Undergraduate
Kansas University, B.A., 1977

Medical School
Kansas University Medical School, 1981

Residency
University of California, Los Angeles, 1981-84

Fellowship
University of California, Los Angeles, 1984-87

1. HIV-1 reservoirs, evolution and selection of drug-resistant virus during effective antiretroviral therapy (ART)

HIV-1 reservoirs: HIV-1 persists even when ART suppresses viral replication for extended periods of time below the limits of detection in blood plasma. Viral persistence is thought to be due to reservoirs of latent virus and/or incompletely suppressed viral replication. In an effort to identify tissue reservoirs, HIV-1 env sequences were examined throughout infection and at autopsy in two subjects. Phylogenetic analysis of virus revealed that blood-borne virus underwent time-ordered evolution. Virus in lymph node, liver and bone marrow collected at autopsy were contemporaneous with blood. In contrast, lung contained genetically diverse viruses grouping with virus in the blood as much as 6 years earlier. Lung viral sequences were also widely dispersed in the phylogenetic trees. The lung tissues of these subjects thus appeared to harbor a persistent viral reservoir during potent antiretroviral therapy. This increased viral diversity and the observation that antiretroviral levels are low in macrophages led us to hypothesize that pulmonary macrophages are a principal long-term reservoir of drug-resistant virus. Studies are underway to track drug-resistance mutations and phylogenetic lineages to define compartmentalized tissues and long-lived reservoirs of HIV infection. We are comparing viral sequences from sequential specimens of blood plasma and peripheral blood mononuclear cells (PBMC) collected from perinatally infected children during mono- and dual-therapy to sequences in blood and pulmonary cells in induced sputa during ART.

HIV-1 replication during effective ART: ART provides long-term effective suppression of HIV-1 replication in 50-80% of people. Multiple factors can contribute to ART failing to suppress viral replication in the remaining 20-50%. We seek to better understand the low-level HIV-1 replication and selection of drug-resistant viruses that potentially occurs among these individuals during apparently effective ART (plasma HIV-1 RNA levels <50c/mL) prior to the time of obvious ART failure. Our projects examine whether low-level replication occurs in specific cell types or tissues that may have lower levels of antiretrovirals. Our studies focus on HIV-1 in various blood cells and in other tissues (e.g. lung and genital tract) before and during effective ART. Phylogenetic relationships, distances from the most recent common ancestor (MRCA) of the viral population and changes in the frequency of drug-resistance mutants are used to evaluate ongoing low-levels of viral replication and selection of drug-resistant virus.

Individuals have been studied during 'standard' HAART, including two nucleoside analogs and a protease or non-nucleoside reverse transcriptase inhibitor, and during 'potent' ART, with four or five drugs from three classes of antiretrovirals. During ART full cycles of viral replication were detected in many children, despite a median plasma HIV-1 RNA <50 copies/mL. Among individuals without evidence of viral replication the HIV-1 DNA level dropped significantly during ART to a low absolute level. These findings suggested that HIV-1 DNA load might be useful in monitoring residual low-level viral replication during ART, which is currently under investigation.

HIV-1 “blips”: Low-level viremias (LLV) (plasma HIV-1 RNA 50–400 c/mL) or “blips” occur episodically during highly active antiretroviral therapy (ART). Several studies have associated LLV with virologic failure of ART, while in other studies LLV did not appear to affect the clinical outcome of ART. To understand the processes leading to LLV and risk-factors predictive of ART-failure, we conducted genetic analyses of LLV to determine whether plasma virions emanated from archived or from newly evolved viral genomes. In our studies LLV sequences most frequently grouped with archived virus from early in the course of infection. These early viral forms were often detected as multiple identical viral sequences, suggesting clonally expanded cells produced the virus. Less frequently, LLV virus evolved over time and included the selection of drug-resistant mutants, indicating viral replication. These studies demonstrated that plasma viremias during effective ART appeared to originate from two distinct processes: (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These two processes would be expected to eventually result in different outcomes, and provide a plausible explanation for the divergent clinical outcomes previously associated with LLV. Current studies aim to further characterize replication and selection of drug-resistance among individuals participating in various antiretroviral treatment regimens prior to ART failure.


