Sequential immunization of macaques with two differentially attenuated vaccines induced long-term virus-specific immune responses and conferred protection against AIDS caused by heterologous simian human immunodeficiency Virus (SHIV(89.6)P).

Four rhesus macaques were sequentially immunized with live vaccines DeltavpuDeltanefSHIV-4 (vaccine-I) and Deltavpu SHIV(PPC) (vaccine-II). The vaccine viruses did not replicate productively in the peripheral blood mononuclear cells (PBMCs) of the vaccinated animals. All four animals developed binding antibodies against both the vaccine-I and -II envelope glycoproteins but neutralizing antibodies only against vaccine-I. They developed vaccine virus-specific CTLs that also recognized homologous as well as heterologous pathogenic SHIVs. Thirty weeks after the last immunization, the vaccinated animals and three unvaccinated control animals were challenged iv with a highly virulent heterologous SHIV(89.6)P. As expected, the three unvaccinated control animals developed large numbers of infectious PBMCs, high plasma viremia, and precipitous loss of CD4(+) T cells. Two controls did not develop any immune response and succumbed to AIDS in about 6 months. The third control animal developed neutralizing antibodies and had a more chronic disease course, but eventually succumbed to AIDS-related complications 81 weeks after inoculation. The four vaccinated animals became infected with challenge virus as indicated by the presence of challenge virus-specific DNA in the PBMCs and RNA in plasma. However, virus in these animals replicated approximately 200- to 60,000-fold less efficiently than in control animals and eventually, plasma viral RNA became undetectable in three of the four vaccinates. The animals maintained normal CD4(+) T-cell levels throughout the observation period of 85 weeks after a transient drop at Week 3 postchallenge. They also maintained CTL responses throughout the observation period. These studies thus showed that the graded immunization schedule resulted in a safe and highly effective long-lasting immune response that was associated with protection against AIDS by highly pathogenic heterologous SHIV(89.6)P.

Use of herpesvirus saimiri-immortalized macaque CD4(+) T cell clones as stimulators and targets for assessment of CTL responses in macaque/AIDS models.

Herpesvirus saimiri (HVS), a nonhuman primate gamma herpes virus, was used to immortalize pig-tailed macaque CD4(+) T lymphocytes. The HVS-immortalized T cell lines were used to develop CD4(+) T cell clones from two animals. Three CD4(+) T cell clones were further characterized for the expression of cell surface markers. All expressed CD2, CD4, CD58, CD69 and CD80 and therefore resembled activated T cells. These clones required exogenous IL-2 for efficient growth and were found to be highly susceptible to infection by the challenge virus, Chimeric simian/human immunodeficiency virus (SHIV(KU-1)). They could also be productively infected not only by the quasispecies of the challenge virus (SHIV(KU-1/PDJ) and SHIV(KU-1/PNA), isolated from macaque PDj and PNa, respectively) but also by a different chimeric simian/human immunodeficiency virus (SHIV(89.6P)) and simian immunodeficiency virus (SIV(MAC239)). The virus-infected CD4(+) T cell clones were also used as stimulators for generation of CTL effectors. These effectors exhibited excellent virus-specific lysis in chromium-release assays when syngenic SHIV(KU-1) infected autologous CD4(+) T cell clones were used as targets. The target cell lysis was virus specific, as uninfected control cells showed no or minimal lysis.

Chronology of genetic changes in the vpu, env, and Nef genes of chimeric simian-human immunodeficiency virus (strain HXB2) during acquisition of virulence for pig-tailed macaques.

