Immunogenicity of a lentiviral-based DNA vaccine driven by the 5'LTR of the naturally attenuated caprine arthritis encephalitis virus (CAEV) in mice and macaques.

Increasing the safety and the efficacy of existing HIV vaccines is one of the strategies that could help to promote the development of a vaccine for human use. We developed a HIV DNA vaccine (Δ4-SHIVKU2) that has been shown to induce potent polyfunctional HIV-specific T cell responses following a single dose immunization of mice and macaques. Δ4-SHIVKU2 also induced protection when immunized macaques were challenged with homologous pathogenic viruses. In the present study, our aim was to examine whether a chimeric HIV DNA vaccine (CAL-Δ4-SHIVKU2) whose genome is driven by the LTR of the goat lentivirus, caprine arthritis encephalitis (CAEV) expresses efficiently the vaccine antigens and induces potent immune responses in animal models for HIV vaccine. Data of radioimmunoprecipitation assays clearly show that this chimeric genome drives efficient expression of all HIV antigens in the construct. In addition, evaluation of the p24 Gag protein in the supernatant of HEK-293-T cells transfected in parallel with Δ4-SHIVKU2 and CAL-Δ4-SHIVKU2 showed no difference suggesting that these two LTRs are inducing equally the expression of the viral genes. Immunization of mice and macaques using our single dose immunization regimen resulted in induction of similar IFN-γ ELISPOT responses in Δ4-SHIVKU2- and CAL-Δ4-SHIVKU2-treated mice. Similar profiles of T cell responses were also detected both in mice and macaques when multiparametric flow cytometry analyses were performed. Since CAEV LTR is not dependent of Tat to drive viral gene expression and is not functional for integration with HIV integrase, this new vector increases the safety and efficacy of our vaccine vectors and vaccination strategy.

Characterization of T-cell responses in macaques immunized with a single dose of HIV DNA vaccine.

The optimization of immune responses (IR) induced by HIV DNA vaccines in humans is one of the great challenges in the development of an effective vaccine against AIDS. Ideally, this vaccine should be delivered in a single dose to immunize humans. We recently demonstrated that the immunization of mice with a single dose of a DNA vaccine derived from pathogenic SHIV(KU2) (Delta4SHIV(KU2)) induced long-lasting, potent, and polyfunctional HIV-specific CD8(+) T-cell responses (G. Arrode, R. Hegde, A. Mani, Y. Jin, Y. Chebloune, and O. Narayan, J. Immunol. 178:2318-2327, 2007). In the present work, we expanded the characterization of the IR induced by this DNA immunization protocol to rhesus macaques. Animals immunized with a single high dose of Delta4SHIV(KU2) DNA vaccine were monitored longitudinally for vaccine-induced IR using multiparametric flow cytometry-based assays. Interestingly, all five immunized macaques developed broad and polyfunctional HIV-specific T-cell IR that persisted for months, with an unusual reemergence in the blood following an initial decline but in the absence of antibody responses. The majority of vaccine-specific CD4(+) and CD8(+) T cells lacked gamma interferon production but showed high antigen-specific proliferation capacities. Proliferative CD8(+) T cells expressed the lytic molecule granzyme B. No integrated viral vector could be detected in mononuclear cells from immunized animals, and this high dose of DNA did not induce any detectable autoimmune responses against DNA. Taken together, our comprehensive analysis demonstrated for the first time the capacity of a single high dose of HIV DNA vaccine alone to induce long-lasting and polyfunctional T-cell responses in the nonhuman primate model, bringing new insights for the design of future HIV vaccines.

Rhesus macaque model of chronic opiate dependence and neuro-AIDS: longitudinal assessment of auditory brainstem responses and visual evoked potentials.

