Wendell Miley

Hematologic and Biochemical Changes Associated with Human T Lymphotropic Virus Type 1 Infection in Jamaica: A Report from the Population-Based Blood Donors Study

Background. A quadrivalent (types 6, 11, 16, and 18) human papillomavirus (HPV) L1 virus-like-particle (VLP) vaccine has been shown to be 95%-100% effective in preventing cervical and genital disease related to HPV-6, -11, -16, and -18 in 16-26-year-old women naive for HPV vaccine types. Because most women in the general population are sexually active, some will have already been infected with ≥ 1 HPV vaccine types at the time vaccination is offered. Here, we assessed whether such infected women are protected against disease caused by the remaining HPV vaccine types. Methods. Two randomized, placebo-controlled trials of the quadrivalent (types 6, 11, 16, and 18) HPV vaccine enrolled 17,622 women without consideration of baseline HPV status. Among women infected with 1-3 HPV vaccine types at enrollment, efficacy against genital disease related to the HPV vaccine type or types for which subjects were naive was assessed. Results. Vaccination was 100% effective (95% confidence interval [Cl], 79%-100%) in preventing incident cervical intraepithelial neoplasia 2 or 3 or cervical adenocarcinoma in situ caused by the HPV type or types for which the women were negative at enrollment. Efficacy for preventing vulvar or vaginal HPV-related lesions was 94% (95% CI,81%-99%). Conclusions. Among women positive for 1-3 HPV vaccine types before vaccination, the quadrivalent HPV vaccine protected against neoplasia caused by the remaining types. These results support vaccination of the general population without prescreening.OBJECTIVE We investigated changes in hematologic and biochemical parameters associated with human T lymphotropic virus type 1 (HTLV-1) infection, antibody titer, and provirus load. Additionally, on a subset of participants, we assessed the epidemiologic relationship of HTLV-1 with Strongyloides stercoralis. METHODS Among volunteer blood donors in Jamaica, HTLV-1 carriers (n=482) were frequency matched with HTLV-1 negative subjects (n=355) by age (+/-5 years), sex, and date of blood donation (+/-3 months). HTLV-1 antibody titer, provirus load, S. stercoralis IgG antibodies, complete blood cell count, blood chemistry, and urinalysis parameters were measured. RESULTS HTLV-1 carriers, compared with HTLV-1-negative individuals, had elevated levels of cleaved lymphocytes (24.5% vs. 16.4%), any lymphocyte abnormalities (atypical, cleaved, and reactive lymphocytes combined, 45.7% vs. 35.4%), and gamma-glutamyl transferase levels (21.2 vs. 19.6 IU/L), as well as lower eosinophil count (2.6% vs. 3.1%). Among carriers, HTLV-1 antibody titer (n=482) was inversely correlated with mean corpuscular volume (r=-0.10) and positively correlated with levels of total protein (r=0.16), phosphorus (r=0.12), and lactate dehydrogenase (r=0.24). HTLV-1-provirus load (n=326) was higher among carriers with cleaved lymphocytes and any lymphocyte abnormalities. Provirus load was inversely correlated with hemoglobin (r=-0.11), mean corpuscular volume (r=-0.15), neutrophil (r=-0.12), and eosinophil (r=-0.19) levels and was positively correlated with lactate dehydrogenase levels (r=0.12). Provirus load was significantly higher among male than female subjects. S. stercoralis antibodies were detected in 35 (12.1%) of 288 participants but were not associated with HTLV-1 status, antibody titer, or provirus load. CONCLUSIONS Markers of HTLV-1 infection (infection status, antibody titer, and provirus load) are associated with hematologic and biochemical alterations, such as lymphocyte abnormalities, anemia, decreased eosinophils, and elevated lactate dehydrogenase levels.

Quantitative proviral DNA and antibody levels in the natural history of HTLV-I infection

The pathogenesis of human T-cell lymphotropic virus type I (HTLV-I) in adult T-cell leukemia/lymphoma (ATL) and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) is poorly understood. We prospectively followed up and evaluated the virologic correlates of infection in transfusion recipients after seroconversion, in asymptomatic carriers, and in ATL and HAM/TSP patients. Proviral DNA levels (copies/105 lymphocytes) were determined by real-time automated polymerase chain reaction and antibody titers by end-point dilution by use of an HTLV-I enzyme-linked immunoassay. In early infection, proviral load was initially elevated (median, 212 copies/105 lymphocytes at time 1) and later decreased (median, 99 copies at time 2, and 27 copies at time 3). Corresponding antibody titers were low at time 1 (1:2154), had significantly increased by time 2 (1:12312), and were stable by time 3 (1:4694). These viral markers were significantly lower in asymptomatic carriers than in HAM/TSP or ATL patients. Therefore, proviral load and antibody titers may be useful as predictive markers of disease among carriers.

