Table 4. Potential vaccine candidates for the prevention of S. pneumoniae infections.

gene

Protein(s)

Strain-to-strain sequence variability^a

Protein location

Protein activity

ID50

of mutant^b

plyA

Pneumolysin

Low

Secreted protein

Kills host cells by assembling pore in

membrane

2×10⁵

pspA

Pneumococcal surface protein

High

Anchored in cell wall; extends out through capsule

Inhibits complement activation

10⁴

rrgB

Pilin

Low

Attachment pilus

Main structural component of pilus that mediates attachment during colonization of human tissues

10⁶

lacG

β-galactosidase

Low

Cytoplasm

Catalyzes hydrolytic cleavage of lactose, producing glucose and galactose

10⁵

ltaS

Lipoteichoic acid synthase

Low

Cell wall

Catalyzes assembly of sugar-glycerol building blocks into species-specific LTA structures

Mutant

not viable

^aThe gene was sequenced in 1000 S. pneumoniae isolated from 1000 infected patients identified in hospitals, and the corresponding protein sequences were deduced. ‘Low’ indicates that there were, on average, 0.5-1% amino acid changes comparing the sequences from any two strains. ‘High’ indicates sequence differences ranging from 5-10%.

^bNull mutants were generated using the pathogenic Bimmel strain of S. pneumoniae. Infectivity was assessed using the procedure described for Question 2c.

8a. (2 pts). Explain why current vaccines [PCV7, PCV13, PPSV23] do not provide full protection against S. pneumoniae infections.

8b. (4 pts) Fredrica has narrowed down her list of possible S. pneumoniae genes to five candidates. Some relevant information about each of the five in the table below. Choose one of these to recommend to Fredrica for her new vaccine. Explain what information led to your selection of this particular antigen for your vaccine and your exclusion of the others (i.e, what you think is its major shortcoming as a vaccine candidate).

8c. (2 pts) Explain why this new vaccine could be superior to currently recommended pneumococcal vaccines.

For her new vaccine, Fredrica must express one of the S. pneumoniae genes in a bacterium that will survive in yogurt. One organism found in most yogurt is L. lactis. Frederica’s plan is to transform L. lactis with a recombinant plasmid that includes the cloned S. pneumoniae gene.

8d. (4 pts). Draw a diagram of a plasmid that could be used for this project.

                 On your drawing indicate the required genes or DNA sequences. Briefly describe the role of each gene or each sequence. Keep in mind that the plasmid needs to work in L. lactis it has to:

be able to replicate,

direct expression of the gene of interest,

it has to be maintained in the L. lactis when it replicates,

it has to include a gene that can be used as a selectable marker for the transformation done in the lab. Also, note that Fredrica absolutely refuses to use an antibiotic-resistance gene as her selectable marker, so what gene can she use to select L. lactis cells that have been transformed with this plasmid? Make sure that you clearly indicate it on your diagram.

8e. (4 pts) Explain: what would a person’s immune system do after she/he ate the SpYogurt containing the genetically engineered L. lactis expressing the gene you chose? What would happen when this person is exposed to S. pneumoniae several months after eating SpYogurt?