Recently, we developed a highly pathogenic variant of simian-human immunodeficiency virus, SHIV-4 (containing the tat, rev, vpu, and env of the HXB2 strain of HIV-1 in a genetic background of SIVmac239), through a series of four bone marrow-bone marrow passages-first in rhesus monkeys and then in pig-tailed macaques [Joag et al. (1996) J. Virol. 70, 3189-3197]. Inoculation of pig-tailed macaques with this pathogenic virus (SHIVKU-1) causes subtotal elimination of CD4(+) T cells and fatal opportunistic infections, usually within 6 months. Genetic characterization of SHIVKU-1 showed that it has a functional vpu gene (the first codon is ATG vs ACG for the vpu of SHIV-4) and several amino acid substitutions in Env and nef [Stephens et al. (1997) Virology 231, 313-321]. Two pig-tailed macaques, PPc and PQc, were the first to develop a severe loss of CD4(+) T cells and the acquired immune deficiency syndrome and were euthanized at 26 and 105 weeks, respectively. In this report, we analyzed the changes that occurred in the vpu, nef, and env (gp120) genes of the virus used to inoculate macaques PPc and PQc and established the chronology of changes that occurred in these viral genes as these two animals lost their CD4(+) T cells and progressed to develop acquired immune deficiency syndrome. Compared with SHIV-4, the virus used to inoculate macaques PPc and PQc had 0, 3, and 0 consensus amino acid changes in the Vpu, gp120, and Nef, respectively. An analysis of the viral sequences amplified from peripheral blood mononuclear cells samples taken at various times after inoculation of PPc revealed that the vpu had not reverted to an open reading frame (closed vpu, ACG) at 4 weeks after inoculation, but by 16 weeks vpu had reverted to an open reading frame (open vpu, ATG). Macaque PQc, which had a longer course of disease, had a closed vpu at 4 and 16 weeks, but by 28 weeks, both closed and open vpu were detected. From 39 to 105 weeks, only an open vpu was detected. In both macaques, the reversion to an open vpu correlated well with the second phase (major) of CD4(+) T cell loss. An analysis of the nef and env sequences isolated from the same times after inoculation revealed an association between the reversion of vpu to an open reading frame and the accumulation of increased numbers of consensus changes in these two viral proteins. These data suggest that the concomitant reversion of vpu to an open reading frame along with increased substitutions in Nef and gp120 were important genetic changes in the viral genome that were responsible for the increased and highly efficient rate of replication of the virus in CD4(+) T cells and macrophages, which in turn led to elimination of the CD4(+) T cells and profound loss of immunocompetence in the infected animals.

Neuroinvasion by ovine lentivirus in infected sheep mediated by inflammatory cells associated with experimental allergic encephalomyelitis.

Maedi Visna Virus (MVV) is a prototypic lentivirus that causes infection only in cells of macrophage lineage, unlike the primate lentiviruses which infect both CD4+ T lymphocytes and macrophages. In primates, the earliest viral invasion is associated with the ability of the virus to infect and activate T cells which convey virus to the brain. Infected monocytes in blood rarely cause CNS infection in absence of activation of CD4+ T cells. In the face of lack of infection or activation of T cells by MVV in sheep, the question arises, how does MVV gain access to the brain to cause the classical lesions of visna? In previous studies on experimental induction of visna, sheep were inoculated with virus directly in the brain. In this study, we asked whether neuroinvasion by MVV would occur if sheep were inoculated with virus in a non-neural site. Nine sheep were inoculated intratracheally and all developed systemic infection when examined 3 weeks later. At this time, five were injected intramuscularly with brain white matter homogenized in Freund's complete adjuvant to induce EAE. None of the four animals inoculated with virus alone developed CNS infection despite typical lentiviral infection in lungs, lymphoid tissues and blood-borne mononuclear cells. In contrast, all five of the sheep injected with brain homogenate developed infection in the brain. Virus was produced by macrophages associated with the EAE lesions. This study illustrated that both activated T cells specific for antigen in the CNS and infected macrophages are essential for lentivirus neuropathogenesis.

Pathogenesis of ovine lentiviral encephalitis: derivation of a neurovirulent strain by in vivo passage.

The lentiviruses of sheep replicate almost exclusively in macrophages and cause chronic interstitial pneumonia, arthritis, and mastitis, but only rarely encephalitis. This study was undertaken to determine whether a non-neurovirulent field strain of ovine lentivirus isolated from joint fluid that replicated productively in lung and joint macrophages could be adapted to enter and replicate in the brain and cause encephalitis. The field isolate was passed seven times sequentially by intracerebral inoculation of sheep. The neuroadapted strain of virus caused severe encephalitis typical of visna in four of four sheep inoculated intracerebrally. The virus replicated to high titers in the brains of these animals and in cultured microglia. The inflammatory response in the brain was characterized by intense infiltrates of macrophages and CD8+ and CD4+ T cells. Many of the perivascular macrophages demonstrated TNF-alpha expression and there was upregulation of MHC Class II antigen expression on both inflammatory cells and endothelium. Inoculation of this neuroadapted virus into the bone marrow of three animals resulted in persistent infection and cell-associated viremia, but not encephalitis. Virus was not detected in brains from these animals, indicating that the virus was not neuroinvasive. These data suggest that neuroinvasiveness and neurovirulence are separate pathogenic determinants, both of which are required for the development of encephalitis during natural infection.

