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Frederica wants to try using yogurt as the delivery mechanism for her vaccine. She will clone...

Frederica wants to try using yogurt as the delivery mechanism for her vaccine. She will clone a gene from S. pneumoniae and express that gene in Lactococcus lactis, an organisms used to make yogurt. Eating the yogurt would serve as the vaccine delivery mechanism. She even has a name for her new vaccine: SpYogurt!

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

gene

Protein(s)

Strain-to-strain sequence variabilitya

Protein location

Protein activity

ID50

of mutantb

plyA

Pneumolysin

Low

Secreted protein

Kills host cells by assembling pore in

membrane

2x105

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?

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Answer #1

8a. current vaccines [PCV7, PCV13, PPSV23] contains the polysaccharides for some of the strains among a very large variety of S. pneumoniae serotypes. PCV7 is effective against 7, PCV13 is effective against 13, PPSV23 is effective against 23. Thus they unable to provise a complete protection against   S. pneumoniae .

8b.

rrgB

Pilin

Low

Attachment pilus

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

106

This protein will be the best option for preparing new vaccine. Because this protein present in the pilus .

Thus it is exposed in the surrounding environment even when the real S. pneumoniae attack occurs. If this protein used as a vaccine and vaccinated person already contains prepared antibody against pilin. Body immune mechanism is able to detect and respond this part quickly. β-galactosidase cannot be considered because it located in cytopalm thus antibody prepared against β-galactosidase took a lot time to recognise antigen.

The activity of this protein is only attachment of bacteria with host cell. It does not play any destructive type of role host system. Thus it is harmless part of bacteria which can be used as vaccine. Pneumolysin cannot be considered because it has a destructive role against host cell.

Along with it has a very low sequence variability rate which helps in vaccination effectiveness. Due to low variability the protein sequence which vaccineation occurred remains nearly similar to actual S. Pneumoniae which attacks. Pneumococcal surface protein cannot be considered because it has a high variability rate.

Moreover, the mutant contains this protein also viable. Lipoteichoic acid synthase cannot be considered because mutants are not viable.

8c. New vaccine conatins pilin protein which present in all the serotypes of S. pneumoniae . Thus it will be a complete protection.

8d.

. Selection marker: Like URA3 (non antibioti selectable marker) Protease dleavage site Multiple cloning site Transcriptional te

8e. after eating the yogurt the body immune system detects the inserted S. Pneumonia protein as an antigen. After recognition immune systems process it through APC and activate B cell through Tcell to produce antibody against the inserted protein.

After when person exposed to the S. pneumoniae the secondary immune repose will be very small. Through memory B cell and T cell immune system easily recognise the antigen protein in S. pneumoniae and destroy it before it cause any harm

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