Peter J. Dailey

Quantitative Image Analysis of HIV-1 Infection in Lymphoid Tissue

Tracking human immunodeficiency virus-type 1 (HIV-1) infection at the cellular level in tissue reservoirs provides opportunities to better understand the pathogenesis of infection and to rationally design and monitor therapy. A quantitative technique was developed to determine viral burden in two important cellular compartments in lymphoid tissues. Image analysis and in situ hybridization were combined to show that in the presymptomatic stages of infection there is a large, relatively stable pool of virions on the surfaces of follicular dendritic cells and a smaller pool of productively infected cells. Despite evidence of constraints on HIV-1 replication in the infected cell population in lymphoid tissues, estimates of the numbers of these cells and the virus they could produce are consistent with the quantities of virus that have been detected in the bloodstream. The cellular sources of virus production and storage in lymphoid tissues can now be studied with this approach over the course of infection and treatment.

Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques.

A substantial risk in using live attenuated, multiply deleted viruses as vaccines against AIDS is their potential to induce AIDS. A mutant of the simian immunodeficiency virus (SIV) with large deletions in nef and vpr and in the negative regulatory element induced AIDS in six of eight infant macaques vaccinated orally or intravenously. Early signs of immune dysfunction were seen in the remaining two offspring. Prolonged follow–up of sixteen vaccinated adult macaques also showed resurgence of chronic viremia in four animals: two of these developed early signs of disease and one died of AIDS. We conclude that this multiply deleted SIV is pathogenic and that human AIDS vaccines built on similar prototypes may cause AIDS.

Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression.

Genetic variants of human and simian immunodeficiency virus (HIV and SIV) that evolve during the course of infection and progression to AIDS are phenotypically and antigenically distinct from their progenitor viruses present at early stages of infection. However, it has been unclear how these late variants, which are typically T-cell tropic, cytopathic and resistant to neutralizing antibodies, influence the development of clinical AIDS. To address this, we infected macaques with cloned SIVs representing prototype variants from early-, intermediate- and late-stage infection having biological characteristics typical of viruses found at similar stages of HIV infection in humans. These studies demonstrate that sequential, phenotypic and antigenic variants represent viruses that have become increasingly fit for replication in the host, and our data support the hypothesis that emerging variants have increased pathogenicity and drive disease progression in SIV and HIV infection.

Retrospective Analysis of Viral Load and SIV Antibody Responses in Rhesus Macaques Infected with Pathogenic SIV: Predictive Value for Disease Progression

The prognostic significance of SIV plasma viral load in macaques has not been well established, primarily owing to the small numbers of animals in experimental groups. In addition, many investigators have noted that animals that fail to develop an anti-SIV humoral response develop disease rapidly. To establish the prognostic significance of viral load and seroconversion, we retrospectively analyzed the plasma viral load and serology data from 74 rhesus macaques infected with SIVmac. Viral load was analyzed at three time points: in the peak (days 7-21), acute (days 30-55), and chronic (days 80-100) periods postinfection. High viral load in the peak and acute phases was associated with more rapid development of disease (p = 0.0086, p = 0.0004, respectively). We defined clinical outcome as rapid ( <1 year) or slow (> or =1 year) progression. When peak and acute viral loads were analyzed together, acute viral load was more strongly associated with rapid progression (p = 0.03). Slow progression was strongly associated with chronic viral loads below the median of 3.47 x 10(5) RNA copies/ml. Despite having preexisting anti-SIV antibodies, 7 of 23 vaccinated animals were rapid progressors. All unvaccinated animals that mounted a humoral response to SIV were slow progressors. Animals that received a formalin-fixed, microencapsulated SIV vaccine prior to infection had lower peak viral loads than unvaccinated animals (p = 0.0005), but developed disease at the same rate. Overall, in naive animals, viral load is an important prognostic indicator of the disease progression rate. We found that viral load measured during the chronic phase (days 80-100) of infection was most closely associated with disease progression. We also found that a formalin-fixed, microencapuslated SIV vaccine reduced viral load without affecting clinical outcome. This latter finding may have implications for the evaluation of HIV-1 human vaccine trials.

