Replicating Ad-recombinants encoding non-myristoylated rather than wild-type HIV Nef elicit enhanced cellular immunity.

OBJECTIVE: To determine if immunization with non-myristoylated nef would elicit enhanced cellular immune responses resulting from improved presentation of Nef peptides by MHC-I on the cell surface, and enhanced T-cell help. DESIGN: The myristoylation site of HIV and SIV Nef is required for several Nef functions that modulate the immune response in an infected host, including downregulation of MHC-I, MHC-II, and CD4, and increased expression of the invariant chain on the cell surface. We constructed replication-competent Ad5- and Ad7-HIV recombinants encoding wild-type nef (nefWT) or a nef mutant (nefNM) lacking 19 amino-terminal amino acids, including the myristoylation site, and sequentially immunized chimpanzees mucosally, first with Ad5-HIVnef recombinants and subsequently with Ad7-HIVnef recombinants. METHODS: Peripheral blood lymphocytes were evaluated over the immunization course for Nef-specific cellular immune responses by interferon (IFN)-gamma ELISPOT and T-cell proliferation assays. Nef-specific CD4 and CD8 memory T cells that produced intracellular IFN-gamma, interleukin-2, and tumor necrosis factor (TNF)-alpha were assessed by flow cytometry. RESULTS: In comparison to immunization with Ad-HIVnefWT, Ad-HIVnefNM elicited statistically significant increases in numbers of IFN-gamma-secreting cells after the Ad7-HIVnefNM immunization and increased T-cell proliferative responses following both Ad5- and Ad7-HIVnefNM immunizations. Nef-specific CD4 and CD8 memory T-cell populations secreting TNF-alpha were also significantly increased in the Ad-HIVnefNM immunization group. CONCLUSIONS: The results support the hypothesis that immunization with Ad-recombinants encoding HIVnefNM rather than HIVnefWT elicits enhanced cellular immunity resulting from improved antigen presentation and greater T-cell help.

Enhanced cellular immunity to SIV Gag following co-administration of adenoviruses encoding wild-type or mutant HIV Tat and SIV Gag.

Among candidate antigens for human immunodeficiency virus (HIV) prophylactic vaccines, the regulatory protein Tat is a critical early target, but has a potential for immune suppression. Adenovirus (Ad) recombinants encoding wild-type HIV Tat (Tat-wt) and a transdominant negative mutant HIV Tat (Tat22) were constructed and administered to mice separately or together with Ad-SIVgag. Immunogenicity and effects on immune responses to the co-administered Gag immunogen were evaluated. Wild-type and mutant Tat recombinants elicited similar Tat-specific cellular and humoral immune responses. Co-administration of either Tat immunogen with Ad-SIVgag induced modest but significant enhancement of Gag-specific interferon-gamma secreting T cells and lymphoproliferative responses. Neither the Ad-recombinant encoding Tat-wt nor Tat22 suppressed induction of anti-Tat or anti-Gag antibodies. Based on the immune responses observed in mice, both recombinants appear to be suitable vaccine candidates. Their contribution to protective efficacy remains to be determined in a non-human primate model.

Replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies.

A major challenge in combating the human immunodeficiency virus (HIV) epidemic is the development of vaccines capable of inducing potent, persistent cellular immunity and broadly reactive neutralizing antibody responses to HIV type 1 (HIV-1). We report here the results of a preclinical trial using the chimpanzee model to investigate a combination vaccine strategy involving sequential priming immunizations with different serotypes of adenovirus (Ad)/HIV-1(MN)env/rev recombinants and boosting with an HIV envelope subunit protein, oligomeric HIV(SF162) gp140deltaV2. The immunogenicities of replicating and nonreplicating Ad/HIV-1(MN)env/rev recombinants were compared. Replicating Ad/HIV recombinants were better at eliciting HIV-specific cellular immune responses and better at priming humoral immunity against HIV than nonreplicating Ad-HIV recombinants carrying the same gene insert. Enhanced cellular immunity was manifested by a greater frequency of HIV envelope-specific gamma interferon-secreting peripheral blood lymphocytes and better priming of T-cell proliferative responses. Enhanced humoral immunity was seen in higher anti-envelope binding and neutralizing antibody titers and better induction of antibody-dependent cellular cytotoxicity. More animals primed with replicating Ad recombinants mounted neutralizing antibodies against heterologous R5 viruses after one or two booster immunizations with the mismatched oligomeric HIV-1(SF162) gp140deltaV2 protein. These results support continued development of the replicating Ad-HIV recombinant vaccine approach and suggest that the use of replicating vectors for other vaccines may prove fruitful.

