Development of reverse transcriptase PCR assays for detection of active human herpesvirus 6 infection.

We developed reverse transcriptase (RT) PCR assays for the detection of mRNA from three spliced genes of human herpesvirus 6 (HHV-6), the immediate-early genes U16/U17 and U89/U90 and the late gene U60/U66. Sequence analysis determined the splicing sites of these genes. The new assays may be instrumental in investigating the association between HHV-6 and disease.

An in vivo model for HIV resistance development.

Treatment of human immunodeficiency virus (HIV-1) with drugs targeted to the reverse transcriptase (RT) rapidly selects for drug-resistant virus. It is essential to develop a suitable animal model that allows the study of the emergence and reversal of drug resistance. A monkey model was previously developed on the basis of a hybrid virus (RT-SHIV) of simian immunodeficiency virus (SIV) with its RT exchanged for HIV-1 RT. In the present study cynomolgus monkeys infected with RT-SHIV were treated with varying doses of the non-nucleoside RT inhibitor nevirapine. The drug was administered for 2-3 weeks, in agreement with clinical experience of resistance development during nevirapine monotherapy. This resulted in the selection of mutants with Y181C and K103N changes in RT, which correspond to the HIV-1 mutations in nevirapine-resistant HIV-1 patients. The mutants coexisted at varying levels with wild-type virus and fluctuations in the proportion of mutants could be closely monitored. Low-dose treatment was not more efficient in induction of mutations than a virus-inhibiting dose. Structured therapy interruptions could be performed. The monkey RT-SHIV infection offers an in vivo model to determine effects of therapies on resistance development.

Protection of human immunodeficiency virus type 2-exposed seronegative macaques from mucosal simian immunodeficiency virus transmission.

At present it is not known which form of immunity would be most effective against infection with human immunodeficiency virus (HIV). To evaluate the possible role of cellular immunity, we examined whether four HIV type 2-exposed but seronegative macaques developed cellular immune responses and determined whether these exposed macaques were resistant to mucosal transmission of simian immunodeficiency virus (SIV). Following intrarectal challenge with SIV, 2 monkeys were protected against detectable SIV replication and another showed suppressed viral replication compared to 14 persistently infected controls. The two protected monkeys demonstrated SIV-specific cytotoxic T lymphocytes before as well as after SIV challenge. Here we provide evidence that activation of the cell-mediated arm of the immune system only, without antibody formation, can control SIV replication in macaques. The results imply that vaccines that stimulate a strong and broad cellular immune response could prevent mucosal HIV transmission.

Reverse transcriptase and corresponding activity-blocking antibody for monitoring SIVsm infection in macaques.

A nonradioactive reverse transcriptase (RT) assay was used to measure RT activity in serum during the viremia peak associated with primary infection and for measuring the generation and maintenance of RT activity-blocking antibody (RTb-ab) titers during and after seroconversion in SIV-infected macaques. The RT assay was compared to an antigen capture immunoassay designed for HIV-2/SIVsm and was found to be approximately 40 times more sensitive in detecting SIVsm in serum from infected macaques. The RT assay detected RT activity in serum corresponding to levels from 3 pg/ml. Earliest detection of viral replication using the RT assay was on day 6-8, with a peak at day 10 (up to 8000 pg/ml). The earliest detection of RTb-ab was seen on day 17-23, with established RTb-ab titers by day 29, followed by increasing titers of 15,000-120,000 by day 62-77. The usefulness of RT and RTb-ab for monitoring the course of SIV infection in monkey models is discussed.

Prevention of simian immunodeficiency virus, SIVsm, or HIV-2 infection in cynomolgus monkeys by pre- and postexposure administration of BEA-005.

OBJECTIVE: To study the possibilities and limitations of postexposure treatment to prevent the establishment of infection after accidental exposure to HIV. DESIGN AND METHODS: The effect of 2,3'-dideoxy-3'-hydroxymethyl cytidine (B1 A-005) was investigated on acute simian immunodeficiency virus (SIV) and HIV-2 infections in macaques in pre- and postexposure treatment experiments. RESULTS: Postexposure treatment with BLA-005 (3 x 10 mg/kg) for as short as 3 days prevented infection with SIVsm after intravenous or rectal inoculation. Infection with HIV-2 could also be blocked by postexposure BFA-005 treatment. CONCLUSION: This study shows that therapeutic intervention can block early systemic and mucosal infections with SIV and HIV-2. Further evaluation is ongoing.

SIV infection of monkey spleen cells including follicular dendritic cells in different stages of disease.

