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.

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.

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 SHIV(KU-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 SHIV(KU-1) and that DNA of both vaccine and SHIV(KU-1) viruses were present 6 mo postchallenge, with minimal replication of SHIV(KU-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 SHIV(KU-1), were reinoculated with SHIV(KU-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 SHIV(KU-1) remain at risk for developing late-onset disease. The primary correlate against AIDS was persistence of the vaccine virus.

Presence of Intact vpu and nef genes in nonpathogenic SHIV is essential for acquisition of pathogenicity of this virus by serial passage in macaques.

Use of the macaque model of human immunodeficiency virus (HIV) pathogenesis has shown that the accessory genes nef and vpu are important in the pathogenicity of simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV). We examined the ability of two nonpathogenic SHIVs, SHIV(PPC) and DeltavpuDeltanefSHIV(PPC), to gain pathogenicity by rapid serial passage in macaques. In this study, each virus was passaged by blood intravenously four times at 4-week intervals in macaques. Animals were monitored for 40 weeks for levels of CD4 T cells and quantitative measures of virus infection. DeltavpuDeltanefSHIV(PPC) maintained a limited phase of productive replication in the four animals, with no loss of CD4(+) T cells, whereas SHIV(PPC) became more pathogenic in later passages, judging by plasma viral load and viral mRNA in lymph nodes, infectious peripheral blood mononuclear cells and CD4(+) T cell loss. The nef, LTR, and env of the SHIV(PPC) viruses underwent numerous mutations, compared to DeltavpuDeltanefSHIV(PPC). This study confirms the seminal role that nef, LTR, and vpu could play in regulation of pathogenesis of HIV infection.