SBIR-STTR Award

We propose to develop a rapid, low cost, threshold assay for the detection of HIV infection of HIV vaccination trial participants. Infection by HIV, and viral load are the usual end points for Phase III HIV vaccination trials. Ongoing Phase III HIV vaccine trials require a simple, and low cost molecular diagnostic test that can detect HIV infection in immunized participants. Indeed, current molecular tests are too complex to be used for screening purposes, immunoassays cannot distinguish between immunization and infection, and CD4 cell counts are unlikely to detect early HIV infection. Although FDA requires the use an approved assay to measure viral load in order to meet the demands for licensure, a low cost, moderate complexity, screening test will greatly facilitate these trials. Indeed all participants in both the placebo and vaccine arms must be screened periodically (e.g., at 3 to 6 month intervals). The assay we propose to develop will use our proprietary helicase dependent amplification (HDA) platform as well as a low cost device specifically designed to perform molecular tests without contaminating the laboratory with amplification products. HDA is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. It is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). We propose to use simulated clinical samples generated with the Armored RNA HIV Quant clone spiked into whole blood from HIV seronegative volunteers at known concentrations. Armored RNA will be isolated from plasma prior to RT-HDA testing. Lateral flow devices have already proven their utility in separating nucleic acid amplification products, and are widely used in several moderate complexity and CLIA-waived tests listed in the FDA device database. Detection of amplicons will be accomplished by using latex particles conjugated to antibodies that bind to the probes used to detect the HDA reaction products. The assay uses a sandwich format to detect haptens (biotin, FITC and Digoxigenin) incorporated into one of the HDA primers and into each of the detection probes. The lateral flow strip used for these assays has two capture zones, and thus allows for the detection of one analyte (HIV) as well as a competitive internal control. Assays that fail to give a band in the lateral flow device window are scored as invalid, while assays with a band in the control line alone are scored as true negative. Assays with bands at both the control and test line position are scored as positives at the threshold value (typically 50 copies of the analyte). Assays with a strong band in the test line but no control line are scored as strong positives (typically over 5000 copies of analyte). We will test the specificity and sensitivity of the HIV test with clinical specimens at Vanderbilt University. Kits will be supplied to Dr. Yi-Wei Tang (Vanderbilt University) so his laboratory can evaluate the assays. We may also supply kits to US Government labs that want to try the test. We will also contract with Assuragen to engineer a clone of Armored RNA for our competitive internal control. Finally, we will compare the performance of HDA and the Roche Amplicor HIV-1 DNA PCR (version 1.5) using a set of Dried Blood Spot (DBS) specimens from the CDC's Proficiency Testing (PT) Program. By the end of Phase I, we will have preliminary data for submission of a pre-IDE to the FDA to kick-off a clinical study to seek regulatory clearance for sale of the assay system for human diagnostics using the Roche Amplicor HIV-1 DNA PCR (version 1.5) assay as a predicate device.Narrative More than 90 % of the 40 million human immunodeficiency virus (HIV)-infected persons live in Third World countries (see www.unaids.org/bangkok2004/GAR2004_html/GAR2004_03_en.htm). An HIV vaccine is the lowest cost medical solution for containing the acquired immuno deficiency syndrome (AIDS) epidemic in developing countries. Ongoing Phase III HIV vaccine trials use protection from infection (or reduced viral load when infection still occurs) as an end point. However, in order to meet the demands for licensure the FDA requires us to use an approved assay to measure viral load. These trials require a simple, and low cost molecular diagnostic test that can detect HIV infection in immunized participants. Current evidence suggests that only the central memory pool of CD4 T cells is affected very early in infection. Since this represents less than 1% of the circulating CD4 T cells, existing low cost CD4 monitoring tests used in the third world cannot replace viral RNA as a surrogate for detecting infection. Moreover, rapid immunodiagnostic assays cannot distinguish between immunization and infection, and current molecular diagnostic assays are