|Acquisition Program: ||Office of the Principal Assistant for Acquisition|| Objective: ||Develop an efficient, cost-effective serum-based multiplex assay platform that will identify vaccine candidates, determine immune responses and serve as a potent diagnostic tool for epidemiological and clinical studies.
|| Description: ||Based on their impact on the military and global health, the United States Department of Defense (DoD) has placed a high priority on the development of vaccines against enteric pathogens causing diarrhea. Enterotoxigenic Escherichia coli (ETEC), Shigella, Campylobacter and norovirus are significant causes of diarrhea and represent significant targets of military, industry, academic and non-governmental (PATH Global Health) vaccine programs. These pathogens cause a high level of morbidity and significantly impact the military in lost manpower days, reduced effectiveness and increased treatment costs. The emerging awareness of post-infection complications further adds to the impact of these infections. In addition, these same pathogens are associated with hundreds of thousands of deaths among children under 5 in the developing world. Specific knowledge regarding the potential risks for infectious diseases in certain areas is critical to preventing disease as well as directing vaccine research efforts. Key areas in the development of successful vaccines are the identification of novel antigen targets, determination of the immunogenicity of a candidate vaccine, development of efficient means to determine prior pathogen exposure, and identification of correlates of protective immunity.
At present, the gold standard immunoassay for detecting prior exposures (non-stool-based) to infectious disease agents is the enzyme linked immunosorbent assay (ELISA). In this relatively laborious assay, antibodies in serum samples are tested for binding to antigens specific for a given enteric pathogen. Such assays have significant limitations with respect to the amount of sample required, technical man-hours (6-24 hours) to complete the assay, and an inability to multiplex in a single well, the latter resulting in increased costs and reagents. Thus, a more efficient assay is desired.
An ideal immunoassay would allow for the assaying of sera from exposed individuals against a panel of pathogen components that would provide valuable information regarding the response to a pathogen-specific vaccine, and the risk for a given infectious disease exposure and associated chronic health outcomes within a population subset. In addition, the flexibility of the platform would allow for customizing the assay for a specific disease agent, which would aid in vaccine discovery, as well as, for testing of immune responses against candidate vaccines undergoing clinical trials. Lastly, the ideal assay system would not be cost-prohibitive and allow for dissemination to and standardization in multiple research sites.
|| ||PHASE I: This phase will demonstrate the feasibility of the immunoassay platform to detect exposure(s) of individuals to ETEC, Shigella, Campylobacter and norovirus utilizing defined historical serum samples archived by NMRC and/or samples from the DoD serum repository. The use of human samples with a known medical history of the targeted infection will allow for determination of the sensitivity and accuracy of the assay in detecting responses induced by natural infection. In addition, this Phase will also evaluate the flexibility of the platform to allow for customizable assays specific to a single pathogen or vaccine that will be tested using existing animal models. Results will be compared using current methodologies (e.g. ELISA) to determine the reproducibility and repeatability of the multiplex assay.
|| ||PHASE II: In this Phase, the immunoassay developed in Phase I will be validated using human serum samples obtained through an ongoing multisite clinical research study (TravMil) based at DoD travel medicine clinics investigating the epidemiology of travel and deployment related infectious disease threats to U.S Department of Defense (DoD) active duty members and beneficiaries. The organism-specific relative risk of traveler’s diarrhea among DoD travelers as determined by pre- and post-travel serological testing and PCR amplification of organism-specific genetic material collected by participants during illness on a stool filter paper card. This phase will further demonstrate the ability of the assay platform to identify and differentiate between exposures to enteric pathogens. Furthermore, this Phase will provide a practical implementation of the assay for use in the clinic and/or field.
|| ||PHASE III: The developed immunoassay would be a significant aid to global health initiatives focused on preventing enteric diseases due to these pathogens. Military, industry, academic and public health institutions would benefit greatly from the described assay in aspects of vaccine research and epidemiological measures. The end-state of this technology would allow for the rapid identification of immune responses to various components of an infectious agent, which would be invaluable in discovering new vaccine candidates and defining exposures to enteric infections of interest. In addition, this assay will allow for using reduced amounts of valuable samples (e.g. clinical specimens) while also significantly increasing throughput while simultaneously reducing work time. Spin-off technologies would include customizing the assay to detect additional infectious agents that have relevance not only to human health, but also veterinary health and/or the food industry.
|| References: ||
1) Porter CK, El Mohammady H, Baqar S, Rockabrand DM, Putnam SD, Tribble DR, Riddle MS, Frenck RW, Rozmajzl P, Kilbane E, Fox A, Ruck R, Lim M, Johnston YJ, Murphy E, Sanders JW. Case series study of traveler's diarrhea in U.S. military personnel at Incirlik Air Base, Turkey. Clin Vaccine Immunol. 2008. Dec;15(12):1884-7.
2) Riddle MS, Tribble DR, Cachafiero SP, Putnam SD, Hooper TI. Development of a travelers' diarrhea vaccine for the military: how much is an ounce of prevention really worth? Vaccine. 2008 May 12;26(20):2490-502.
3) Riddle MS, Sanders JW, Putnam SD, Tribble DR. Incidence, etiology, and impact of diarrhea among long-term travelers (US military and similar populations): a systematic review. Am J Trop Med Hyg. 2006 May;74(5):891-900.