2. HIV-1 evolution and compartmentalization in the female genital tract

We hypothesize that the greatest reservoir of HIV-1 in women infected during sex is within the genital tract. Viral genetic diversity is predicted to be greatest in the genital tract virus and would likely contribute to evolution of drug-resistant virus. Local inflammation due to infections could also enhance the conditions for viral replication in the genital tract and increase the likelihood of that drug-resistant virus is selected. Furthermore, antiretrovirals may not achieve therapeutic levels in the genital tract, which would also favor viral replication and selection of drug-resistant viral variants. We hypothesize that HIV-1 RNA shedding will occur intermittently from the female genital mucosa, similar to viral RNA 'blips' in the plasma. Evolution of drug resistance mutations will be detectable in genital tract virus of women with subsequent failure of ART. Whereas, the genital and plasma HIV-1 RNA detected in 'blips' among women who continue to exhibit suppression of replication will be 'wild type'.

In this project we are characterizing the frequency, quantity and genotype of HIV-1 RNA shed from the genital tract of women before and during effective HAART. Using specimens from women with undetectable plasma virus (<50 copies/mL), we are characterizing the HIV-1 pol and env genomes in the genital RNA and in provirus from cytobrush and biopsies of the uterine cervix. In addition, we are evaluating whether mutations associated with drug resistance are detected first in the blood or genital tract and the timing of interchange between these viruses.


3. Prevention of mother-to-child-transmission of HIV-1 infection

Etiology of mastitis: Breastfeeding accounts for nearly half of mother-to-child transmission of HIV-1 (MTCT). Safe and affordable alternatives to breastfeeding are not available for the majority of infants born to HIV-1 infected women. Numerous studies support the role of mastitis in increasing breastfeeding transmission, likely from the increase in breast milk HIV-1 load associated with inflammation of the lactating breast. Clinical mastitis, characterized by pain, redness and swelling, occurs in up to one-third of lactating women. Subclinical mastitis, without symptoms, but with an increased breast milk sodium ([Na+]), is even more prevalent and has also been associated with increases breast milk HIV-1 RNA and MTCT. There are no published studies elucidating the etiologies of subclinical mastitis, nor mastitis in HIV-1 infected women. Veterinary studies of mastitis implicate a broad range of bacteria, mycobacteria, fungi, mycoplasmas and herpes group viruses. We hypothesize that mastitis, especially subclinical mastitis, in HIV-1 infected women is also caused by a variety of organisms. In addition, that mastitis increases milk HIV-1 load within the breast, independent of the blood, presumably by immune stimulation of latently infected cells. By identifying the infectious agents associated with high levels of HIV-1 in breast milk, treatment and prophylaxis strategies can be developed that may reduce HIV-1 transmission to breastfeeding infants. Studies are in progress to examine associations between breast milk virus, cells and [Na+] and blood virus; and, to determine the infectious etiologies of mastitis in HIV-1 infected women.

Evaluation of HIV-1 specific immunization of infants by breastfeeding from infected mothers during nevirapine chemoprophylaxis: Considerable data indicate that HIV-1 specific humoral immunity can protect from infection and that cellular immunity modulates HIV-1-associated immune depletion. We hypothesize that when HIV-1 infection of infants is prevented by administration of nevirapine to breastfeeding infants, sufficient exposure to virus will result in HIV-1 specific immunization. We are evaluating whether HIV-1 specific primed-T cell and IgA responses in infants can be induced by repeated exposure to virus in maternal breast milk. If currently available drug regimens provide simultaneous prophylaxis of breastfeeding infants from HIV-1 infection and allow induction of HIV-1-specific immune responses, this would provide a rationale for studies to evaluate the protective effect of these immune responses after the prophylaxis is discontinued. If protective immunity can be induced during a short period of chemoprophylaxis while breastfeeding, this would provide the basis for a novel strategy for the immunization of infants in countries where breastfeeding remains their primary source of nutrition, and of HIV-1 infection.


4. Defining reservoirs of nevirapine-resistant HIV-1 in women and infants selected by peripartum nevirapine chemoprophylaxis regimens

Nevirapine chemoprophylaxis is moderately effective in reducing mother-to-infant HIV-1 transmission at a low cost. Nevirapine, however, rapidly selects for drug resistant HIV-1 because only a single base mutation, (i.e. K103N, Y181C and G190A), confers high-level resistance. Drug-resistant mutants, once selected, appear to persist in reservoirs. When antiretrovirals with cross-resistance are prescribed, these mutants proliferate rapidly and can foreshorten the benefits of ART even when started months after single-dose nevirapine (note: first-line ART in most countries includes nevirapine).

