: 728-1214, covering Duffy_binding domain 2

3d)

Yes!

• Biological process: cytoadherence to microvasculature, mediated by symbiont protein
• Biological process: pathogenesis
• Cellular component: host cell plasma membrane
• Cellular component: infected host cell surface knob
• Molecular function: cell adhesion molecule binding
• Molecular function: host cell surface receptor binding

All these examples support that these proteins are involved in binding the infected erythrocytes to the endothelial cells (as described in the exercise).

 

4 - Prediction of B-cell epitopes in a membrane protein

4a) The PDB entry is 2WAU and it's a crystal structure (X-ray).

4b) FASTA sequence for the Duffy Binding domain covered by the 3D structure:

>2WAU_1|Chains A, B|ERYTHROCYTE MEMBRANE PROTEIN 1 (PFEMP1)|PLASMODIUM FALCIPARUM (36329)
ICNKYKNINVNMKKNNDDTWTDLVKNSSDINKGVLLPPRRKNLFLKIDESDICKYKRDPKLFKDFIYSSAISEVERLKKV
YGEAKTKVVHAMKYSFADIGSIIKGDDMMENNSSDKIGKILGDGVGQNEKRKKWWDMNKYHIWESMLSGYKHAYGNISEN
DRKMLDIPNNDDEHQFLRWFQEWTENFCTKRNELYENMVTACNSAKCNTSNGSVDKKECTEACKNYSNFILIKKKEYQSL
NSQYDMNYKETKAEKKESPEYFKDKCNGECSCLSEYFKDETRWKNPYETLDDTEVKNNCMCK

4c) The sequence interval was 2333-2634. This means that the first postion in the new FASTA file corresponds to position 2333 in the original sequence.

4d) It's possible to convert from the coordinates in the FASTA files to the full length sequence by adding 2332. In the table below the epitopes have been named by their starting position as well as numbered.

EPITOPE     POSITIONS    LENGTH     ORIG_POSITIONS
#1 ep_5       5 to  19       15     2337 to 2351
#2 ep_23     23 to  29        7     2381 to 2388
#3 ep_105   105 to 116       11     2437 to 2448
#4 ep_192   192 to 222       30     2535 to 2564
#5 ep_238   238 to 247       10     2570 to 2580
#6 ep_293   293 to 297        5     2626 to 2630

 

5 - Visualization of epitopes

5a) Invisible positions:

Chain A: 2333-2349 and 2540-2546
Chain B: 2333-2348 and 2535-2549

This means that the first epitope (pos 5-19, orig pos 2337 to 2351) and the 4th epitope (pos 192 to 222, orig pos 2535 to 2564) are partially invisible.

5b)

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