Immunoaffinity enriched spleen follicular dendritic cells (FDCs), lymphocytes, and macrophages from SIVsm-inoculated cynomolgus monkeys (Macaca fascicularis) at different stages of disease were compared for latent and productive SIV infection. Analysis of FDCs by in situ hybridization, electron microscopy, and coculture assays indicated that comparatively high levels of virus were associated with the FDC fraction. Polymerase chain reaction (PCR) and RT-PCR results revealed that the levels for SIVpol DNA did not correlate with the level of env mRNA in the various cell subsets, suggesting differences in latency. Limiting dilution assays for spliced env mRNA showed a 10-100-fold higher amount of env mRNA in FDCs than in other spleen cell subsets early during SIV infection. At late stages of disease, the number of productively infected FDCs significantly decreased in parallel with a marked reduction of the FDC network and follicular involution. Our findings indicate that destruction of FDCs probably reflects a cytopathic effect of SIV and/or the activity of specific antiviral cytotoxic T lymphocytes.

Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is the major target for antiretroviral therapy of the acquired immunodeficiency syndrome (AIDS). While some inhibitors exhibit activity against most retroviral RTs, others are specific for the HIV-1 enzyme. To develop an animal model for the therapy of the HIV-1 infection with RT inhibitors, the RT of the simian immunodeficiency virus (SIV) was replaced by the RT of HIV-1. Macaques infected with this SIV/HIV-1 hybrid virus developed AIDS-like symptoms and pathology. The HIV-1-specific RT inhibitor LY300046.HCl, but not zidovudine [3'-azido-3'-deoxythymidine (AZT)] delayed the appearance of plasma antigenemia in macaques infected with a high dose of the chimeric virus. Infection of macaques with the chimeric virus seems to be a valuable model to study the in vivo efficacy of new RT inhibitors, the emergence and reversal of drug resistance, the therapy of infections with drug-resistant viruses, and the efficacy of combination therapy.

Thymic immunopathology and progression of SIVsm infection in cynomolgus monkeys.

Thymuses from 22 cynomolgus monkeys infected with simian immunodeficiency virus (SIVsm) developed characteristic cortical and medullary changes including formation of B-cell follicles (8/21) and accumulation of virus immune complexes. Advanced thymic histopathology was correlated with more pronounced immunodeficiency. SIVsm provirus was detected by polymerase chain reaction (PCR) in most (16/18) thymuses and spliced viral env mRNA in 3 (3/7) thymuses with advanced histopathologic changes indicative of thymic SIVsm replication. By combined in situ hybridization (ISH) and immunohistochemistry, viral RNA was localized mainly to the follicular dendritic network, macrophages, multinucleated giant cells, and lymphocytes of the medullary regions. Latent infection by an Epstein-Barr-related herpesvirus (HVMF1) was also found by PCR and by ISH in medullary regions of three (3 of 8) thymuses with B-cell follicles, suggestive of an inductive role for B-cell proliferation in these thymuses. In a control group of HIV-2-infected nonimmunosuppressed monkeys, no comparable thymic changes were observed. Our results indicate that SIV, and probably by analogy HIV, can have direct and diverse pathogenic effects on the thymus that are important in the development of simian (human) AIDS.

Simian immunodeficiency virus (SIVsm) infection of cynomolgus monkeys: effects on follicular dendritic cells in lymphoid tissue.

We studied follicles in sections of lymph nodes and spleen from cynomolgus monkeys (Macaca fascicularis) after infection with simian immunodeficiency virus (SIVsm), by (immuno)histology and (immunogold) electron microscopy. Also isolated follicular dendritic cells (FDC) were investigated. Histology showed ranged from follicular hyperplasia to follicle fragmentation. FDC showed desmin and vimentin, characteristic of mesenchymal cells. Except for two animals who got experimental chemotherapy in the first postinfection period, the cells expressed SIV gag p28 protein. Electron microscopy showed SIVsm-like particles in the germinal centers. A number of cell types in the germinal center, including FDC, showed tubuloreticular structures, indicative of alpha-interferon synthesis during an antiviral response. In immunogold electron microscopy, SIV p28 label was observed on the surface of FDC, on SIVsm-like particles, and in the cytoplasm of macrophages. A relatively high density of CD8-positive cells (T cytotoxic-suppressor phenotype) was observed around and in germinal centers, especially areas depleted of FDC. Cells immunoreactive for serine esterase granzyme-B, a protein occurring in granules of cytotoxic cells, occurred around germinal centers, but not in germinal centers at areas where FDC and SIV p28 label localized. This argues against a role of cytotoxic T cells in mediating follicle destruction.

Penetration of zidovudine and 3'-fluoro-3'-deoxythymidine into the brain, muscle tissue, and veins in cynomolgus monkeys: relation to antiviral action.