A quantification of human cells using an ERV-3 real time PCR assay.

A novel approach to quantifying human cells using a real time PCR assay was developed. The target sequence used in the assay is a 135 bp segment within the unique 1.7 kb Hind III / Pst I fragment of the ERV-3 envelope gene. ERV-3 is a full-length human endogenous retrovirus present in known copy number in all human cells. The detection range of ERV-3 by real time PCR is from 10(6) to 10(1). The precision described, sensitivity and specificity of the assay indicate that the ERV-3 sequence is an accurate cell quantitation marker. The quantitative ERV-3 assay enables simple, fast, and reproducible detection and quantitation of the cell number. The assay can be used to determine the sample DNA conditions and also it can be used to adjust the quantitative DNA measurements of other target gene assays relative to the number of cell equivalents.

Provirus Load in Breast Milk and Risk of Mother-to-Child Transmission of Human T Lymphotropic Virus Type I

In a prospective study of 101 mother-child pairs in Jamaica, we examined the association of provirus load in breast milk and the risk of mother-to-child transmission of human T lymphotropic virus type I. The provirus load in breast milk was a strong predictor of risk of transmission to children (relative risk, 2.34/quartile), after adjustment for other known risk factors. The risk of transmission increased from 4.7/1000 person-months when the provirus load in breast milk was <0.18% to 28.7/1000 person-months when it was >1.5%. Provirus detection in maternal breast milk predicted transmission months before infection in children was detected by serologic testing.

Inhibition of cGAS DNA Sensing by a Herpesvirus Virion Protein.

Invading viral DNA can be recognized by the host cytosolic DNA sensor, cyclic GMP-AMP (cGAMP) synthase (cGAS), resulting in production of the second messenger cGAMP, which directs the adaptor protein STING to stimulate production of type I interferons (IFNs). Although several DNA viruses are sensed by cGAS, viral strategies targeting cGAS are virtually unknown. We report here that Kaposis sarcoma-associated herpesvirus (KSHV) ORF52, an abundant gammaherpesvirus-specific tegument protein, subverts cytosolic DNA sensing by directly inhibiting cGAS enzymatic activity through a mechanism involving both cGAS binding and DNA binding. Moreover, ORF52 homologs in other gammaherpesviruses also inhibit cGAS activity and similarly bind cGAS and DNA, suggesting conserved inhibitory mechanisms. Furthermore, KSHV infection evokes cGAS-dependent responses that can limit the infection, and an ORF52 null mutant exhibits increased cGAS signaling. Our findings reveal a mechanism through which gammaherpesviruses antagonize host cGAS DNA sensing.

Reactivation of Kaposi’s sarcoma-associated herpesvirus by natural products from Kaposi’s sarcoma endemic regions

Kaposis sarcoma (KS) and its causative agent, Kaposis sarcoma associated herpesvirus (KSHV/HHV‐8), a gamma2 herpesvirus, have distinctive geographical distributions that are largely unexplained. We propose the “oncoweed” hypothesis to explain these differences, namely that environmental cofactors present in KS endemic regions cause frequent reactivation of KSHV in infected subjects, leading to increased viral shedding and transmission leading to increased prevalence of KSHV infection as well as high viral load levels and antibody titers. Reactivation also plays a role in the pathogenesis of KSHV‐associated malignancies. To test this hypothesis, we employed an in vitro KSHV reactivation assay that measured increases in KSHV viral load in KSHV infected primary effusion lymphoma (PEL) cells and screened aqueous natural product extracts from KS endemic regions. Of 4,842 extracts from 38 countries, 184 (5%) caused KSHV reactivation. Extracts that caused reactivation came from a wide variety of plant families, and extracts from Africa, where KSHV is highly prevalent, caused the greatest level of reactivation. Time course experiments were performed using 28 extracts that caused the highest levels of reactivation. The specificity of the effects on viral replication was examined using transcriptional profiling of all viral mRNAs. The array data indicated that the natural extracts caused an ordered cascade of lytic replication similar to that seen after induction with synthetic activators. These in vitro data provide support for the “oncoweed” hypothesis by demonstrating basic biological plausibility.