Our work characterizes the effects of opiate (morphine) dependence on auditory brainstem and visual evoked responses in a rhesus macaque model of neuro-AIDS utilizing a chronic continuous drug delivery paradigm. The goal of this study was to clarify whether morphine is protective, or if it exacerbates simian immunodeficiency virus (SIV)-related systemic and neurological disease. Our model employs a macrophage tropic CD4/CCR5 coreceptor virus, SIV(mac)239 (R71/E17), which crosses the blood-brain barrier shortly after inoculation and closely mimics the natural disease course of human immunodeficiency virus infection. The cohort was divided into three groups: morphine only, SIV only, and SIV + morphine. Evoked potential (EP) abnormalities in subclinically infected macaques were evident as early as 8 weeks postinoculation. Prolongations in EP latencies were observed in SIV-infected macaques across all modalities. Animals with the highest cerebrospinal fluid viral loads and clinical disease showed more abnormalities than those with subclinical disease, confirming our previous work (Raymond et al., J Neurovirol 4:512-520, 1998; J Neurovirol 5:217-231, 1999; AIDS Res Hum Retroviruses 16:1163-1173, 2000). Although some differences were observed in auditory and visual evoked potentials in morphine-treated compared to morphine-untreated SIV-infected animals, the effects were relatively small and not consistent across evoked potential type. However, morphine-treated animals with subclinical disease had a clear tendency toward higher virus loads in peripheral and central nervous system tissues (Marcario et al., J Neuroimmune Pharmacol 3:12-25, 2008) suggesting that if had been possible to follow all animals to end-stage disease, a clearer pattern of evoked potential abnormality might have emerged.

Longitudinal study to assess the safety and efficacy of a live-attenuated SHIV vaccine in long term immunized rhesus macaques.

Live-attenuated viruses derived from SIV and SHIV have provided the most consistent protection against challenge with pathogenic viruses, but concerns regarding their long-term safety and efficacy have hampered their clinical usefulness. We report a longitudinal study in which we evaluated the long-term safety and efficacy of DeltavpuSHIV(PPC), a live virus vaccine derived from SHIV(PPC). Macaques were administered two inoculations of DeltavpuSHIV(PPC), three years apart, and followed for eight years. None of the five vaccinated macaques developed an AIDS-like disease from the vaccine. At eight years, macaques were challenged with pathogenic SIV and SHIV. None of the four macaques with detectable cellular-mediated immunity prior to challenge had detectable viral RNA in the plasma. This study demonstrates that multiple inoculations of a live vaccine virus can be used safely and can significantly extend the efficacy of the vaccine, as compared to a single inoculation, which is efficacious for approximately three years.

Nef modulates the immunogenicity of Gag encoded in a non-infectious HIV DNA vaccine.

Gag-CD8+ T cell responses are associated with immune control of HIV infection. Since during HIV infection Nef impairs T cell responses, we evaluated whether deletion of nef from a non-infectious HIV DNA vaccine (Delta4 Nef+), creating Delta5 Nef(-), would affect its immunogenicity. When compared with Delta4, mice injected with Delta5 developed significantly lower CD8+ T cell responses to Gag, but no significant change in the responses to Env was observed. In vitro, deletion of Nef abrogated the induced cell death, production of virus-like particles and release of Gag from transfected cells. Thus, the effect of Nef in causing extrusion of Gag might adjuvant the CD8+ T cell responses to Gag in DNA vaccine.

Roles of MCP-1 in development of HIV-dementia.

The encephalopathy caused by HIV, known clinically as HIV-associated dementia (HAD) and pathologically as HIV encephalitis (HIVE), results from intense infiltration of mononuclear cells, productive replication of the virus in monocyte-derived macrophages/microglia, abortive replication in astrocytes and activation of macrophages/microglia and astrocytes leading to neuronal degeneration in the brains of infected persons. Recent findings have suggested that development of HAD is based more on the activation process than on direct evidence of virus replication in the brain. Since HAD is based on the encephalitic process, major studies have been directed to the mechanisms regulating the inflammatory process. Monocyte chemoattractant protein 1, MCP-1, is a chemokine that is implicated in this process and also in the development of activation in the brain. In this review, we have attempted to identify mechanisms that induce expression of MCP-1 in the brain and the role that it plays in recruitment of mononuclear cells from blood to brain and in the activation processes of inflammatory and neural cells that lead to development of degenerative changes in the neuronal population.