Studies are underway to provide insight into the establishment, modification and persistence of drug-resistant viral reservoirs as related to therapies administered to reduce mother-to-child-transmission of HIV-1. This information is critical to the development of global programs that synergize strategies for the reduction of mother-to-child-transmission of HIV-1 with the treatment of HIV-1 disease in adult women.

We hypothesize that recently selected drug-resistant virus is highly represented in short-lived lymphocytes. That without prolonged selection few mutants will enter long-lived reservoirs, and that most mutants in short-lived cells will decay to clinically insignificant levels. To evaluate these hypotheses we are quantifying the selection of NVP-resistant viruses in women exposed to “single-dose” NVP, the persistence of these mutants and whether the frequency of mutants at the initiation of ART is predictive of the efficacy of HAART in Thai and Mozambican women.



5. Development of a rapid and inexpensive test for drug-resistant HIV-1 suitable for use in resource poor settings

A sensitive, specific and high-throughput oligonucleotide ligation assay (OLA) for detection of genotypic HIV-1 resistance to nucleoside, non-nucleoside reverse transcriptase inhibitors and protease inhibitors has been developed and evaluated. This ligation-based assay uses differentially modified oligonucleotides specific for wild-type and mutant sequences. The assay allows sensitive and simple detection of both genotypes in a single well of a microtiter plate. The OLA detects genetic subpopulations more often than consensus sequencing. Reproducible and semiquantitative detection of the mutant and the wild-type genomes by the OLA was observed by analysis of wild-type and mutant plasmids mixtures containing as little as 5% of a mutant genotype in a background of wild-type genome. This rapid, simple, economical and highly sensitive assay provides a practical alternative to dideoxy-sequencing for genotypic evaluation of HIV-1 resistance to antiretrovirals. Currently work is ongoing to optimize the assay for testing of non-B subtypes and transfer technology to Peru and Mozambique.


6. Determining the prevalence of HIV-1 drug resistance in recently diagnosed individuals in Seattle, WA by dideoxynucleotide sequencing and an oligonucleotide ligation assay

Specimens from persons newly diagnosed with HIV-1 infection are evaluated for evidence of drug-resistance. Detection of mutant virus by two assays, consensus dideoxy-sequencing and the oligoligase assay, are ongoing. This study will evaluate whether transmitted drug-resistance mutations tend to 'regress', as commonly observed with mutations selected by ART when the selective pressure provided by drugs are removed. Whether drug-resistant variants are transmitted and persist at levels below the usual limit of detection is relevant to optimizing the treatment of HIV-1 infected individuals.


7. Clinical trials for HIV-1 and other viral infections in children, adolescents and pregnant women

We participate in NIH-funded multi-centered trials to prevent and treat HIV-1 infections (the Pediatric AIDS Clinical Trials Group (PACTG)/International Maternal Pediatric Adolescent AIDS Trials (IMPAACT) Networks) and to treat other viral infections in children (Collaborative Antiviral Study Group (CASG)). We have made major contributions to these networks through enrollment of subjects into trials and participation in protocol development, committee work and in contributions to the scientific agenda. We continue to have an active role in these networks and offer individuals living in the NW access to these protocols.

Rapid Economical Assays:

Beck and Frenkel. Manual for Genotyping Kits for the Detection of HIV-1 pol Drug-Resistance Mutations by an Oligonucleotide Ligation Assay. Protocol Version 1.4, July 2007.

Edelstein RE et al. An oligonucleotide ligation assay (OLA) detecting mutations in the HIV-1 pol gene associated with resistance to zidovudine, didanosine and lamivudine. J Clin Microbiol 36:569-72,1998.

Beck IA, Mahalanabis M, Pepper G, Wright A, Hamilton S, Langston E, Frenkel LM. 2002. A rapid and sensitive oligonucleotide ligation assay (OLA) for the detection of mutations in human immunodeficiency virus type 1 associated with high-level resistance to protease inhibitors. J Clin Microbiol 40: 1413-9, 2002.

Ellis GM, Mahalanabis M, Beck IA, Pepper G, Wright A, Hamilton S, Holte S, Naugler WE, Pawluk DM, Li CC, Frenkel LM. Comparison of Oligonucleotide Ligation Assay (OLA) and Consensus Sequencing for Detection of Drug-Resistant Mutants of Human Immunodeficiency Virus Type 1 in Peripheral Blood Mononuclear Cells and Plasma. J Clin Microbiol 42:3670-4, 2004.