Cynomolgus monkeys had microdialysis probes implanted under ketamine anesthesia into peripheral veins, thigh muscles, and the brain in order to sample the extracellular fluid for the concentrations of unbound nucleoside analogs. A dose of 25 mg of zidovudine or 3'-fluoro-3'-deoxythymidine (FLT) per kg was administered subcutaneously to each of three animals. Relatively high antiviral concentrations of FLT and zidovudine were present in peripheral tissues and in the brain. It was found that the concentration of zidovudine in the brain was approximately one-third of that in muscle and veins; the same relation was observed for FLT. The in vivo unbound concentrations of both drugs in the brain, muscle, and venous blood exceeded those reported to inhibit human immunodeficiency virus replication in vitro. In addition, in a correlative study we found that the appearance of p24 antigen in sera of monkeys infected with simian immunodeficiency virus was significantly delayed by both compounds (15 mg/kg three times daily for 9 days after infection). Thus, we have shown that the extracellular concentrations of unbound FLT and zidovudine in the brain and peripheral tissues attained with in vivo antiviral doses exceed in vitro antiviral concentrations.

Long-term tolerance and efficacy of 3'-azidothymidine and 3'-fluorothymidine treatment of asymptomatic monkeys infected with simian immunodeficiency virus.

Macaques chronically infected with simian immunodeficiency virus were treated with zidovudine (20 mg/kg of body weight per day for 9 weeks) or 3'-fluorothymidine (5 mg/kg of body weight per day for 9 weeks or three doses of 2 mg/kg per day for 24 days). Hematological changes in the treated animals included macrocytic anemia and leukopenia. Determination of antiviral effects in this model requires improved assay methods.

A monkey model for Epstein Barr virus-associated lymphomagenesis in human acquired immunodeficiency syndrome.

High-grade malignant nonHodgkin's lymphomas--five lymphoblastic, three pleomorphic, and two immunoblastic--developed in 10/25 cynomolgus monkeys (Macaca fascicularis) followed for up to 746 d after infection with simian immunodeficiency virus, strain SIVsm. These lymphomas were shown to be associated with an Epstein-Barr (EB)-like cynomolgus B-lymphotropic herpesvirus (CBLV) by electron microscopy, by Southern blot hybridization with probes against human EBV, and by the expression of antigens corresponding to EBV-associated nuclear antigens (EBNAs) involved in human B cells transformation. Southern blot demonstration of immunoglobulin gene rearrangements and homogeneous EBV episomes indicated that all the lymphomas were CBLV-associated monoclonal B cell proliferations. Our findings suggest that these tumors correspond to the EBV-associated malignant lymphomas in acquired immunodeficiency syndrome with respect to clinical, morphological, phenotypic, and genotypic characteristics. The particular susceptibility of SIVsm immunodeficient cynomolgus monkeys for CBLV-associated lymphomagenesis appears therefore a useful model for EBV-associated lymphomas in humans.

Prevention of HIV-2 and SIV infections in cynomolgus macaques by prophylactic treatment with 3'-fluorothymidine.

The aim of this study was to determine the usefulness of human immunodeficiency virus type 2 (HIV-2) for in vivo evaluation of antiviral drugs in monkeys and to study if prophylactic treatment with 3'-fluorothymidine (FLT) could prevent infection against a low challenge dose of HIV-2 or simian immunodeficiency virus (SIV). Protection against infection was assessed by virus isolation and polymerase chain reaction (PCR) on monkey peripheral blood mononuclear cells (PBMC) as well as by antibody and viral antigen assays. Prophylactic treatment with FLT 3 x 5 mg/kg/day, starting 8 h prior to virus inoculation, prevented HIV-2 infection in 3 of 8 monkeys. In another experiment 2 of 4 monkeys resisted 2-10 monkey infectious doses (MID50) of SIV with the same prophylactic treatment. All control animals (HIV-2 n = 8, SIV n = 4) became infected. Thus, FLT treatment prevented HIV-2 and SIV infection in 5 of 12 animals.

Clinical features and predictive markers of disease progression in cynomolgus monkeys experimentally infected with simian immunodeficiency virus.