Contrast Study of CdZnTe Detectors for Digital Mammography.

Experiments have been performed with the aim of optimizing the image quality parameters of CdZnTe detectors for digital mammography. A geometrical breast phantom has been designed, and the dependence of the contrast resolution of a planar CdZnTe detector on the phantom thickness has been experimentally determined. Specifically, the detected signal and noise contributions were measured and related to phantom thickness. The results of this study indicate that the CdZnTe detectors exhibit a high contrast resolution. On the other hand, the dynamic range of this detector can be improved significantly by further implementation of the data acquisition electronics.

Variations in lentiviral gene expression in monocyte-derived macrophages from naturally infected sheep.

Seventy-nine 1-year-old lambs from three individual farms and a feedlot were examined for natural lentivirus infection. We used three different methods to detect infection and to identify the stage of the ovine lentivirus life cycle in blood-derived macrophages. Cytopathic infectious virus was obtained from 14/14 Border Leicester animals obtained from a naturally infected flock. Neither virus particles, virus proteins, virus specific antibodies nor viral DNA were detected in samples from 34 lambs from two South Kansas City farms. However, among 31 feedlot lambs, we identified 11 infected animals. Specific viral proteins were immunoprecipitated from macrophages of one animal, but no infectious cytopathic virus was isolated from these cells. Cells from ten of the other feedlot animals harboured viral DNA but neither viral particles nor proteins could be detected by our techniques. Thus, in these naturally infected animals, the virus life cycle either proceeded to completion, subject to differentiation of infected precursor cells in blood, or remained arrested at the DNA stage despite maturation of monocytes to macrophages. Sequence analysis of the env gene of viral genomes from two of the ten feedlot sheep showed sequences distinct from those of known ovine and caprine lentiviruses. Surprisingly, these sequences have a higher identity (of nucleotide and derived amino acid sequences) to caprine arthritis-encephalitis virus than to the ovine prototype, maedi-visna virus. These data suggest that the ovine and caprine lentiviruses found in North American sheep may have a common ancestral genotype that is closely related to the caprine virus.

Restrictive type of replication of ovine/caprine lentiviruses in ovine fibroblast cell cultures.

Caprine arthritis-encephalitis virus (CAEV) is a natural lentivirus pathogen of goats. CAEV, like all members of the ovine/ caprine lentivirus family, has an in vivo tropism for cells of the monocyte/macrophage cell lineage and activation of viral gene expression is observed only following differentiation of monocytes to macrophages. In addition to cells of the monocyte/ macrophage lineage, CAEV and the closely related maedi visna virus of sheep (MVV) can also replicate productively in fibro-epithelial cells derived from synovial membrane of goats (GSM). However, these viruses varied greatly in their ability to replicate in fibroblasts. We studied the biological and biochemical properties of CAEV and maedi-visna virus (MVV) of sheep following inoculation into the three ovine/caprine cell types. Our data showed no substantial differences in virus titers, viral protein biosynthesis, or processing of the viral proteins between CAEV and MVV following inoculation into primary macrophages and GSM cells. However, unlike MVV, CAEV failed to replicate productively in ovine fibroblasts (sheep choroid plexus cells). This correlated with a specific but abnormal proteolytic cleavage of the envelope glycoprotein of the virus. This abnormal proteolytic cleavage represents a novel type of host cell restriction of lentivirus replication.

Initial characterization of viral sequences from a SHIV-inoculated pig-tailed macaque that developed AIDS.

