Testing preexisting antibodies prior to AAV gene transfer therapy: rationale, lessons and future considerations.

Given the increasing number of gene transfer therapy studies either completed or underway, there is growing attention to the importance of preexisting adaptive immunity to the viral vectors used. The recombinant viral vectors developed for gene transfer therapy share structural features with naturally occurring wild-type virus. Antibodies generated against viral vectors obtained through a previous exposure to wild-type virus can potentially compromise transgene expression by blocking transduction, thereby limiting the therapeutic efficacy of the gene transfer therapy; they may also pose potential safety concerns. Therefore, systemic gene transfer delivery requires testing patients for preexisting antibodies. Two different assays have been used: (1) binding assays that focus on total antibodies (both neutralizing and non-neutralizing) and (2) neutralizing assays that detect neutralizing antibodies. In this review we focus on adeno-associated virus-based gene therapies, describing the immune response that occurs to naturally occurring adeno-associated viruses, the implications for patients with this exposure, the assays used to detect preexisting immune responses, and strategies to circumvent preexisting adaptive immunity to expand the patient base that could benefit from such therapies.

Nuclear Transit and HIV LTR Binding of NF-κB Subunits Held by IκB Proteins: Implications for HIV-1 Activation.

No effective therapy to eliminate the HIV latently infected cell reservoir has been developed. One approach, "shock and kill", employs agents that activate HIV, subsequently killing the activated infected cells and/or virus. Shock and kill requires agents that safely and effectively activate HIV. One class of activation agents works through classical NF-κB pathways, but global NF-κB activators are non-specific and toxic. There exist two major IκBs: IκBα, and IκBε, which hold activating NF-κB subunits in the cytoplasm, releasing them for nuclear transit upon cell stimulation. IκBα was considered the main IκB responsible for gene expression regulation, including HIV activation. IκBε is expressed in cells constituting much of the latent HIV reservoir, and IκBε knockout mice have a minimal phenotype, suggesting that IκBε could be a valuable target for HIV activation and reservoir depletion. We previously showed that targeting IκBε yields substantial increases in HIV expression. Here, we show that IκBε holds c-Rel and p65 activating NF-κB subunits in the cytoplasm, and that targeting IκBε with siRNA produces a strong increase in HIV expression associated with enhanced c-Rel and p65 transit to the nucleus and binding to the HIV LTR of the activating NF-κBs, demonstrating a mechanism through which targeting IκBε increases HIV expression. The findings suggest that it may be helpful to develop HIV activation approaches, acting specifically to target IκBε and its interactions with the NF-κBs.

Apoptosis-induced activation of HIV-1 in latently infected cell lines.

BACKGROUND: Despite much work, safe and effective approaches to attack and deplete the long-lived reservoir of cells latently infected with HIV-1 remain an elusive goal. Patients infected with HIV-1 treated with cytotoxic agents or bone marrow transplantation can experience decreases in the reservoir of HIV-1 latently infected cells. Other viruses capable of long-term latency, such as herpesviruses, can sense host cell apoptosis and respond by initiating replication. These observations suggest that other viruses capable of long-term latency, like HIV-1, might also sense when its host cell is about to undergo apoptosis and respond by initiating replication. RESULTS: Pro-monocytic (U1) and lymphoid (ACH-2) HIV-1 persistently infected cell lines were treated with cytotoxic drugs - doxorubicin, etoposide, fludarabine phosphate, or vincristine - and activation of latent HIV-1 was evaluated using assays for HIV-1 RNA and p24 production. Both cell lines showed dose-dependent increases in apoptosis and associated HIV-1 activation following exposure to the cytotoxic agents. Pretreatment of the cells with the pan-caspase inhibitor Z-VAD-FMK prior to exposure to the cytotoxic agents inhibited apoptosis and viral activation. Direct exposure of the latently infected cell lines to activated caspases also induced viral replication. HIV-1 virions produced in association with host cell apoptosis were infectious. CONCLUSIONS: The results indicate that latent HIV-1 can sense when its host cell is undergoing apoptosis and responds by completing its replication cycle. The results may help explain why patients treated with cytotoxic regimens for bone marrow transplantation showed reductions in the reservoir of latently infected cells. The results also suggest that the mechanisms that HIV-1 uses to sense and respond to host cell apoptosis signals may represent helpful new targets for approaches to attack and deplete the long-lived reservoir of cells latently infected with HIV-1.

