Improving Safety of the Blood Supply from Transmission of HIV/AIDS and Other Emerging Blood Borne Viral and Biodefense Agents by Developing Sensitive Diagnostic Tools and Investigating Disease Pathogenesis
Principal Investigator: Indira Hewlett, PhD
Office / Division / Lab: OBRR / DETTD / LMV
HIV diagnostics and pathogenesis
HIV/AIDS is a global public health concern, with 33 million infections worldwide and several million deaths a year.
The virus is very diverse and new strains continue to emerge and spread rapidly worldwide. This poses challenges to diagnosing infected individuals and developing new vaccines and therapies. Therefore, it is imperative that FDA determine if newly developed tests for screening the blood supply for HIV accurately detect all the existing and emerging strains of the virus in blood samples. Our ongoing studies in Africa will help FDA in its global collaborations aimed at ensuring there are accurate HIV diagnostic tests and safe and effective vaccines available to respond to the changing population of AIDS viruses.
Our laboratory studies the factors that cause variations in the response of humans to HIV diversity. These studies will provide new insights that will support development of improved diagnosis, disease monitoring, drug design, and vaccines. We also helped to produce a new FDA document that offers guidance to blood donor facilities in identifying donors at increased risk for being infected with the variety of HIV-1 called "group O." This will help to screen out donors who might be infected based on having spent time in certain parts of Africa or having received blood transfusions or engaged in sexual activity with residents or former residents in those areas.
Novel detection technologies
Although testing for HIV has reduced the risk of transmitting the virus through donated blood, there are still three challenges to keeping the blood supply safe: 1) existing tests are performed only on plasma, while many agents are found mostly in blood cells; 2) testing requires large volumes and labor intensive instrumentation; and 3) existing tests cannot readily be modified to detect simultaneously the increasing number of infectious agents that pose new threats to the blood supply.
The goals of our work are to 1) develop new, improved diagnostic tools for testing blood and plasma and rapidly detecting emerging pathogens that threaten public health; and 2) provide FDA product reviewers with improved scientific understanding of novel technologies that might be applied by manufacturers, enhancing the review and evaluation of new diagnostic tests for blood-borne pathogens and bioterrorism agents.
Pandemic influenza and blood safety
Each year the FDA helps industry and international health agencies choose which influenza viruses will be represented by that year's "flu shot." FDA research also contributes to tests that determine the safety and potency of the vaccine. In addition to seasonal influenza there are other types of influenza of concern to FDA and other public health agencies.
H5N1 strains of avian influenza (bird flu) infect humans with a mortality rate close to 50%, much higher than the rate for seasonal influenza. Researchers have detected H5N1 in the blood of severely ill persons using very sensitive methods that can identify the genetic material of these pathogens. In 2009 another influenza virus called "swine flu" or H1N1, caused several hundred deaths in the US and spread quickly throughout the world.
Our group is developing assays as part of influenza pandemic preparedness. In collaboration with the FDA's Center for Devices and Radiological Health we are studying how the disease progresses, and how that progression is linked to the level of influenza viruses in the blood or nasal secretions. The major goal of this study is to determine whether influenza virus is present in blood in the asymptomatic phase, that is, when the infected individual is not showing evidence of disease. These data would help regulatory and public health officials to develop a blood screening policy that addresses donor deferral and product shortage issues during a pandemic if the virus is detected in the the blood of potential donors during the pre-symptomatic phase. In addition we are studying the correlation of the levels of flu virus genetic material with the ability of the virus to infect humans using animals as models of this infection. The laboratory also contributed significantly to developing a document that provides industry with guidance and recommendations for the assessment of donor suitability and blood product safety with regard to pandemic influenza
XMRV and blood safety
In 2006, the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) was identified and reported to be associated with certain cases of prostate cancer and, more recently, scientists have shown an association with chronic fatigue syndrome (CFS). XMRV has also been found in about 4% of healthy individuals.
The results of various studies suggest that people infected with XMRV carry the virus in their blood stream. This raises public health concerns that such individuals could spread the virus through blood donations. Therefore, our laboratory is developing sensitive ways to test for XMRV in blood and to test the accuracy of assays used by different laboratories to diagnose infection with XMRV. This work will help to prepare blood collection centers for testing blood for this virus in the event that it becomes necessary in order to ensure the safety of the blood supply.