In this study, we report on the derivation of a pathogenic SIV-HIV chimeric virus (SHIV) and the initial characterization of the viral sequences from the first (macaque PPc) of a series of pig-tailed macaques that developed CD4+ T cell loss and AIDS. Viral genes were amplified by PCR from the brain, lymphoid, and kidney tissues and their sequences compared to the original SHIV used to initiate passages in macaques. Our results show that the vpu gene, which was nonfunctional in the original SHIV, now coded for functional protein in macaque PPc. The tat and rev genes had no consensus changes but the nef gene had 4-5 consensus changes, depending on the tissue examined. The gp 120 gene had the highest number of nucleotide and amino acid substitution rates that varied from 0.64% to 1.44% and 1.17% to 3.71%, respectively, again depending on the tissue examined. These results suggest that a constellation of changes accumulated at the genomic level during the derivation of a SHIV that was pathogenic for pig-tailed macaques.

Development of transgenic sheep that express the visna virus envelope gene.

The ovine lentiviruses cause encephalitis, pneumonia, and arthritis in sheep worldwide. Visna virus is a prototype of this family and the pathogenesis and molecular biology of the virus has been well characterized. The envelope proteins of visna virus are responsible for binding of virus to host cells and for causing cell fusion. The surface glycoprotein also elicits cellular and humoral immune responses to the virus, the former being thought to be responsible for eliminating infected cells as well as causing inflammatory lesions. In this study, transgenic sheep were constructed that expressed the envelope genes of visna virus under the control of the visna LTR to investigate the role of the env gene in the pathogenesis of lentiviral disease in its natural host. Three transgenic lambs were identified that contain the env transgene and express the envelope glycoproteins. These transgenic animals have remained healthy and expression of the viral gene has had no obvious deleterious effect. Expression of the visna envelope protein was demonstrated by cell fusion mediated by the envelope gene as well as by immunoprecipitation of the envelope proteins with monoclonal antibodies and immunofluorescence analyses of Env protein in cells. The target cell for visna virus replication in infected animals is the monocyte/macrophage. In natural infection, the level of viral gene expression in these cells increases with cell maturation. In the transgenic sheep, monocytes did not express the envelope glycoproteins until they differentiated into macrophages in vitro. Expression of the env mRNA in macrophages was quantitated by an RNase protection assay. In addition to expression in macrophages, the transgene was expressed by fibroblasts isolated from skin of the transgenic sheep. Expression of both the Env and Rev proteins was detected by immunoprecipitation and immunofluorescence. Two of the three lambs responded immunologically to the expression of the transgene by producing binding antibodies to the envelope glycoproteins. Thus, these transgenic sheep provide a model to study whether a lentivirus glycoprotein will prevent infection or modulate disease in its natural host after virus challenge.

Phase-shifting holographic interferometry for breast cancer detection.

Using a mild pressure stressing device and a pulsed ruby-laser system, we apply phase-shifted holographic interferometry to the detection of breast cancer. We compare a four-step algorithm and a Carré algorithm for their ability to evaluate the interferograms. The results show the feasibility of holographic interferometry in the detection of anomalies in the human female breast.

Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes.

The lentiviruses of sheep, goats, and horses cause chronic multiorgan disease in which macrophages are highly permissive for viral replication. Monocytes, which mature into macrophages, are thought to be latently infected with lentivirus, but the extent to which other leukocytes are infected is unknown. Dendritic cells have not been studied separately from monocytes and T-cell subsets have not been examined in previous attempts to identify infected cells in peripheral blood mononuclear cells (PBMC). We found no evidence of T-cell tropism using an animal-passaged, pathogenic ovine lentivirus. Phytohemagglutinin-stimulated infectious PBMC produced 20-fold less virus than differentiated macrophages, and cocultivation of infectious PBMC with fresh, uninfected phytohemagglutinin blasts did not facilitate virus replication. Furthermore, central lymph cells, the best in vivo source of purified lymphocytes, lacked virus and did not yield virus upon in vitro cultivation. In contrast, cultivated blood-derived macrophages were highly permissive for viral replication. To identify the latently infected PBMC, PBMC from infected sheep were selectively depleted of monocytes and B cells by passage over nylon wool and then of nonadherent cells bearing CD4, CD8, T19, gamma delta T-cell receptor, CD45RA, or major histocompatibility complex class II antigens by panning. Removal of adherent monocytes and B cells or of adherent cells and the three major T-cell subsets (CD4+, CD8+, T19+) did not decrease the infectivity of PBMC. The richest sources of infected cells in fresh PBMC were CD45RA+ and major histocompatibility complex class II+ nonadherent cells, which are three characteristics of dendritic cells. Thus, the dendritic cell, and not the monocyte or the CD4+ cell, is probably the predominant infected cell type in blood.

