Darlene Sheffer

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.

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

Abstract: 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.

A Noninfectious Simian/Human Immunodeficiency Virus DNA Vaccine That Protects Macaques against AIDS

ABSTRACT Simian/human immunodeficiency virus SHIVKU2 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 (ΔrtSHIVKU2) 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 106 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.

Protection Against Late-Onset AIDS in Macaques Prophylactically Immunized with a Live Simian HIV Vaccine Was Dependent on Persistence of the Vaccine Virus

This is a 5-year follow-up study on 12 macaques that were immunized orally with two live SHIV vaccines, six with V1 and six with V2. All 12 macaques became persistently infected after transient replication of the vaccine viruses; all were challenged vaginally 6 mo later with homologous pathogenic SHIVKU-1. Two of the V1 group developed full-blown AIDS without evidence of vaccine virus DNA in tissues. The data on the 10 vaccinated survivors showed that all 10 became infected with SHIVKU-1 and that DNA of both vaccine and SHIVKU-1 viruses were present 6 mo postchallenge, with minimal replication of SHIVKU-1. During the following 5 years, these animals remained persistently infected, but with only one of the two viruses. Six animals eliminated their vaccine virus after variable periods of time and four of these succumbed to reactivation of the challenge virus and AIDS. Five years after challenge, four latently infected animals, two with V2 and two with SHIVKU-1, were reinoculated with SHIVKU-1. This resulted in transient superinfection and the animals promptly returned to their prechallenge status. Immunosuppression of the four animals 1 year later with Abs to CD8+ lymphocytes resulted in transiently productive replication of their respective latent viruses, and upon recovery of CD8+ lymphocytes, they reverted to their latent virus status. The major finding was that of eight animals that eliminated the vaccine virus, six developed AIDS. The two others harboring SHIVKU-1 remain at risk for developing late-onset disease. The primary correlate against AIDS was persistence of the vaccine virus.

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 Freunds 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.

Characterization of T-Cell Responses in Macaques Immunized with a Single Dose of HIV DNA Vaccine

ABSTRACT 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 SHIVKU2 (Δ4SHIVKU2) 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 Δ4SHIVKU2 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.

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.

P17-01. HIV-specific immune responses induced by a single dose of HIV DNA vaccine in Rhesus macaques

Background DNA vaccine is one of the most promising strategies for development of an efficacious HIV vaccine. Current HIV DNA vaccines induce potent immune responses (IR) in rodents but their immunogenicity remains weak in human and non-human primates (NHP). On the other hand, the MRKAd-5 based HIV vaccine that induces strong HIV-specific T cell responses failed to control HIV infection in human clinical trial. Therefore, strategies that enhance the immunogenicity of HIV DNA vaccines together with a strong commitment to better characterize the vaccine-induced IR are needed. Such comprehensive studies are critical in defining correlates of immune protection.