These tools will also help public health officials and researchers to collect data on the prevalence and distribution of XMRV and to study infection and transmission of the virus.
Therefore, we plan to develop in-house tests to detect the virus, the immune response to the virus, and its genetic material. Our laboratory also hopes to study the prevalence of XMRV in other parts of the world, such as Cameroon in African, where similar types of viral infections are known to be prevalent. We also plan to investigate the ability of the virus to infect hematopoietic cells, which are the parent cells that give rise to the various blood cells.
We obtained plasma and viruses representing major new strains from Cameroon, where all diverse HIV strains are prevalent and new strains continue to emerge. Our laboratory uses these samples to evaluate new antibody/antigen and nucleic acid tests (NAT) designed to detect and quantify these strains. We use gene sequencing to identify strains, tropism, and drug resistance. Correlation of sequencing data with cell-based phenotyping for tropism will provide FDA with valuable experience with this emerging class of products. The laboratory now plans to use new, high throughput, ultra-deep sequencing techniques to characterize virus genetic material.
Although most samples were detected by FDA licensed assays, NAT assays only inconsistently detected some CRF02_AG and CRF01_AE specimens. Therefore, we tested these strains in a collaborative study. New reference panels of these viruses were prepared to help FDA evaluate licensed and new NAT assays to detect them.
Our laboratory is also using in vitro cell culture systems and molecular genomics to study the impact of strain diversity, sex hormone effects, host genetics and cell signaling pathways on viral pathogenesis.
Novel detection technologies
We use nanoparticles in ELISA rapid or microarray formats as proof-of-concept strategies to improve assays. Gold nanoparticles coupled with silver enhanced detection of HIV-1 p24 antigen by approximately 100-150-fold compared to the conventional ELISA format. Fluorescent europium (Eu+) nanoparticles (NP) reduced assay time while achieving similar levels of sensitivity. Europium nanoparticles also enhanced detection of anthrax 50-100 fold, allowing earlier detection of the toxin than was previously possible. We showed that nanoparticles offer remarkable improvements in the sensitivity of protein-based assays for pathogens and might provide improved diagnostic tools for pathogens in the future.
Our laboratory is also evaluating non-PCR nanoparticle-based genomic microarray NAT techniques that achieve PCR-like detection limits without using enzymes or PCR amplification. West Nile Virus (WNV) RNA detection was comparable to PCR using these arrays. We detected Ebola, Marburg and Lassa viruses with a high degree of sensitivity using this approach and successfully used it for multiplexed detection and genotyping of major influenza viruses, including H1N1, H3N2 and H5N1. The signals in this assay could be visually read by enhancement with silver staining allowing adaptation to rapid detection formats. Our work clearly demonstrates the potential for nanotechnology to provide new, rapid and sensitive diagnostics for pathogens.
Pandemic influenza and blood safety
We determined the presence of virus in the blood of ferrets and mice infected with different doses of H5N1 virus, collecting blood, lungs, nasal washes, nasal turbinates and brain at different time points. Sensitive taqMan and virus culture assays are being used to measure virus in blood and the various tissues. We are planning to inoculate H5N1 and 2009 H1N1 intravenously into ferrets and mice to determine transmissibility and pathogenesis of virus if transmitted through the intravenous route, simulating transfusion.
Our preliminary findings indicate that viremia in H5N1 infection can be detected at the onset of symptoms and is generally associated with fatal outcomes in ferrets. Similar studies are underway with H1N1 infection. In a separate study we tested plasma from 300 blood donors using sensitive Taqman assays for H1N1 and have so far found no positive samples in this sample set. These findings are important in our understanding of influenza viremia and its detectability in blood donors.
XMRV and blood safety
In 2006, the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) was identified and reported to be associated with certain cases of prostate cancer and, more recently, with chronic fatigue syndrome (CFS). XMRV has also been found in about 4% of healthy individuals.
Studies have shown that XMRV infects T and B cells, and transient viremia has been detected in animals after infection with XMRV. In addition, virus from plasma was able to infect susceptible cells, suggesting that infectious virus is present in the blood stream. This raises public health concerns about the safety of the blood supply. It is therefore critical to develop sensitive test methods and well-characterized reference reagents to standardize assays from different labs and ensure the accuracy of diagnostic findings. The reference panels will be used for lot release testing of kits in the event testing of the blood supply becomes necessary. These tools will also be needed to perform studies of prevalence, distribution, virus infectivity, and transmission, to clarify and corroborate the findings of XMRV that have been reported so far. Current data on prevalence in the general population is primarily derived from a restricted geographic locations in the US; additional studies are needed to examine prevalence on other populations.