Comparison of the effects of pathogenic simian human immunodeficiency virus strains SHIV-89.6P and SHIV-KU2 in cynomolgus macaques.

Factors explaining why human immunodeficiency virus (HIV) enhances the risk of reactivated tuberculosis (TB) are poorly understood. Unfortunately, experimental models of HIV-induced reactivated TB are lacking. We examined whether cynomolgus macaques, which accurately model latent TB in humans, could be used to model pathogenesis of HIV infection in the lungs and associated lymph nodes. These experiments precede studies modeling the effects of HIV infection on latent TB. We infected two groups of macaques with chimeric simian-human immunodeficiency viruses (SHIV-89.6P and SHIV-KU2) and followed viral titers and immunologic parameters including lymphocytes numbers and phenotype in the blood, bronchoalveolar lavage cells, and lymph nodes over the course of infection. Tissues from the lungs, liver, kidney, spleen, and lymph nodes were similarly examined at necropsy. Both strains produced dramatic CD4(+) T cell depletion. Plasma titers were not different between viruses, but we found more SHIV-89.6P in the lungs. Both viruses induced similar patterns of cell activation markers. SHIV-89.6P induced more IFN-gamma expression than SHIV-KU2. These results indicate SHIV-89.6P and SHIV-KU2 infect cynomolgus macaques and may be used to accurately model effects of HIV infection on latent TB.

Effect of morphine on the neuropathogenesis of SIVmac infection in Indian Rhesus Macaques.

Morphine is known to prevent the development of cell-mediated immune (CMI) responses and enhance expression of the CCR5 receptor in monocyte macrophages. We undertook a study to determine the effect of morphine on the neuropathogenesis and immunopathogenesis of simian immunodeficiency virus (SIV) infection in Indian Rhesus Macaques. Hypothetically, the effect of morphine would be to prevent the development of CMI responses to SIV and to enhance the infection in macrophages. Sixteen Rhesus Macaques were divided into three experimental groups: M (morphine only, n = 5), VM (morphine + SIV, n = 6), and V (SIV only, n = 5). Animals in groups M and VM were given 2.5 mg/kg of morphine sulfate, four times daily, for up to 59 weeks. Groups VM and V were inoculated with SIVmacR71/17E 26 weeks after the beginning of morphine administration. Morphine prevented the development of enzyme-linked immunosorbent spot-forming cell CMI responses in contrast to virus control animals, all of which developed CMI. Whereas morphine treatment had no effect on viremia, cerebrospinal fluid viral titers or survival over the time course of the study, the drug was associated with a tendency for greater build-up of virus in the brains of infected animals. Histopathological changes in the brains of animals that developed disease were of a demyelinating type in the VM animals compared to an encephalitic type in the V animals. This difference may have been associated with the immunosuppressive effect of the drug in inhibiting CMI responses.

Protection of macaques against AIDS with a live attenuated SHIV vaccine is of finite duration.

Using background data that live vaccines against several viral pathogens are effective in inducing life-long protection against disease, we undertook studies in macaques to determine the duration of protection that two live SHIV vaccines could induce against AIDS. Earlier studies had established that macaques immunized once with a live vaccine and challenged 6 months later were protected, and that other macaques given two sequential inoculations of live vaccines were protected for at least 1 year. Protection was associated with persistence of the vaccine viruses. In this study, we sought to determine whether the duration of protection in macaques given a single inoculation of replication competent live vaccines would extend beyond 3 years. Two groups of four rhesus macaques were inoculated with two live SHIV vaccines, respectively. The viruses replicated transiently in all animals but at the 3-year time point, PCR analysis of PBMC did not detect DNA of either virus in any of the animals, and all were negative for CMI responses in the blood. All 8 animals succumbed to disease when challenged with pathogenic viruses.

Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef.

Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes.

Phenotypic and functional analysis of immune CD8+ T cell responses induced by a single injection of a HIV DNA vaccine in mice.

HIV DNA vaccines are potent inducers of cell-mediated immune (CMI) response in mice but elicit poor HIV-specific IFN-gamma-producing T cells in monkeys and humans. In this study, we performed kinetic analyses on splenocytes of BALB/c mice that were immunized by a single injection with a unique DNA vaccine. Using IFN-gamma-ELISPOT and multiparametric FACS analysis, we characterized the induced CMI response. We found that the response was detectable for at least 63 wk. ELISPOT detection of IFN-gamma-producing T cells showed a profile with two waves separated by a long period of minimal response. Multiparametric FACS analysis showed two populations of CD3(+)CD8(+) T cells that were specific for all HIV Ags. These cells had similar robust proliferation abilities and contained granzyme B. However, only a few produced IFN-gamma. Both IFN-gamma-producing and non-IFN-gamma-producing HIV-specific CD8(+) T cells were detected in the early stage (week (W)1 and W2 postimmunization (PI)), in the prolonged intermediate period of minimal response (W4-W26 PI), and in the final late phase of increased response (W30-W63 PI). Our longitudinal characterization showed that both subsets of cells underwent expansion, contraction, and memory generation/maintenance phases throughout the lifespan of the animal. Altogether, these findings bring insight to the heterogeneity of the immune T cell response induced by a single immunization with this DNA and strengthen the concept that used of the IFN-gamma-ELISPOT assay alone may be insufficient to detect critical T cell responses to candidate HIV vaccines.

Upregulation of expression of platelet-derived growth factor and its receptor in pneumonia associated with SHIV-infected macaques.

BACKGROUND: HIV-associated pulmonary disorders are the most frequent cause of AIDS-related deaths. Rhesus macaques infected with SIV-HIV (SHIV) recapitulate the human HIV-1 lung disease and provide an excellent working model to study the pathogenesis of the human syndrome. Lungs of macaques with SHIV-associated pneumonia have pathology involving macrophage and T cell infiltration that is often accompanied with concurrent opportunistic infections. OBJECTIVE: To explore the relationship between SHIV-associated respiratory disease and the expression of platelet-derived growth factor (PDGF) B chain (PDGF-B) and its cognate receptors, PDGF-Ralpha and PDGF-Rbeta, which have been implicated in chronic inflammatory processes. METHODS: Lung tissues from 10 SHIV-infected rhesus macaques were evaluated for pathological changes and correlation of these lesions with PDGF-B/PDGF-R expression by real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. RESULTS: Virus-associated pneumonia was associated with virus replication in macrophages in the lungs, enhanced recruitment of macrophages and mononuclear cells into the organ, and, occasionally, fibrosis. These changes were accompanied by upregulation of PDGF-B and its cognate receptors in the diseased tissue. Confocal microscopy identified SHIV-infected macrophages as one of the major cell types expressing PDGF-B and PDGF-Ralpha/beta in the affected lungs. CONCLUSION: These results suggest that PDGF and its cognate receptors play a critical role in the pathogenesis of pulmonary disease associated with this virus.

Immunoprophylaxis against AIDS in macaques with a lentiviral DNA vaccine.