Use of biostereometric analysis as a screen for breast cancer.

Biostereometric analysis, a photographic surface imaging technology that is noninvasive, was tested for its effectiveness in selecting breast pathology in a sample of 1000 female subjects, including 80 with cancers, 635 normals, and 285 with benign breast conditions. These individuals were recruited from the population of women undergoing routine mammographic examination. The project was designed specifically to determine whether biostereometric analysis could identify the individuals in the sample with malignant breast disease in the hope of providing information to aid in future development of a breast cancer screening protocol. The overall sensitivity of the method for cancers of all sizes was 76%. Biostereometric analysis was 85% sensitive for selection of cancers in the subjects over 50 years of age, and identified 80% (4 of 5) of the subjects with clinically confirmed breast cancers less than 1 cm in size. The method was 69% specific, but identified benign breast disease in only 51% of cases.

Breast volume measurement of 598 women using biostereometric analysis.

A study of the volumes of the right and left breasts of 598 subjects was undertaken using biostereometric analysis. This measurement uses close-range stereophotogrammetry to characterize the shape of the breast, and is noncontact, noninvasive, accurate, and rapid with respect to the subject involvement time. Using chi-square tests, volumes and volumetric differences between breast pairs were compared with handedness, perception of breast size by each subject, age, and menstrual status. No significant relationship was found between the handedness, age, or menstrual status of the subject and the breast volume. Several groups of subjects were accurate in their perception of breast size difference. Analysis did confirm the generally accepted clinical impression of left-breast volume dominance. These results are shown to be consistent with those of a previous study using 248 women.

Modulation of lentivirus replication by antibodies: Fc portion of immunoglobulin molecule is essential for enhancement of binding, internalization, and neutralization of visna virus in macrophages.

Antibodies to visna virus neutralized the virus in fibroblasts and macrophages but specifically enhanced the binding, penetration, and uncoating of the virus in the latter cells. F(ab')2 fragments of the immune antibody neutralized the virus in fibroblasts but did not enhance the early stages of the virus life cycle in macrophages. Furthermore, these fragments did not neutralize infectivity in macrophages but delayed the appearance of infectious virus in cells after the inoculation of preincubated virus-F(ab')2 complexes.

Biostereometric analysis for breast cancer detection.

A measurement technique has been developed for noninvasive breast cancer detection. The process involves the use of close-range stereophotogrammetry as a data acquisition device for the determination of breast surface concavities. We report the methodology used to detect these surface depressions, the rationale for the study, and our preliminary findings.

Breast volume measurement of 248 women using biostereometric analysis.

A study of volumes of the right and left breasts of 248 subjects was undertaken using biostereometric analysis. This measurement technique uses close-range stereophotogrammetry to characterize the shape of the breast and is noncontact, noninvasive, accurate, and rapid with respect to the subject involvement time. Volumes and volumetric differences between breast pairs were compared, using chi-square tests, with handedness, perception of breast size by each subject, age, and menstrual status. No significant relationship was found between the handedness of the subject and the larger breast volume. Several groups of subjects based on age and menstrual status were accurate in their perception of breast size difference. Analysis did not confirm the generally accepted clinical impression of left breast volume dominance. Although a size difference in breast pairs was documented, neither breast predominated.

Right and left breast volume and volume distribution comparisons in normal and tumor-containing breasts.

A measurement technique involving the use of close-range stereophotogrammetry has been developed for the study of breast volume and volume distribution (shape) comparison. This biostereometric method has been shown to be able to measure volume to within 0.63% of that determined by water displacement. This study describes the rapid and non-invasive data acquisition methodology as well as the mathematical algorithms used to determine breast volume. Additionally, the paper discusses the application of this process to documenting the presence or absence of total volume and volume distribution differences existing in normal breasts and those containing benign and malignant tumors.