Patient-derived XMRV has been shown to be infectious, and both cell-associated and cell-free transmission of the virus is possible, and a study in rhesus macaques indicated a viremic phase. These findings point to a new transfusion threat that needs further investigation.
Therefore, we proposed to 1) develop in-house Taqman, antigen and antibody assays to detect the virus and its immune responses; 2) develop reference panels for detection of nucleic acid, and antibody, 3) study prevalence in other settings including Cameroon in Africa where other retroviral infections are known to be prevalent; and 4) investigate infectivity and tropism of the virus for hematopoietic cells in cell culture systems and appropriate animal models.
We have developed sensitive Taqman assays based on primers from the gag region to make it specific for XMRV. In addition, we are obtaining infectious clones, virus isolates, recombinant proteins, and antibodies for further characterization in a collaborative study based on sequence analysis and evaluation of reactivity using tests developed at CBER and other laboratories in a collaborative study.
For the nucleic acid test (NAT) panel, we provided laboratories with virus preparations diluted in plasma for testing. Results of all labs will be evaluated statistically to obtain a consensus value. For antibody panels, reactivity of candidate materials will be tested in multiple laboratories to determine the suitability of the material for use as a reference reagent. The assays will be used to test samples previously obtained from donors in Cameroon for the presence of XMRV sequences by NAT or antibodies by ELISA. Various T and B cell lines, monocytes, and PBMCs will be infected with different concentrations of the virus to evaluate infectivity. Transmissibility of cell-free and cell-associated virus between lymphoid cells and indicator cells will be studied. Rhesus macaque studies will be performed to study the natural history of the virus and secondary transmission through transfusion. Samples collected at various time points after infection can also serve as useful reagents to assess the sensitivity of assays and as future reference reagents.
The proposed studies are expected to provide the following outcomes: 1) optimizing in-house assays for studies of XMRV prevalence, transmission, and pathogenesis; 2) developing reference reagents for standardization of XMRV assays based on NAT or antibody and animal derived seroconversion panels for sensitivity evaluation; 3) identifying seroprevalence in persons from different geographic regions; 4) gaining new knowledge of cell tropism, transmissibility, and infectivity for blood cells using in vitro cell culture systems; and 5) evaluating infectivity, transmissibility, and pathogenesis of the virus using susceptible animal models.
- Mol Cell Biochem 2019 Dec;462(1-2):41-50
The effects of MAPK p38alpha on AZT resistance against reactivating HIV-1 replication in ACH2 cells.
Wang X, Zhao J, Ragupathy V, Hewlett I
- BMC Res Notes 2019 Nov 15;12(1):745
Exploring the immunomodulatory role of depot medroxyprogesterones acetate and endogenous progesterone levels in HIV infected and uninfected women.
Mnqonywa N, Abbai N, Ragupathy V, Ramjee G, Hewlett I, Moodley D
- EBioMedicine 2019 May;43:307-16
Development and validation of plasma miRNA biomarker signature panel for the detection of early HIV-1 infection.
Biswas S, Haleyurgirisetty M, Lee S, Hewlett I, Devadas K
- AIDS Res Hum Retroviruses 2019 Apr;35(4):396-401
Cross-subtype detection of HIV-1 capsid p24 antigen using a sensitive europium nanoparticle assay.
Haleyur Giri Setty MK, Kurdekar A, Mahtani P, Liu J, Hewlett I
- Sci Adv 2018 Nov 21;4(11):eaar6280
Streptavidin-conjugated gold nanoclusters as ultrasensitive fluorescent sensors for early diagnosis of HIV infection.
Kurdekar AD, Avinash Chunduri LA, Manohar CS, Haleyurgirisetty MK, Hewlett IK, Venkataramaniah K
- Wiley Interdiscip Rev Nanomed Nanobiotechnol 2018 Sep-Oct;10(5):e1512
Application of nanotechnology in biosensors for enhancing pathogen detection.