too complex, and expensive for most developing country laboratories. Although transporting samples, using dry blood spots (DBS), to laboratories that can perform complex molecular tests offers a solution to this problem, a simple, and low cost molecular diagnostic test is preferable from the standpoint of the patient, and medical ethics (i.e., the sooner an infection is detected the sooner the patient could receive treatment with antiviral drugs). Once a participant has been identified as infected, a blood sample can be collected and analyzed with a high complexity, FDA approved viral load assay. BioHelix's proprietary helicase dependent amplification (HDA) platform is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. HDA is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). Indeed, HDA can be performed in a simple $100 water bath. To illustrate the potential of this technology for the third world, we currently sell HDA reagents as part of a teaching kit for use in high schools (sold through Carolina Biological Supply). BioHelix is combining this HDA technology with a low cost (~$2-$3.75) disposable device specifically designed to perform molecular tests without contaminating the laboratory with amplicons. The device uses a lateral flow system to detect amplification products. Lateral flow devices are common in many moderately complex, and CLIA-waived tests listed on the Food and Drug Administration (FDA) device database. We believe this combination of characteristics makes our proposed product ideal for the HIV vaccine trial sites. In addition, recent evidence suggests that more than half of the newly acquired infections are caused by acutely infected individuals who are often seronegative. A low cost nucleic acid screening test will help identify those people and reduce the spread of the HIV epidemic as well as benefit participants in HIV vaccination trials who become infected with the virus.

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We propose to develop a rapid, low cost, threshold assay for the detection of HIV infection of HIV vaccination trial participants. The assay we propose to develop will use our proprietary helicase dependent amplification (HDA) platform as well as a low cost device specifically designed to perform molecular tests without contaminating the laboratory with amplification products. HDA is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. It is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). We propose to use a lateral flow device to detect amplification products. Such devices have already proven their utility in separating nucleic acid amplification products, and are widely used in several "moderate complexity" and CLIA-waived tests listed in the FDA device database. Detection of amplicons will be accomplished by using latex particles conjugated to antibodies that bind to the probes used to detect the HDA reaction products. The assay uses a sandwich format to detect haptens (biotin, FITC and Digoxigenin) incorporated into one of the HDA primers and into each of the detection probes. The lateral flow strip used for these assays has two capture zones, and thus allows for the detection of one analyte (HIV) as well as a competitive internal control. Assays that fail to give a band in the lateral flow device window are scored as invalid, while assays with a band in the control line alone are scored as true negative. Assays with bands at both the control and test line position are scored as positives at the threshold value (typically 200 copies of the analyte). Assays with a strong band in the test line but no control line are scored as strong positives (typically over 5000 copies of analyte). Preliminary studies suggest a limit of detection of 100 copies/mL plasma when nucleic acids extracted from plasma samples with a sequence-specific capture method are used as templates for RT-HDA. We propose to further test the specificity and sensitivity of the HIV test with clinical specimens obtained from 5 high-risk patient recruitment sites. Assays will be performed at BioHelix, and at Vanderbilt University. Kits will be supplied to Dr. Yi-Wei Tang (Vanderbilt University) so his laboratory can evaluate the assays. In addition, we propose to perform a validation study at the National Institute for Communicable Diseases (NICD) of South Africa to gain clearance to sell kits in South Africa. Finally, we may also contract with Assuragen to engineer a clone of Armored RNA for our competitive internal control. By the end of Phase II, we will have preliminary data for submission of an IDE to the FDA to kick-off a clinical study to seek pre market approval (PMA) for sale of the assay system for human diagnostics in the US.