We earlier reported that immunization of macaques with a reverse transcriptase-deleted SHIV(KU2) (DeltartSHIV(KU2)) plasmid that contained HIV-1(HXB2) env and SIV gag-nef induced protection against AIDS caused by challenge virus SHIV89.6P with a heterologous env. We further deleted vif and integrase from DeltartSHIV(KU2) and substituted the 3'LTR with SV40 poly A sequences, creating Delta4SHIV(KU2) (M) and a parallel construct containing gag-nef of HIV-1(SF2), Delta4SHIV(KU2) (H). Six macaques received two intramuscular injections of the (M) DNA, and another six received three injections of the (H) DNA. Three of the latter group received two post-challenge boosts with (M) DNA vaccine. Seven virus control macaques were inoculated with SHIV89.6P. All twelve immunized macaques were challenged with SHIV89.6P virus, and CMI responses were measured by ELISPOT assays. Virus control animals all developed progressive infection, whereas vaccinated macaques from both groups controlled virus replication, with plasma viral loads dropping to undetectable levels between weeks 6 and 126 p.i. This DNA vaccine was efficacious even though it encoded Env, Gag, and Nef that were genetically distinct from the proteins in the challenge virus. The DNA vaccine induced broad-based protection without using viral proteins to boost the immunity.

Therapy of "SHIV" infected macaques with liposomes delivering antisense interleukin-4 DNA.

BACKGROUND/OBJECTIVES: To explore the effects of antisense (AS) interleukin (IL)-4 on virus replication and CD8+ T-cell responses in lymph nodes and blood of macaques infected with simian human immunodeficiency virus, SHIV(89.6)P. METHODS: Six macaques were inoculated with simian human immunodeficiency virus (SHIV(89.6)P). Seven days later, four of the animals were given 1 mg AS IL-4 plasmid complexed with Megafectin liposome, intravenously, and two of these received a second injection of the same material on day 9. All six macaques were killed at 2 weeks post infection (pi) and monitored for viral RNA and CD8+ T cells in blood and lymph nodes by real-time reverse transcriptase-polymerase chain reaction, flow cytometry and immunohistochemistry. RESULTS: In contrast to the lymph nodes from virus control animals, the lymph nodes of AS IL-4-treated animals had a significant reduction in viral loads and reduced depletion of cells from the nodes. There was an increase in CD8+ T cells in the nodes, and many of the cells expressed granzyme B, suggesting functional activation. This trend of virus reduction and increased CD8+ T cell numbers was also reflected in blood. CONCLUSIONS: The therapeutic effect of the AS IL-4 suggests indirectly that the acute immunosuppressive disease caused by SHIVs is mediated, in part, by IL-4 that causes enhanced virus replication by suppressing anti-viral CD8+ T-cell responses, and that this effect was reduced by treatment of the animals with AS IL-4.

CXCL10-induced cell death in neurons: role of calcium dysregulation.

Chemokines play a key role in the regulation of central nervous system disease. CXCL10 over-expression has been observed in several neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease and HIV-associated dementia. More recent studies by others and us have shown that CXCL10 elicits apoptosis in fetal neurons. The mechanism of CXCL10-mediated neurotoxicity, however, remains unclear. In this study, we provide evidence for the direct role of Ca(2+) dysregulation in CXCL10-mediated apoptosis. We demonstrate that treatment of fetal neuronal cultures with exogenous CXCL10 produced elevations in intracellular Ca(2+) and that this effect was modulated via the binding of CXCL10 to its cognate receptor, CXCR3. We further explored the association of intracellular Ca(2+) elevations with the caspases that are involved in CXC10-induced neuronal apoptosis. Our data showed that increased Ca(2+), which is available for uptake by the mitochondria, is associated with membrane permeabilization and cytochrome c release from this compartment. The released cytochrome c then activates the initiator active caspase-9. This initiator caspase sequentially activates the effector caspase-3, ultimately leading to apoptosis. This study identifies the temporal signaling cascade involved in CXCL10-mediated neuronal apoptosis and provides putative targets for pharmaceutical intervention of neurological disorders associated with CXCL10 up-regulation.

Antigen expression kinetics and immune responses of mice immunized with noninfectious simian-human immunodeficiency virus DNA.

