TB Peptides SOW_8-6.pdf

Statement of Work Synthesis and Pooling of Peptides Spanning the Entire Tuberculosis (TB) Proteome

Developing a more effective TB vaccine will require an understanding of the mechanisms that confer protection against Tuberculosis (TB). Intravenous (IV) immunization with Bacille Calmette- Guérin (BCG) prevents TB infection and disease in rhesus macaques. Identifying the antigens within TB responsible for inducing higher CD4 and CD8 T cell responses following IV BCG will provide critical information for understanding how protection is mediated. To accomplish this, the Vaccine Research Center (VRC) will use overlapping peptides that span the entire TB proteome to screen IV BCG vaccinated macaques and/or humans. Due to the size of TB proteome, a method of screening consisting of 20 amino acid peptides that overlap each other by 10 amino acids will be used. This will result in a total of ~132,000 peptides, which will be combined (pooled) according to the Open Reading Frame (ORF) to which each peptide belongs.

1. Scope:

Peptide synthesis services to create, validate, and pool the ~132,000 peptides that span the entire TB proteome.

2. Requirements:

A. The Contractor must receive and log the electronic document containing 132,098 peptide sequences provided by the Government. Electronic documents containing peptide sequences will remain confidential.

B. The Contractor must review the amino acid sequences of the peptides and report if there are any overly difficult peptides (based on hydrophobicity) and propose alternative breakpoints (for the 20 overlapping by 10) if found before the project begins.

C. The Contractor must synthesize greater than or equal to 5mg of all peptides contained within the electronic document with no additional high-performance liquid chromatography (HPLC). Crude purities are expected to be >30%.

D. The Contractor must validate all peptides by mass spectrometry (MS) to ensure proper synthesis of requested sequence. If upon MS validation sequence errors are found in peptides the contractor must re-synthesize those peptides and re-validate by MS.

E. The Contractor must combine peptides by antigen into approximately 4,400 pools

(containing between 1 and 70 peptides each) according to the electronic document provided by the Government. No MS validation will be performed on pooled peptides.

F. The contractor shall supply the peptide pools in lyophilized form, in a 96-well format.

Each 96-well format plate (of 48 total) must comprise 92 wells of peptide pools and 4 empty wells. Each plate will be replicated to between 5 and 20 copies (as defined by the pooling strategy agreed upon by Contractor and the Government).

G. Final Deliverables: Contractor will supply the Government with 5-20 exact duplicates of each of the 48 plates; each will contain 92 wells of lyophilized peptide pools; each pool comprises >5mg of between 1 and 70 peptides.

3. Reporting requirements:

A. Content: The Contractor must generate validation reports for each peptide, that consists of:

a. Peptide sequence synthesized.

b. Purity of synthesized peptide.

c. Documentation of MS validation for each synthesized peptide.

B. Style: The Contractor must:

a. Prepare the draft report in Microsoft Word.

b. Provide the final report in PDF format.

C. Review and Revision: The Contractor must:

a. Provide the draft report electronically to VRC/NIAID for review.

b. Make any requested revisions and return the amended report to VRC/NIAID for a final review.

c. Once authorized by VRC/NIAID, provide this final audited, signed report electronically to VRC/NIAID.

d. Provide a Word file with the text of the report.

D. Delivery schedule:

a. The Contractor must complete the project within 18 months of initiation

b. The Contractor must provide the entire project in two shipments when complete.

The first shipment will comprise one set of plates of 300 peptides pooled as specified by the Government (“Pilot run”). The second shipment will comprise all other sets of plates.