Sposito AJ, Kurdekar A, Zhao J, Hewlett I
- J Clin Microbiol 2018 Jul 26;56(8):e02045-17
Comparison of detection limits of 4th generation combination HIV antigen/antibody, p24 antigen and viral load assays on diverse HIV isolates.
Stone M, Bainbridge J, Sanchez AM, Keating SM, Pappas A, Rountree W, Todd C, Bakkour S, Manak M, Peel SA, Coombs RW, Ramos EM, Shriver MK, Contestable P, Nair SV, Wilson DH, Stengelin M, Murphy G, Hewlett I, Denny TN, Busch MP, EQAPOL Program
- J Biol Chem 2018 Jul 27;293(30):11687-708
PTAP motif duplication in the p6 Gag protein confers a replication advantage on HIV-1 subtype C.
Sharma S, Arunachalam PS, Menon M, Ragupathy V, Satya RV, Jebaraj J, Ganeshappa Aralaguppe S, Rao C, Pal S, Saravanan S, Murugavel KG, Balakrishnan P, Solomon S, Hewlett I, Ranga U
- PLoS One 2018 Apr 17;13(4):e0195661
Distinctive variation in the U3R region of the 5' long terminal repeat from diverse HIV-1 strains.
Mbondji-Wonje C, Dong M, Wang X, Zhao J, Ragupathy V, Sanchez AM, Denny TN, Hewlett I
- Sci Rep 2018 Feb 7;8(1):2546
Differentially expressed host long intergenic noncoding RNA and mRNA in HIV-1 and HIV-2 infection.
Biswas S, Haleyurgirisetty M, Ragupathy V, Wang X, Lee S, Hewlett I, Devadas K
- PLoS One 2018 Jan 26;13(1):e0191916
Modulation of HIV replication in monocyte derived macrophages (MDM) by steroid hormones.
Devadas K, Biswas S, Ragupathy V, Lee S, Dayton A, Hewlett I
- Sci Rep 2017 Aug 2;7(1):7149
Femtogram level sensitivity achieved by surface engineered silica nanoparticles in the early detection of HIV infection.
Chunduri LAA, Kurdekar A, Haleyurgirisetty MK, Bulagonda EP, Kamisetti V, Hewlett IK
- J Cell Physiol 2017 Jul;232(7):1746-53
Differences in activation of HIV-1 replication by superinfection with HIV-1 and HIV-2 in U1 cells.
Wang X, Sun B, Mbondji C, Biswas S, Zhao J, Hewlett I
- RSC Adv 2017;7(32):19863-77
Fluorescent silver nanoparticle based highly sensitive immunoassay for early detection of HIV infection.
Kurdekar AD, Chunduri LAA, Chelli SM, Haleyurgirisetty MK, Bulagonda EP, Zheng JW, Hewlett IK, Kamisetti V
- Microfluid Nanofluidics 2016 Dec;20(12):167
Development of carbon dot based microplate and microfluidic chip immunoassay for rapid and sensitive detection of HIV-1 p24 antigen.
Chunduri LAA, Haleyurgirisetty MK, Patnaik S, Bulagonda PE, Kurdekar A, Liu J, Hewlett IK, Kamisetti V
- Biosens Bioelectron 2016 Dec 15;86:150-5
Sensitive detection of influenza viruses with Europium nanoparticles on an epoxy silica sol-gel functionalized polycarbonate-polydimethylsiloxane hybrid microchip.
Liu J, Zhao J, Petrochenko P, Zheng J, Hewlett I
- J Mol Endocrinol 2016 Oct;57(3):185-99
Progesterone augments cell susceptibility to HIV-1 and HIV-1/HSV-2 co-infections.
Ragupathy V, Xue W, Tan J, Devadas K, Gao Y, Hewlett I
- PLoS One 2016 Sep 22;11(9):e0163175
Sensitive detection and simultaneous discrimination of influenza A and B viruses in nasopharyngeal swabs in a single assay using next-generation sequencing-based diagnostics.
Zhao J, Liu J, Vemula SV, Lin C, Tan J, Ragupathy V, Wang X, Mbondji-Wonje C, Ye Z, Landry ML, Hewlett I
- Microfluid Nanofluidics 2016 Jul;20(7):99
Comparative performance evaluation of carbon dot-based paper immunoassay on Whatman filter paper and nitrocellulose paper in the detection of HIV infection.