In a previous report we demonstrated that three injections of an rt-deleted noninfectious genome of the simian-human immunodeficiency virus SHIV(KU2) induced protection against AIDS in macaques (D. K. Singh, Z. Liu, D. Sheffer, G. A. Mackay, M. Smith, S. Dhillon, R. Hegde, F. Jia, I. Adany, and O. Narayan, J. Virol 79:3419-3428, 2005). To make this DNA safer, we deleted two more genes, the integrase gene and vif, along with the 3' long terminal repeat. We also replaced the gag, pro, and nef genes (SIVmac239 origin) with those of human immunodeficiency virus (HIV) type 1 strain SF2. The resultant construct, designated delta4SHIV(KU2) DNA, was used in this study to evaluate gene expression and immunogenicity in BALB/c mice. DNA-transfected human embryonic kidney epithelial cells (HEK 293) produced all of the major viral proteins and released p24 in the supernatant for 12 days. Inoculation of the vaccine DNA into the gastrocnemius muscles resulted in intense mononuclear cell infiltration at the inoculated sites and the production of viral p24 in myocytes, in infiltrating mononuclear cells, and in cells in the spleen and draining lymph nodes between 3 and 10 days postinoculation. Expression of p24 in the muscle cells peaked at day 7 and became undetectable after day 12. The same 12-day period of expression of p24 was observed in mice that were given a second injection 4 weeks after the first. Evaluation of immune responses in BALB/c mice revealed that the DNA induced enzyme-linked immunospot and antigen-specific proliferative cell-mediated immunity responses. The responses were stronger in mice that were coinjected with a second plasmid expressing granulocyte-macrophage colony-stimulating factor. Since new waves of viral antigen production could be induced with each boosting injection of the vaccine DNA, this DNA could be a safe and efficient agent to induce long-term protection against HIV.

Active simian immunodeficiency virus (strain smmPGm) infection in macaque central nervous system correlates with neurologic disease.

Simian immunodeficiency virus strain smmPGm can induce neuropathology in macaques and is a model for the development of human HIV-related brain injury. For quantitative studies of proviral presence and expression in the central nervous system (CNS), we inoculated 8 macaques intravenously with the virus. Three animals were necropsied 2 to 4 weeks after development of infection, and we obtained lymphoid tissue biopsies from 5 animals before 5 weeks after infection. Peak plasma viral loads averaged 10 viral RNA Eq/mL at week 2, whereas cerebrospinal fluid viral loads peaked at 10 viral RNA Eq/mL. The proviral DNA loads and viral gag mRNA expression in tissues were quantified by real-time polymerase chain reaction. Two animals developed neurologic disease characterized by meningoencephalitis and meningitis. Proviral DNA levels in CNS tissues of these animals at necropsy revealed 10 and 10 copies/microg of DNA, respectively, whereas viral RNA expression in the CNS reached 100 to 1000 times higher levels than those seen in early necropsies. In sharp contrast, in 2 animals necropsied at later times without CNS disease, virus mRNA expression was not detected in any CNS tissue. Our results are consistent with the hypothesis that active virus expression in the CNS is strongly correlated with neurologic disease and that the event occurs at variable periods after infection.

Simian human immunodeficiency virus-associated pneumonia correlates with increased expression of MCP-1, CXCL10, and viral RNA in the lungs of rhesus macaques.