Route of Simian Immunodeficiency Virus Inoculation Determines the Complexity but Not the Identity of Viral Variant Populations That Infect Rhesus Macaques

ABSTRACT A better understanding of the host and viral factors associated with human immunodeficiency virus (HIV) transmission is essential to developing effective strategies to curb the global HIV epidemic. Here we used the rhesus macaque-simian immunodeficiency virus (SIV) animal model of HIV infection to study the range of viral genotypes that are transmitted by different routes of inoculation and by different types of viral inocula. Analysis of transmitted variants was undertaken in outbred rhesus macaques inoculated intravenously (IV) or intravaginally (IVAG) with a genetically heterogeneous SIVmac251 stock derived from a well-characterized rhesus macaque viral isolate. In addition, we performed serial IV and IVAG passage experiments using plasma from SIV-infected macaques as the inoculum. We analyzed the V1-V2 region of the SIV envelope gene from virion-associated RNA in plasma from infected animals by the heteroduplex mobility assay (HMA) and by DNA sequence analysis. We found that a more diverse population of SIV genetic variants was present in the earliest virus-positive plasma samples from all five IV SIVmac251-inoculated monkeys and from two of five IVAG SIVmac251-inoculated monkeys. In contrast, we found a relatively homogeneous population of SIV envelope variants in three of five monkeys inoculated IVAG with SIVmac251 stock and in two monkeys infected after IVAG inoculation with plasma from an SIV-infected animal. In some IVAG-inoculated animals, the transmitted SIV variant was the most common variant in the inoculum. However, a specific viral variant in the SIVmac251 stock was not consistently transmitted by IVAG inoculation. Thus, it is likely that host factors or stochastic processes determine the specific viral variants that infect an animal after IVAG SIV exposure. In addition, our results clearly demonstrate that the route of inoculation is associated with the extent and breadth of the genetic complexity of the viral variant population in the earliest stages of systemic infection.

Hepatitis C virus: quantitation and distribution in liver.

The optimal method for viral quantitation and the most appropriate site for determining viral load in patients with chronic hepatitis C virus (HCV) infection are unknown. We developed a method for measuring HCV RNA in the liver with the following features: 1) efficient extraction of RNA from tissue (89% of RNA recovered); 2) accurate amplification using branched DNA with strong concordance between a single sample tested on multiple occasions either in the same or in different runs; 3) good sensitivity (95%) and specificity (100%). HCV RNA was detected in as little as 2 mg of tissue, and viral load determined in a needle biopsy was representative of viral load in other parts of the liver. Within individual livers, 68% of the samples quantitated were within 1.5‐fold of the geometric mean, and 95% were within 2.2‐fold of the geometric mean. The mean ratio of virus in the liver and serum was 103, range 17.4–286. A delay of 30 minutes before freezing the liver tissue resulted in a reduction in the measured viral load in some, but not all instances. A sensitive, specific, and reproducible method for quantitating HCV RNA in the liver has been developed. Measurement of viral load at one site was representative of viral load at other sites. While hepatic HCV RNA levels are consistently greater than serum levels, the ratio of liver to serum viral load varies widely. The clinical use of measurement of viral load in the liver remains to be defined. J. Med. Virol. 51:217–224, 1997.

The Liver Is a Major Organ for Clearing Simian Immunodeficiency Virus in Rhesus Monkeys