Virus Markers Associated with Vertical Transmission of Human T Lymphotropic Virus Type 1 in Jamaica

In a prospective study involving 150 mothers and their offspring in Jamaica, we examined maternal viral factors associated with the risk of transmission of human T lymphotropic virus type 1 (HTLV-1). Overall, the incidence of HTLV-1 infection among children was 8.3 occurrences per 1000 person-months. A higher maternal provirus level (odds ratio [OR], 1.9 per quartile) and a higher HTLV-1 antibody titer (OR, 2.2 per quartile) were independently associated with transmission to children, whereas the presence of anti-Tax antibody was not. Higher maternal antibody titers also were associated with older age at infection among children who were breast-fed for </=12 months, which suggests that passively transferred maternal antibodies confer protection against infection while they persist. These data imply that mothers who have high provirus loads should be encouraged not to breast-feed. Alternatively, the successful reduction of maternal provirus loads or maintenance of passive antibody levels in infants during breast-feeding may lower the risk of transmission.

Real-Time PCR Assay for Detection and Quantification of Hepatitis B Virus Genotypes A to G

ABSTRACT The detection and quantification of hepatitis B virus (HBV) DNA play an important role in diagnosing and monitoring HBV infection as well as assessing therapeutic response. The great variability among HBV genotypes and the enormous range of clinical HBV DNA levels present challenges for PCR-based amplification techniques. In this study, we describe the development, evaluation, and validation of a novel real-time PCR assay designed to provide accurate quantification of DNA from all eight HBV genotypes in patient plasma specimens. A computer algorithm was used to design degenerate real-time PCR primers and probes based upon a large number (n = 340) of full-length genomic sequences including HBV genotypes A to H from Europe, Africa, Asia, and North and South America. Genotype performance was tested and confirmed using 59 genotype A to G specimens from two commercially available worldwide genotype panels. This assay has a dynamic range of at least 8 log10 without the need for specimen dilution, good clinical intra- and interassay precision, and excellent correlation with the Bayer Diagnostics VERSANT HBV DNA 3.0 (branched DNA) assay (r = 0.93). Probit analysis determined the 95% detection level was 56 IU/ml, corresponding to 11 copies per PCR well. The high sensitivity, wide linear range, good reproducibility, and genotype inclusivity, combined with a small sample volume requirement and low cost, make this novel quantitative HBV real-time PCR assay particularly well suited for application to large clinical and epidemiological studies.

Real-time polymerase chain reaction assay for cell-associated HTLV type I DNA viral load.

We have developed a quantitative real-time PCR assay for HTLV-I DNA. This assay approach uses real-time monitoring of fluorescent signal generation as a consequence of Taq-mediated amplification of specific target sequences to allow real-time kinetic analysis of amplicon production. This kinetic approach yields excellent sensitivity and an extremely broad linear dynamic range, and ensures that quantitation is based on analysis during the exponential phase of amplification, regardless of the input template copy number. The HTLV-I DNA assay has a nominal threshold sensitivity of 10 copy Eq/reaction, although single-copy plasmid template can be detected at frequencies consistent with statistical prediction. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 14% (coefficient of variation) for control templates over a range of 10(1) to 10(6) copy Eq/reaction and 25%, based on studies of extraction and analysis of replicate aliquots of PBMC specimens from HTLV-I-infected subjects. The primer/probe combination targets tax sequences conserved across described HTLV-I and HTLV-II isolates. Parallel quantitation in the same samples of an endogenous sequence present at a known copy number per cell allows normalization of results for potential variation in DNA recovery. Availability of this assay should facilitate studies of basic pathogenesis and clinical evaluation of HTLV-I and HTLV-II infection, as well as assessment of therapeutic approaches.

Detection of antibodies to Kaposi’s Sarcoma-Associated Herpesvirus: a new approach using K8.1 ELISA and a newly developed recombinant LANA ELISA

Detection of antibodies to Kaposis sarcoma-associated herpesvirus (KSHV or Human herpesvirus 8) is a topic of ongoing controversy. KSHV expresses multiple antigens and host responses are highly variable. We have previously described an algorithm for determining KSHV infection based on K8.1 ELISA and LANA immunofluorescence assay (IFA). Here we describe the development of a recombinant ELISA for LANA and an improved testing strategy using ELISAs for LANA and K8.1. We assessed mammalian and baculovirus expression systems for the production of full-length recombinant LANA. We evaluated the performance of LANA ELISAs using human serum samples from several sources including blood donors and clinical patients diagnosed with Kaposis sarcoma and compared them to LANA IFA. Both LANA ELISAs exhibited comparable sensitivity and specificity to LANA IFA but showed considerably greater reliability. The LANA ELISA can thus be used in conjunction with the previously described K8.1 ELISA to enable the highly sensitive and specific detection of antibodies to KSHV. Use of this testing strategy will provide a more accurate and reliable diagnostic assessment of KSHV status.