Pulmonary disorders are the most frequent cause of death in HIV-1-infected individuals with AIDS and remain important even in the current era of potent antiretroviral therapy. Macaques infected with Simian/Human Immunodeficiency Virus (SHIV) develop pulmonary disease and concurrent opportunistic infections similar to those observed in HIV-infected individuals, thereby providing an excellent working model to elucidate the pathogenesis of the human lung disease. Since chemokines play a crucial role in the recruitment of inflammatory cells to tissues, we investigated the relationship between respiratory disease and the levels of chemokines, monocyte chemotactic protein-1 (MCP-1) and CXCL10, in the lungs of SHIV-infected rhesus macaques. We found that lung pathology in infected macaques was closely associated with overexpression of MCP-1 and CXCL10. In addition, these chemokines could, in part, be responsible for the recruitment of inflammatory cells infiltrating into the diseased lungs as demonstrated by chemotactic assays. Lung pathology and increased levels of MCP-1 and CXCL10 correlated with high viral loads in the lung parenchyma. Using confocal microscopy, we identified SHIV-infected macrophages as the major producers of MCP-1 and CXCL10 in the diseased lungs. These data suggest that chemokine overexpression plays an important role in the pathogenesis of SHIV-associated pulmonary disease in macaques.

A noninfectious simian/human immunodeficiency virus DNA vaccine that protects macaques against AIDS.

Simian/human immunodeficiency virus SHIV(KU2) replicates with extremely high titers in macaques. In order to determine whether the DNA of the viral genome could be used as a vaccine if the DNA were rendered noninfectious, we deleted the reverse transcriptase gene from SHIVKU2 and inserted this DNA (DeltartSHIVKU2) into a plasmid that was then used to test gene expression and immunogenicity. Transfection of Jurkat and human embryonic kidney epithelial (HEK 293) cells with the DNA resulted in production of all of the major viral proteins and their precursors and transient export of a large quantity of the Gag p27 into the supernatant fluid. As expected, no infectious virus was produced in these cultures. Four macaques were injected intradermally with 2 mg of the DNA at 0, 8, and 18 weeks. The animals developed neutralizing antibodies and low enzyme-linked immunospot assay (E-SPOT) titers against SHIVKU2. These four animals and two unvaccinated control animals were then challenged with heterologous SHIV89.6P administered into their rectums. The two control animals developed viral RNA titers exceeding 10(6) copies/ml of plasma, and these titers were accompanied by the loss of CD4+ T cells by 2 weeks after challenge. The two control animals died at weeks 8 and 16, respectively. All four of the immunized animals became infected with the challenge virus but developed lower titers of viral RNA in plasma than the control animals, and the titers decreased over time in three of the four macaques. The fourth animal remained viremic and died at week 47. Whereas the control animals failed to develop E-SPOT responses, all four of the immunized animals developed anamnestic E-SPOT responses after challenge. The animal that died developed the highest E-SPOT response and was the only one that produced neutralizing antibodies against the challenge virus. These results established that noninfectious DNA of pathogenic SHIV could be used as a vaccine to prevent AIDS, even though the immunological assays used did not predict the manner in which the challenge virus would replicate in the vaccinated animals.

Inhibition of pathogenic SHIV replication in macaques treated with antisense DNA of interleukin-4.

Interleukin-4 is implicated in the pathogenesis of HIV-induced AIDS and causes enhancement of replication of virus strains that use the CXCR4 (X4) coreceptor. In this study, we explored the effects of interleukin-4 (IL-4) antisense (AS) DNA on replication of X4, simian human immunodeficiency viruses, SHIV(KU-2) and SHIV89.6P. AS IL-4 oligomer caused inhibition of virus replication in cultures of CD4+ T cells and macrophages derived from macaques. Plasmid expressing AS IL-4 DNA was also effective in abrogating virus replication in macrophage cultures. Relevance of these cell culture studies was confirmed in vivo by treating SHIV89.6P-infected macaques with AS IL-4 DNA. Six macaques were inoculated with the virus, and 4 were treated with AS IL-4 DNA. This resulted in a significant decrease in viral RNA concentrations in the liver, lungs, and spleen tissues that are all sites of virus replication in macrophages. This is the first demonstration of effective inhibition of an HIV-like virus in tissues by AS DNA of a cytokine. In the present era of increasing resistance of HIV to antiviral compounds, exploration of adjunct therapies directed at host responses in combination with antiretroviral drugs may be of value for the treatment of AIDS.