OBJECTIVE: To study the pathogenicity of simian immunodeficiency virus (SIVsm) in cynomolgus monkeys in order to establish an animal model for human AIDS. METHODS: Thirty-three cynomolgus monkeys were monitored for more than 2 years following experimental infection with SIVsm. RESULTS: All the macaques became SIV-infected, as demonstrated by virus recovery from peripheral blood lymphocytes and by the appearance of viral antibodies. SIVsm was found to be pathogenic, killing 29 out of the 33 monkeys (88%) within 26 months. Clinically, infected monkeys developed lymphadenopathy, splenomegaly, diarrhoea, weight loss, neurological symptoms and a remarkably high incidence (39%) of malignant lymphomas. All lymphomas were high-grade malignant and of B-cell origin. Disease progression was associated with low CD4+ lymphocyte count, involution of initially hyperplastic follicular B-cell areas in lymph nodes, reappearance of viral antigen in serum, loss of anti-Gag antibodies and development of systemic giant cell disease in 55% of the monkeys. CONCLUSIONS: There are many similarities between SIVsm-induced AIDS in cynomolgus monkeys and human AIDS with regard to clinical, virological, immunological and pathological manifestations.

Simian immunodeficiency virus (SIVsm) isolation from blood and brain of experimentally infected macaques.

The possibility of using simian immunodeficiency virus (SIV)-infected macaques to study pathogenic events linked to HIV infection of the brain prompted us to investigate some of the virological features in SIV-infected macaques. Nine cynomolgus macaques were inoculated with SIVsm and killed at different times. We successfully isolated virus from the blood of all the animals and from the brains of eight. These results point to the early and regular spread of this lentivirus to the brain. Neutralizing activity was studied in the serum and cerebrospinal fluid specimens obtained from these macaques against a selected group of isolates. Cerebrospinal fluid did not show any neutralizing activity. Our findings integrate the observations from HIV-1 infection in man and indicate that SIV infection of macaques is a useful model for studying pathogenic events of brain infection.

Monocyte/macrophage giant cell disease in SIV-infected cynomolgus monkeys.

A non-opportunistic, generalized giant cell disease (GCD) was found in 12 out of 25 (48%) cynomologus monkeys infected with SIVsm. Most organs were affected notably the lymph nodes (LN), spleen, gut, liver, lungs and CNS. The multinucleated GC varied considerably in cell size and in the number and cytoplasmic distribution of the nuclei. Immunohistochemically most GC expressed SIV antigens and markers of mononuclear phagocytes (CD68), CD4 and also occasionally the T-cell markers CD45RO, CD43 and CD2. Monkeys with GCD had more pronounced immunosuppression with lower CD4-cell counts, more often demonstrable SIV antigen in the blood and LN and had been infected for a longer time period, as compared to monkeys without GCD. These findings show that SIV infection in cynomolgus monkeys is frequently associated with extensive formation of multinucleated GC of macrophage origin, which appears to be related to the pathogenesis of the infection and the degree of immunosuppression.

Antiviral effects of 3'-fluorothymidine and 3'-azidothymidine in cynomolgus monkeys infected with simian immunodeficiency virus.

An acute infection with simian immunodeficiency virus (SIVSM) in cynomolgus monkeys was used to evaluate the antiviral effects of 3'-fluorothymidine (FLT) and 3'-azidothymidine [zidovudine (ZDV)]. Neither compound prevented the infection despite dosing prior to virus inoculation. FLT was about ten times more potent than ZDV in delaying the appearance of SIVSM antigen in the monkeys. The serum half-life of FLT was longer than that of ZDV and ZDV was bound to plasma proteins to about 60% while FLT was virtually unbound. It is proposed that the in vivo difference in potency between ZDV and FLT could, at least partly, be explained as the combined effects of a longer plasma half-life and a higher free concentration of FLT and possibly a higher intracellular concentration of the triphosphate of FLT.

Malignant lymphomas in cynomolgus monkeys infected with simian immunodeficiency virus.

Malignant lymphomas were observed in 38% (9 of 24) of simian immunodeficiency virus (SIV)-infected cynomolgus monkeys (Macaca fascicularis) 5 to 15 months after inoculation with SIV strain SMM3. Lymphomagenesis in the SIV-infected monkeys was not related directly to the SIV-infectious dose given. All SIV-infected animals developed severe immunodeficiency. No significant difference in immunodeficiency was observed between tumor-bearing and non-tumor-bearing animals. In contrast, no lymphomas were observed in a comparable group of HIV-2-infected monkeys, which did not develop immunodeficiency; nor did the noninfected control monkeys. All 9 SIV-related tumors were high-grade B-cell lymphoblastic or pleomorphic lymphomas with extranodal, disseminated growth. Most tumors showed marked infiltration by monocytes and CD8+ T lymphocytes. Occasional tumor infiltrating cells showed immunohistochemical reaction for SIV. The cells of two tumors were established in vitro and shown to be of B-cell phenotype. The tumor cell cultures showed no reverse transcriptase activity and no evidence of virus infection by electron microscopy. Our observations indicate that SIV-induced immunodeficiency in cynomolgus monkeys also mimics HIV infection and AIDS in humans with regard to increased lymphomagenesis and type of lymphomas.