Public Health Relevance:
More than 90 % of the 40 million human immunodeficiency virus (HIV)-infected persons live in Third World countries (see www.unaids.org/bangkok2004/GAR2004_html/GAR2004_03_en.htm). Unfortunately, performing HIV molecular tests in resource poor settings is difficult. BioHelix's proprietary helicase dependent amplification (HDA) platform is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. HDA is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). Indeed, HDA can be performed in a simple $100 water bath. To illustrate the potential of this technology for the third world, we currently sell HDA reagents as part of a teaching kit for use in high schools (sold through Carolina Biological Supply). BioHelix is combining this HDA technology with a low cost (~$2- $3.75) disposable device specifically designed to perform molecular tests without contaminating the laboratory with amplicons. The device uses a lateral flow system to detect amplification products. Lateral flow devices are common in many moderately complex, and CLIA-waived tests listed on the Food and Drug Administration (FDA) device database. We believe this combination of characteristics makes our proposed product ideal for the HIV vaccine trial sites. A low cost nucleic acid screening test will help identify those people and reduce the spread of the HIV epidemic as well as benefit participants in HIV vaccination trials who become infected with the virus. An estimated 16 to 22 million persons aged 18-64 years are tested annually for HIV in the United States (see http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5347a4.htm). Most of the near patient testing relies on rapid tests like OraQuick while most hospital screening programs use enzyme immuno assay (EIA). Walensky et al. (2008) recently reported that using western blot alone as a confirmation test for oral rapid tests provides conclusive HIV status in only 50% of patients at first follow-up. Such patients are typically asked to return for testing in ~ 1 month. In contrast, performing an HIV-1 RNA test to confirm the rapid test, when a western blot fails to do so, improves this rate to 96.2% in the first round of testing. Considering that our assay system relies of minimal instrumentation we feel our test can offer an additional layer of confirmation for OraQuick positives at the near patient setting and in the developing world and thus relieve the need for asking patients to return for testing in 1 month.

Public Health Relevance:
Narrative More than 90 % of the 40 million human immunodeficiency virus (HIV)-infected persons live in Third World countries (see www.unaids.org/bangkok2004/GAR2004_html/GAR2004_03_en.htm). Unfortunately, performing HIV molecular tests in resource poor settings is difficult. BioHelix's proprietary helicase dependent amplification (HDA) platform is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. HDA is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). Indeed, HDA can be performed in a simple $100 water bath. To illustrate the potential of this technology for the third world, we currently sell HDA reagents as part of a teaching kit for use in high schools (sold through Carolina Biological Supply). BioHelix is combining this HDA technology with a low cost (~$2- $3.75) disposable device specifically designed to perform molecular tests without contaminating the laboratory with amplicons. The device uses a lateral flow system to detect amplification products. Lateral flow devices are common in many moderately complex, and CLIA-waived tests listed on the Food and Drug Administration (FDA) device database. We believe this combination of characteristics makes our proposed product ideal for the HIV vaccine trial sites. A low cost nucleic acid screening test will help identify those people and reduce the spread of the HIV epidemic as well as benefit participants in HIV vaccination trials who become infected with the virus. An estimated 16 to 22 million persons aged 18-64 years are tested annually for HIV in the United States (see http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5347a4.htm). Most of the near patient testing relies on rapid tests like OraQuick while most hospital screening programs use enzyme immuno assay (EIA). Walensky et al. (2008) recently reported that using western blot alone as a confirmation test for oral rapid tests provides conclusive HIV status in only 50% of patients at first follow-up. Such patients are typically asked to return for testing in ~ 1 month. In contrast, performing an HIV-1 RNA test to confirm the rapid test, when a western blot fails to do so, improves this rate to 96.2% in the first round of testing. Considering that our assay system relies of minimal instrumentation we feel our test can offer an additional layer of confirmation for OraQuick positives at the near patient setting and in the developing world and thus relieve the need for asking patients to return for testing in 1 month.