Targeting IκB proteins for HIV latency activation: the role of individual IκB and NF-κB proteins.

Latently infected cell reservoirs represent the main barrier to HIV eradication. Combination antiretroviral therapy (cART) effectively blocks viral replication but cannot purge latent provirus. One approach to HIV eradication could include cART to block new infections plus an agent to activate latent provirus. NF-κB activation induces HIV expression, ending latency. Before activation, IκB proteins sequester NF-κB dimers in the cytoplasm. Three canonical IκBs, IκBα, IκBß, and IκBε, exist, but the IκB proteins' role in HIV activation regulation is not fully understood. We studied the effects on HIV activation of targeting IκBs by single and pairwise small interfering RNA (siRNA) knockdown. After determining the relative abundance of the IκBs, the relative abundance of NF-κB subunits held by the IκBs, and the kinetics of IκB degradation and resynthesis following knockdown, we studied HIV activation by IκB knockdown, in comparison with those of known HIV activators, tumor necrosis factor alpha (TNF-α), tetradecanoyl phorbol acetate (TPA), and trichostatin A (TSA), in U1 monocytic and J-Lat 10.6 lymphocytic latently infected cells. We found that IκBα knockdown activated HIV in both U1 and J-Lat 10.6 cells, IκBß knockdown did not activate HIV, and, surprisingly, IκBε knockdown produced the most HIV activation, comparable to TSA activation. Our data show that HIV reactivation can be triggered by targeting two different IκB proteins and that IκBε may be an effective target for HIV latency reactivation in T-cell and macrophage lineages. IκBε knockdown may offer attractive therapeutic advantages for HIV activation because it is not essential for mammalian growth and development and because new siRNA delivery strategies may target siRNAs to HIV latently infected cells.

Risk for HIV-1 infection is not associated with repeat-region polymorphism in the DC-SIGN neck domain and novel genetic DC-SIGN variants among North Indians.

BACKGROUND: Several genetic factors have been related to HIV-1 resistance, the homozygosity for a mutation in CCR5 gene (CCR5Delta 32 allele) is presently considered the most relevant one. The C-type lectin, DC-SIGN efficiently binds and transmits HIV-1 to susceptible cell in trans thereby augmenting the infection. A potential association of the DC-SIGN neck domain repeats polymorphism and risk of HIV-1 infection is currently under debate. METHODS: Genetic risk association study was conducted in HIV-1 exposed seronegative (HES; n=50) individuals, HIV-1 seronegative (HSN; n=314) healthy control and HIV-1 infected seropositive patients (HSP; n=190) for polymorphism in neck domain of DC-SIGN gene. The DC-SIGN genotypes were identified by PCR from DNA extracted from peripheral blood and confirmed by sequencing. Fisher exact or chi(2) test was used for statistical analysis. RESULTS: One HSN and HSP individual who were heterozygous (7/8) with respect to DC-SIGN repeat regions were found. The DC-SIGN neck repeat polymorphism among North Indian individuals was not associated with susceptibility to HIV-1 infection. Furthermore, inheritance study of heterozygous mutation (7/8) in HSN individual's family showed that one parent, two brothers, one sister and one daughter were heterozygous (7/8) for DC-SIGN mutant allele. Sequence analyses of DC-SIGN exon 4 repeat region of randomly selected 25 North Indian individuals from HSP, HSN and HES revealed four conserved intronic mutations. These mutations were at nucleotide position 1283, 1306, 1308 upstream and 1906 downstream of the DC-SIGN exon 4 repeat region when compared with the wild type sequence (NCBI Acc. No. AF209479). CONCLUSION: The polymorphism in DC-SIGN neck repeats region was rare and not associated with HIV-1 susceptibility among North Indians. Sequencing analysis of DC-SIGN gene confirmed four novel genetic variants in intronic region flanking exon 4 coding region.