Kurdekar A, Chunduri LAA, Bulagonda EP, Haleyurgirisetty MK, Kamisetti V, Hewlett IK
- AIDS Res Hum Retroviruses 2016 Jun;32(6):612-9
Novel time-resolved fluorescence europium nanoparticle immunoassay for detection of Human Immunodeficiency Virus-1 group O viruses using microplate and nicrochip platforms.
Haleyur Giri Setty MK, Liu J, Mahtani P, Zhang P, Du B, Ragupathy V, Devadas K, Hewlett IK
- PLoS One 2016 Jun 17;11(6):e0157340
Genetic characterization of a panel of diverse HIV-1 isolates at seven international sites.
Hora B, Keating SM, Chen Y, Sanchez AM, Sabino E, Hunt G, Ledwaba J, Hackett J Jr, Swanson P, Hewlett I, Ragupathy V, Vikram Vemula S, Zeng P, Tee KK, Chow WZ, Ji H, Sandstrom P, Denny TN, Busch MP, Gao F
- J Med Virol 2016 Jun;88(6):1092-7
High sensitivity detection of HIV-1 using two genomic targets compared with single target PCR.
Shah K, Ragupathy V, Saga A, Hewlett I
- Viruses 2016 May 2;8(5):121
Identification of host micro RNAs that differentiate HIV-1 and HIV-2 infection using genome expression profiling techniques.
Devadas K, Biswas S, Haleyurgirisetty M, Ragupathy V, Wang X, Lee S, Hewlett I
- Biochem Biophys Res Commun 2016 May 13;473(4):926-30
IL-1beta and IL-18 Inhibition of HIV-1 Replication in Jurkat Cells and PBMCs.
Wang X, Mbondji-Wonje C, Zhao J, Hewlett I
- Viruses 2016 Apr 12;8(4):96
Current approaches for diagnosis of influenza virus infections in humans.
Vemula SV, Zhao J, Liu J, Wang X, Biswas S, Hewlett I
- AIDS Res Hum Retroviruses 2016 Apr;32(4):381-5
Monitoring HIV-1 group M subtypes in Yaounde, Cameroon reveals broad genetic diversity and a novel CRF02_AG/F2 infection.
Courtney CR, Agyingi L, Fokou A, Christie S, Asaah B, Meli J, Ngai J, Hewlett I, Nyambi PN
- Vaccine 2016 Apr 12;34(17):2035-43
Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing.
Mee ET, Preston MD, CS533 Study Participants, Minor PD, Schepelmann S
- Viruses 2016 Feb 2;8(2):33
Pandemic influenza A (H1N1) virus infection increases apoptosis and HIV-1 replication in HIV-1 infected Jurkat cells.
Wang X, Tan J, Biswas S, Zhao J, Devadas K, Ye Z, Hewlett I
- PLoS One 2016 Jan 28;11(1):e0147421
Analysis of host gene expression profile in HIV-1 and HIV-2 infected T-cells.
Devadas K, Biswas S, Haleyurgirisetty M, Wood O, Ragupathy V, Lee S, Hewlett I
- Virus Res 2015 Dec 2;210:337-43
Identification and characterization of a highly pathogenic H5N1 avian influenza A virus during an outbreak in vaccinated chickens in Egypt.
Amen O, Vemula SV, Zhao J, Ibrahim R, Hussein A, Hewlett IK, Moussa S, Mittal SK
- Sensors 2015 Jun 24;15(7):14864-70
Non-invasive optical sensor based approaches for monitoring virus culture to minimize BSL3 laboratory entry.
Ragupathy V, Setty MK, Kostov Y, Ge X, Uplekar S, Hewlett I, Rao G
- Emerg Infect Dis 2015 Mar;21(3):400-8
Nanomicroarray and multiplex next-generation sequencing for simultaneous identification and characterization of influenza viruses.
Zhao J, Ragupathy V, Liu J, Wang X, Vemula SV, El Mubarak HS, Ye Z, Landry ML, Hewlett I
- Viruses 2015 Feb 5;7(2):543-58
HIV-1 induced nuclear factor I-B (NF-IB) expression negatively regulates HIV-1 replication through interaction with the long terminal repeat region.
Vemula SV, Veerasamy R, Ragupathy V, Biswas S, Devadas K, Hewlett I