Melvin AJ, Alarcon J, Velasquez C, Rodriguez C, Piscoya J, Giraldo A, Dinh P, Frenkel LM. Rapid HIV-1 Testing of Women Presenting in Late Pregnancy with Unknown HIV Status in Lima, Perú. AIDS Resear Hum Retrovir 20: 1046-52, 2004.

Beck IA, Drennan KD, Melvin AJ, Mohan KM, Hertz AM, Alarcón J, Piscoya J, Velázquez C, Frenkel LM. Simple, sensitive, and specific detection of HIV-1 subtype B DNA in dried blood samples for infant diagnosis in the field. J Clin Microbiol 39:29-33, 2001.

Li CC, Beck IA, Seidel KD, Frenkel LM. Persistence of Human Immunodeficiency Virus Type 1 Subtype B DNA in Dried Blood Samples on FTA® Filter Paper. J Clin Microbiol 42:3847-9, 2004.

Li CC, Beck IA, Seidel KD, Coombs RW, Frenkel LM. Detection and quantification of HIV-1 p24Ag in dried whole blood and plasma on filter paper stored under various conditions. J Clin Microbiol 43:3901-5, 2005.

HIV-1 Mother-to-Child-Transmission:

Frenkel LM, et al. Genetic evaluation of suspected cases of transient HIV-1 infection of infants. Science 280:1073-7, 1998.

HIV-1 Evolution during Antiretroviral Therapy (ART):

Frenkel LM, Wang Y, Learn GH, Melvin AJ, Ellis GM, McKernan JL, De Vange SM, Mohan KM, Holte S, Sylva GL, Naugler WE, Mahalanabis M, Beck IA, Lewis PA, Heath LM, Tobin NH, Pawluk D, Mullins JI. Multiple Viral Genetic Analyses Detect Low-level Human Immunodeficiency Virus type-1 Replication During Effective HAART. J Virol 77:5721-30, 2003.

Tobin NH, Learn GH, Holte SE, Wang Y, Melvin AJ, McKernan JL, Pawluk DM, Mohan KM, Lewis PF, Mullins JI and Frenkel LM. Evidence that Low-level Viremias During Effective HAART Result from Two Processes: Expression of Archival Virus and Replication of Virus. J Virol 79:9625-34, 2005.

Neonatal Herpes Simplex Virus:

Diamond C, Mohan K, Hobson A, Frenkel L, Corey L. Viremia in neonatal herpes simplex virus infections. Pediatr Infect Dis J 18:487-9, 1999.

Kimberlin DW, Lin CY, Jacobs RF, Powell DA, Frenkel LM, Gruber WC, Rathore M, Bradley JS, Diaz PS, Kumar M, Arvin AM, Gutierrez K, Shelton M, Weiner LB, Sleasman JW, de Sierra TM, Soong SJ, Kiell J, Lakeman FD, Whitley RJ and the National Institute of Allergy and Infectious Disease Collaborative Antiviral Study Group. Natural history of neonatal Herpes Simples Virus infection in the acyclovir era. Pediatrics 108:223-9, 2001.

Kimberlin DW, Lin CY, Jacobs RF, Powell DA, Corey L, Gruber WC, Rathore M, Bradley JS, Diaz PS, Kumar M, Arvin AM, Gutierrez K, Shelton M, Weiner LB, Sleasman JW, de Sierra TM, Weller S, Soong SJ, Kiell J, Lakeman FD, Whitley RJ and the National Institute of Allergy and Infectious Disease Collaborative Antiviral Study Group (Frenkel, LM). Safety and efficacy of high-dose intervenous acyclovir in the management of neonatal Herpes Simplex Virus infections. Pediatrics 108:230-8, 2001.

Editorials:

Frenkel LM, Mullins JI. Should patients with drug-resistant HIV-1 continue to receive antiretroviral therapy? (editorial) N Engl J Med 344:520-2, 2001.

Frenkel LM and Tobin NH. Understanding HIV-1 Drug Resistance. (editorial) Therapeutic Drug Monitoring 26:116-21, 2004.

Frenkel, LM. Challenges in the Diagnosis and Management of Neonatal Herpes Simplex Virus Encephalitis. (editorial) Pediatrics 115: 795-797, 2005.