B08 - HT942523Q0045 Amend 0001_FINAL.docx

HT942523Q0045

SECTION SF 30 BLOCK 14 CONTINUATION PAGE

SUMMARY OF CHANGES

SECTION SF 30 - BLOCK 14 CONTINUATION PAGE

The following have been added by full text:

QUESTIONS FROM OFFERORS

Questions from Offerors

Q1: Following the link you sent, www.sam.gov, I found all relevant documents, however, I did not find any official form or similar thing to enter proposal items or data. Does this mean that we can send our proposal format and the signed documents (Organizational Conflict of Interest, Non-Disclosure Statement) by email directly to you?

A1: Proposals shall be submitted through the Solicitation Module of the PIEE suite as outlined in the Instructions to Offerors. Refer to Page 30, Instructions to Offerors, within the solicitation for specific instructions on how to submit proposals and what should be included in each proposal.

Q2: I found in the PWS in section 3 the corresponding performance requirements, which are well defined. Would a regular quote be sufficient in this case a proposal, or do we need to embed this into a broader proposal format?

A2: Refer to Page 30, Instructions to Offerors, within the solicitation for specific instructions on how to submit proposals and what should be included in each proposal.

Q3: 3.1. Task 1 (Milestone 1) - Three hybridomas for Ferret IgM (Deliverable 5). The Contractor shall conduct/accomplish three different hybridomas generated on rats and selected based on Ferret IgM specific reactivity.

The background of the proposal states “These monoclonal antibodies will be used to do basic FACs analysis and eventually flow cytometry cell sorting for these cells”. Are these cells ferret peripheral blood mononuclear cells (PBMCs).

How are the “different hybridomas” to be defined

In what targets and assay formats should IgM (or IgG) specific reactivity be demonstrated (e.g., cell membrane associated IgM (or IgG) on ferret PBMCs by flow cytometry, soluble IgM (or IgG) by ELISA)?

In the case of cell membrane associated IgG or IgM, do the antibodies need to work in the presence of other antibodies to phenotype ferret cells, (e.g., antibodies to b-cell receptor proteins CD79alpha or CD79beta)?

A3: The monoclonal antibodies should be different cell culture isolates. We are NOT asking the company to check H&L chain sequence, discover the specific epitope, isotype should be IgG (but can be any subtype). The monoclonals against IgM should bind IgM, and the monoclonals anti IgG should bind IgG. USAMRIID will perform the FACS and Western Blots analysis, but the company should verify it is binding the antigen used for immunization. Also have a semiquantitative or quantitate assessment of affinity/avidity (ELISA based or similar).

Q4: 3.1.1. The Contractor shall synthesize and clone the Ferret IgM Fc region based on the sequenced provided by the PI.

The goal (referring to 3.1.2) seems to be a DNA vector expressing Ferret IgM (or IgG Fc). Is this the case?

Should soluble IgM (or IgG) Fc be produced and purified to verify expression and provide protein?

A4: The ideal would be to use a DNA-based vaccine. However, if the contractor prefers to immunize using soluble Fc region (either IgM or IgG) that would also be acceptable. USAMRIID will provide the target sequence (one for ferret IgM FcR and one for ferret IgG FcR).

Q5: 3.1.2. The Contractor shall perform the genetic immunization of rats with the Ferret IgM Fc region (from task 3.1.1).

Contractor can generate hybridomas from both mice and rats, with the former being a lower cost option. Is there a specific rationale or mandate for the use of the rat that would inform the immunization strategy or hybridoma selection?

Commonly, immunizations would be performed with protein antigen plus adjuvant. While immunization using a DNA vector (genetic) can work, combining DNA injections with protein injections would typically result in a higher frequency of specific hybridomas. Is USAMRDC open to alternative immunization strategies?

A5: USAMRIID prefers rats, but other rodents (e.g. mouse) are also acceptable. USAMRIID is open to alternative immunization protocol that might result in better results by the contractor experience. The PWS has been updated accordingly.

Q6: 3.1.3. The Contractor shall perform the fusion of rat lymphocytes from the immunized rodents to generate the hybridomas and perform the screening for positive hybridomas (antibodies targeting the ferret IgM Fc region).

What would be considered a positive hybridoma (e.g., what type of target and in what assay (see 3.1))?

A6: Positive hybridomas would be those that can specifically recognize the target antigen provided by USAMRIID (target sequence, one for ferret IgM FcR and one for ferret IgG FcR). The company should test that the monoclonal antibodies for IgM are not cross-reacting with IgG, and vice versa.

Q7: 3.1.4. The Contractor shall scale the selected (task 3.1.3) hybridomas.

What would be considered scaling (e.g., further culture, cryopreserve, supernatant production)?

A7: Cryopreserve enough quantity (minimum 24 vials of 1ml each) of the six different hybridomas (3 for each target). This will ensure we can preserve frozen hybridomas in the N2 tank bank while expanding the hybridoma cells in house. USAMRIID would be responsible of expanding the hybridoma and purify the monoclonal antibodies, however, supernatant from the hybridoma culture should be provided to perform preliminary tests. The PWS has been updated accordingly.

Q8: 3.1.5. The Contractor shall subclone the selected rat hybridomas (task 3.1.4).

Are there any specific properties other than clonality?

A8: No, this step is just to ensure the clonality of the hybridoma.

Q9: 3.1.6. The Contractor shall deliver the three hybridomas to the USAMRIID PI, 14 business days prior to the end of the contract.

Contractor would expect to obtain many hybridomas. What criteria would be applied to select the three hybridomas to be delivered (see 3.1 comment)?

In what form and quantity should hybridomas be delivered (e.g., cryopreserved vials, number of each, supernatant, V-domain sequences, purified Ab)?

A9: See responses A3, A6 and A7. Selection of hybridomas should be based on specificity for the target antigen and affinity. If more than 3 (three) hybridomas perform “equally”, selection of the three hybridomas can be random. The PWS has been updated accordingly.

Q10: Above questions 3 through 9 are applicable to below and do not require an answer for Ferret IgG if covered above.

3.2. Task 2 (Milestone 2) - Three hybridomas for Ferret IgG (Deliverable 6). The Contractor shall conduct/accomplish three different hybridomas generated on rats and selected based on Ferret IgG specific reactivity.

3.2.1. The Contractor shall synthesize and clone the Ferret IgG Fc region based on the sequenced provided by the PI.

3.2.2. The Contractor shall perform the genetic immunization of rats with the Ferret IgG Fc region (from task 3.2.1).

3.2.3. The Contractor shall perform the fusion of rat lymphocytes from the immunized rodents to generate the hybridomas and perform the screening for positive hybridomas (antibodies targeting the ferret IgG Fc region).

3.2.4. The Contractor shall scale the selected (task 3.2.3) hybridomas.

3.3.5. The Contractor shall subclone the selected rat hybridomas (task 3.2.4).

3.2.6. The Contractor shall deliver the three hybridomas to the USAMRIID PI, 14 business days prior to the end of the contract.

A10: IgM and IgG hybridomas should be generated equally (other than the target antigen). Refer to answers 3 through 9 above. Paragraphs 3.2.2., 3.2.4. and 3.2.6. have been updated accordingly.

SECTION SF 1449 - CONTINUATION SHEET

TABLE OF CONTENTS

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Exhibit/Attachment Table of Contents

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Exhibit/Attachment Table of Contents

13-APR-2023

13-APR-2023

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