Project Terms:
1H-Thieno(3,4-d)imidazole-4-pentanoic acid, hexahydro-2-oxo-, (3aS-(3aalpha,4beta,6aalpha))-; 5-Isothiocyanatofluorescein; AIDS Virus; AIDS test; AIDS/HIV test; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Active Follow-up; Age; Antibodies; Assay; Automation; Bathing; Baths; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological; Biological Assay; Biotin; Blood; Blood Plasma; Blotting, Western; Buffers; Card-20(22)-enolide, 3,12,14-trihydroxy-, (3beta,5beta,12beta)-; Characteristics; Clinical; Clinical Research; Clinical Study; Communicable Diseases; Complex; Contracting Opportunities; Contracts; DNA; DNA Helicases; DNA Unwinding Proteins; DNA unwinding enzyme; Data; Data Banks; Data Bases; Databank, Electronic; Databanks; Database, Electronic; Databases; Deoxynucleotide-triphosphate[{..}]DNA deoxynucleotidyltransferase (RNA-directed); Deoxyribonucleic Acid; Detection; Developing Countries; Developing Nations; Devices; Diagnostic; Digoxigenin; Drug Formulations; EC 2.7.7.49; Educational process of instructing; Engineering; Engineerings; Enzymes; Epidemic; Equilibrium; Ethics Committees, Research; FDA; FITC; Fluorescein-5-isothiocyanate; Food and Drug Administration; Food and Drug Administration (U.S.); Formulation; Formulations, Drug; Gene Products, RNA; HIV; HIV test; HIV vaccine; HIV-1; HIV-I; HIV/AIDS Vaccines; HIV1; HOSP; HTLV-III; Haptens; History; Hospitals; Hour; Human; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human immunodeficiency virus 1; Human immunodeficiency virus test; Human, General; Hydrogen Oxide; IRBs; Immunodeficiency Virus Type 1, Human; Incubated; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases and Manifestations; Infectious Disorder; Institutes; Institutional Review Boards; Instrumentation, Other; Journals; LAV-HTLV-III; Laboratories; Lanadigenin; Lateral; Latex; Latex Particles; Less-Developed Countries; Less-Developed Nations; Life; Lymphadenopathy-Associated Virus; Magazine; Man (Taxonomy); Man, Modern; Marketing; Methods; Microbeads; Microspheres; Molecular; Molecular Interaction; Multi-Institutional Clinical Trial; Multi-center clinical study; Multi-center clinical trial; Multi-site clinical study; Multi-site clinical trial; Nucleic Acids; Oral; PCR; Paper; Participant; Patient Recruitments; Patients; Performance; Persons; Phase; Plasma; Polymerase Chain Reaction; Position; Positioning Attribute; Process; Programs (PT); Programs [Publication Type]; Publications; RNA; RNA Transcriptase; RNA, Non-Polyadenylated; RNA, Viral; RNA-Dependent DNA Polymerase; RNA-Directed DNA Polymerase; Reaction; Reagent; Recording of previous events; Reporting; Republic of South Africa; Research Ethics Committees; Research Resources; Research Specimen; Resources; Reticuloendothelial System, Blood; Reticuloendothelial System, Serum, Plasma; Reverse Transcriptase; Revertase; Ribonucleic Acid; Sales; Sampling; Scientific Publication; Screening procedure; Sensitivity and Specificity; Serum, Plasma; Site; Solutions; South Africa; Specimen; Spottings; Strepavidin; Streptavidin; System; System, LOINC Axis 4; Teaching; Technology; Testing; Third-World Countries; Third-World Nations; Time; Tube; USFDA; Under-Developed Countries; Under-Developed Nations; Union of South Africa; United States; United States Food and Drug Administration; Universities; Vaccination; Viral Diseases; Virus; Virus Diseases; Virus-HIV; Viruses, General; Vitamin H; Walking; Water; Western Blotting; Western Blottings; Western Immunoblotting; aged; balance; balance function; base; clinical data repository; clinical data warehouse; coenzyme R; cost; data repository; design; designing; follow-up; helicase; high risk; high school; human T cell leukemia virus III; human T lymphotropic virus III; human immunodeficiency virus vaccine; improved; instrument; instrumentation; internal control; manufacturing facility; migration; multi center clinical study; multi center clinical trial; multi site clinical study; multi site clinical trial; prevent; preventing; programs; protein blotting; public health relevance; relational database; screening; screenings; validation studies; viral RNA; viral infection; virus RNA; virus infection