4) Flores J, DuPont HL, Jiang ZD, Belkind-Gerson J, Mohamed JA, Carlin LG, Padda RS, Paredes M, Martinez-Sandoval JF, Villa NA, Okhuysen PC. Enterotoxigenic Escherichia coli heat-labile toxin seroconversion in US travelers to Mexico. J Travel Med. 2008 May-Jun;15(3):156-61.
5) Jertborn M, Ahren C, Perlman DM, Kaijser B, Svennerholm AM. Serum antibody responses to bacterial enteropathogens in Swedish travelers to south-east Asia. Scand J Infect Dis. 1990;22(6):699-704
6) Walz SE, Baqar S, Beecham HJ, Echeverria P, Lebron C, McCarthy M, Kuschner R, Bowling S, Bourgeois AL, Scott DA. Pre-exposure anti-Campylobacter jejuni immunoglobulin a levels associated with reduced risk of Campylobacter diarrhea in adults traveling to Thailand. Am J Trop Med Hyg. 2001 Nov;65(5):652-6.
7) Hyams KC, Malone JD, Bourgeois AL, Hawkins R, Hale TL, Murphy JR, 1995. Serum antibody to
lipopolysaccharide antigens of Shigella species among U.S. military personnel deployed to Saudi Arabia and Kuwait during Operations Desert Shield and Desert Storm. Clin Diagn Lab Immunol 2: 700-3.
|Keywords: ||immunoassays, vaccines, enteric pathogens|
Questions and Answers:
Q: Do you want assay that work in field too, or in lab only?
A: Lab only.
Q: Does a green card affect eligibility for the SBIR award?
A: As long as the person has a green card they can work on an SBIR project.
Each proposer must qualify as a small business for research or research and development purposes as defined in Section 2.0 and certify to this on the Cover Sheet of the proposal. In addition, a minimum of two-thirds of the research and/or analytical work in Phase I must be carried out by the proposing firm. For Phase II, a minimum of one-half (50%) of the research and/or analytical work must be performed by the proposing firm. The percentage of work is usually measured by both direct and indirect costs,
although proposers planning to subcontract a significant fraction of their work should verify how it will be measured with their DoD contracting officer during contract negotiations. For both Phase I and II, the primary employment of the principal investigator must be with the small business firm at the time of the award and during the conduct of the proposed effort. Primary employment means that more than one-half of the principal investigator's time is spent with the small business. Primary employment
with a small business concern precludes full-time employment at another organization. For both Phase I and Phase II, all research or research and development work must be performed by the small business concern and its subcontractors in the United States.
Q: Will DoD supply specific strains of ETEC, Shigella, Campylobacter and Norovirus, or is the awardee required to use outbreak strains or ATCC strains for the study?
A: Applicants should write proposals with the assumption that several antigens will be provided to them by NMRC following the awarding of a grant. Additional antigens may need to be obtained from commercial vendors.
Q: How many human sera samples for each pathogen are required to be tested with the multiplex immunoassay platform?
A: The applicant's should put together a sample size estimate based on the phase of study which would provide for the reasonable evaluation of test performance characteristics experimental test with a 'gold standard'.
Q: How many animal sera samples for each pathogen are required to be tested with the multiplex immunoassay platform (animal models)?
A: There is no exact requirement in number of sera samples. Investigators should consider the phase of development and perform a sample size estimate which would afford a reasonable assessment of test performance compared to gold standard.
Q: Is the awardee required to use a commercial ELISA to compare the results of the multiplex platform with each sera sample or does the sponsor have a specific recommendation?
A: NMRC has archived samples from previous human challenge and mouse experiments with defined seroconversion. These samples will be provided at the discretion of NMRC to validate multiplex platforms. It is likely that a CRADA or MTA will need to be developed between both parties.
Q: 1. Can you please list suitable multiplex assay platforms you have in mind?
2. Can you provide details on minimum detectable levels required?
A: Q1: Luminex or equivalent bead-based assays in addition to novel platforms such as microfluidic or optical detection systems.
Q2: At a minimum, should be able to detect a 4-fold increase in antigen specific titer compared to base-line.
Q: What level of biosafety containment is expected for a Phase I award?
What about Phase II?
A: BSL 2.
Q: What "Government Certification" should be obtained by contractors that propose the use of biohazardous materials? Can information be provided regarding who to contact to obtain this certification?
A: If selected for award, the small business will receive instructions on completing Environmental and Safety requirements.
Q: Can you give more details about the antigens NMRC will provide for Phase I? (How many antigens overall and per pathogen; nature of the antigen - e.g. recombinant - purified - whole organism; etc)?
A: Antigens will include recombinant and purified proteins, polysaccharide, whole cell extracts, LPS.
Antigens per pathogen:
ETEC: 1-7 (recombinant, purified, whole organism)
Campy: 2-3 (recombinant, purified)
Shigella: 3 (purified, recombinant)
Salmonella: TBD, likely recombinant or peptide
Q: 1. What are the vaccines against enteric pathogens? Are they bacterial antigens or serum antibodies?
2. If the vaccines are the antigens, do you mean to identify a antigen combination (multiplex antigen or vaccine) that has immune response to pathogens listed in the solicitation?
A: A1: Current enteric (diarrheal) vaccines under development by DoD or in which the DoD are ETEC, Campylobacter, Shigella and Norovirus. Multiple approaches are being taken with these vaccines including live-attenuated, subunit recombinant protein, purified protein, VLP, and polysaccharide-protein conjugates.
A2: In general, components of the each of vaccine constructs will be used as antigens, though there will also be antigens (whole organisms, or whole organism extracts) which may be used as antigens as well for purposes of immune response/epidemiology.