ABSTRACT Infection with human or simian immunodeficiency virus (SIV) is characterized by the rapid turnover of both viral particles and productively infected cells. It has recently been reported that the clearance of SIV in vivo is exceedingly fast, with half-lives on the order of minutes. The underlying mechanism or site responsible for this rapid clearance, however, remains unknown. To investigate this issue, we chose to infuse infectious SIVmac239 grown from autologous peripheral blood mononuclear cells that were radioactively labeled by [35S]methionine and [35S]cysteine. This approach eliminates from the viral membrane alloantigens that may have a significant impact on viral clearance. In addition, this approach also permits identification of the sites of viral clearance by measuring the radioactive intensity, even if degradation of SIV RNA occurs in tissues. We now report that the half-life of infused SIV in blood is extremely close to estimates from a previous study, in which unlabeled SIV grown in a heterologous cell line was used. The allogeneic effect due to the presence of human antigens on the surfaces of virions may, therefore, play a minimal role in the high rate of virion clearance. Moreover, close to 30% of infused radioactivity was found in the liver and measureable amounts were detected in the lungs (5.4%), lymph nodes (3.0%), and spleen (0.4%). The detection of a significant proportion of infused virus in the liver suggests that viral clearance from circulation is mediated by a common, nonspecific mechanism, such as the phagocytic functions of the reticuloendothelial system. The rapid clearance and degradation of exogenously infused virions may pose a major obstacle for gene therapy with viral vectors, unless strategies to overcome the rapid in vivo elimination of these particles are developed.

Hepatitis C virus RNA quantification in right and left lobes of the liver in patients with chronic hepatitis C

Summary. Quantification of hepatitis C virus RNA in liver tissue is likely to be useful in the study of the natural history, pathogenesis, progression and treatment of hepatitis C virus‐associated liver disease. Quantitative measurements of hepatitis C virus RNA in liver biopsy samples using the branched DNA (bDNA) signal amplification assay were carried out. The aims of this study were threefold: first, to assess the level of hepatitis C virus RNA in biopsy samples from the right and left lobes of the liver; second, to evaluate the correlation between hepatitis C virus RNA levels in serum and liver; and third, to investigate the relationship between serum and liver hepatitis C virus RNA levels and the severity of hepatic histology in non‐cirrhotic patients with chronic hepatitis C. There was a strong correlation (r= 0.92, P < 0.01) between hepatitis C virus RNA levels in the right and left lobes of the liver as well as a strong correlation between hepatitis C virus RNA levels in liver and serum (r= 0.82, P < 0.01). However, there was no significant correlation between the severity of hepatic histology and levels of hepatitis C virus RNA in serum and liver among patients with chronic active hepatitis classified according to Knodells hepatic activity index (KI). Our results indicate that hepatitis C virus RNA quantification from a single liver biopsy is representative of both lobes in patients with chronic hepatitis, and suggest that serum hepatitis C virus RNA levels are a meaningful reflection of hepatitis C virus RNA levels in the liver.

Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFalpha, IL-2, IL-4, IL-6, IL-10, and IFNgamma. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNgamma mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFalpha or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Outpatient biopsies of the palatine tonsil: Access to lymphoid tissue for assessment of human immunodeficiency virus RNA titers

OBJECTIVES Our objective was to assess the feasibility of using tonsillar lymphoid biopsy specimens obtained on an outpatient basis to quantitate a patients lymphoid human immunodeficiency virus (HIV) RNA titers. DESIGN A pilot cohort study was performed. PATIENTS We evaluated ten HIV-seropositive patients who ranged in age from 26 to 48 years and had CD4+ cell counts ranging from 110 to 833 at enrollment. MAIN OUTCOME MEASURES The main outcome measures were tolerance and safety of outpatient tonsil biopsies and quantitation of HIV RNA titers in tonsillar lymphoid biopsy specimens, plasma, and peripheral blood mononuclear cells determined by a new method of HIV RNA signal amplification with branched DNA probes. RESULTS Outpatient tonsil biopsies were well tolerated and were performed without complications. Nine of 10 tonsil biopsies from the HIV-seropositive patients examined were positive for significant concentrations of HIV RNA, ranging from 106 to 101 HIV RNA equivalents per gram of tissue. All of the HIV RNA-positive tonsillar lymphoid specimens had HIV RNA titers that were 101 to 104 times greater than those recovered from plasma (per milliliter) of the same patient obtained at the time of biopsy. CONCLUSIONS Sufficient tonsillar tissue can be obtained in an outpatient clinic setting to quantitate lymphoid HIV titers by the new branched-DNA signal amplification method with relative ease and without complication. The biopsy method described here affords ready access to the lymphoreticular system, which may help to advance our understanding of the pathogenesis of myriad immune diseases without the need for excisional node biopsies.