HIV-2 derived lentiviral vectors: gene transfer in Parkinson's and Fabry disease models in vitro.

Lentiviral vectors are prime candidate vectors for gene transfer into dividing and non-dividing cells, including neuronal cells and stem cells. For safety, HIV-2 lentiviral vectors may be better suited for gene transfer in humans than HIV-1 lentiviral vectors. HIV-2 vectors cross-packaged in HIV-1 cores may be even safer. Demonstration of the efficacy of these vectors in disease models will validate their usefulness. Parkinson's disease and Fabry disease provide excellent models for validation. Parkinson's disease is a focal degeneration of dopaminergic neurons in the brain with progressive loss of ability to produce the neurotransmitter dopamine. Current treatment entails administration of increasing doses of L-dopa, with attendant toxicity. We explore here the hypothesis that gene transfer of aromatic acid decarboxylase (AADC), a key enzyme in the pathway, will make neuronal cells more efficiently convert L-dopa into dopamine. Fabry disease on the other hand is a monogenic inherited disease, characterized by alpha-galactosidase A (AGA) deficiency, resulting in glycolipid accumulation in several cell types, including fibroblasts. Animal models for preclinical investigations of both of these diseases are available. We have designed monocistronic HIV-1 and HIV-2 vectors with the AADC transgene and monocistronic and bicistronic HIV-2 vectors with the AGA and puromycin resistance transgenes. They were packaged with either HIV-2 cores or HIV-1 cores (hybrid vectors). Gene transfer of AADC gene in neuronal cells imparted the ability on the transduced cells to efficiently convert L-dopa into dopamine. Similarly, the AGA vectors induced Fabry fibroblasts to produce high levels of AGA enzyme and caused rapid clearance of the glycolipids from the cells. Both monocistronic and bicistronic vectors were effective. Thus, the insertion of a second gene downstream in the bicistronic vector was not deleterious. In addition, both the self-packaged vectors and the cross-packaged hybrid vectors were effective in gene transfer.

Human immunodeficiency virus type 2 lentiviral vectors: packaging signal and splice donor in expression and encapsidation.

Retroviral vectors provide the means for gene transfer with long-term expression. The lentivirus subgroup of retroviruses, such as human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), possesses a number of regulatory and accessory genes and other special elements. These features can be exploited to design vectors for transducing non-dividing as well as dividing cells with the potential for regulated transgene expression. Encapsidation of the transgene RNA in lentiviral vectors is determined by the leader sequence-based multipartite packaging signal. Embedded in the packaging signal is a major splice donor site that, this study shows, is not by itself essential for transgene expression or encapsidation. We designed HIV-2 vectors that contained all the sequence elements thought to be necessary and sufficient for vector RNA encapsidation. Unexpectedly, despite abundant expression, only a small fraction of the transgene RNA was encapsidated and the titre of the vector was low. Redesign of the vector with a mutant splice donor resulted in increased vector RNA encapsidation and yielded vectors with high titre. Inefficient encapsidation by the conventionally designed vector was not due to suboptimal Rev responsive element (RRE)-Rev function. Varying the length of RRE in the vector did not change vector RNA encapsidation, nor did the introduction of a synthetic intron into the mutant vector. The vector RNA with the intact splice donor may have been excessively spliced, decreasing the amount of packageable RNA. A titre of 10(5) transducing units (TU)/ml was readily obtained for vectors with the neo or GFP transgene, and the vector could be concentrated to a titre of 1-5x10(7) TU/ml.