Alternate NF-κB-Independent Signaling Reactivation of Latent HIV-1 Provirus.

Current combination antiretroviral therapies (cART) are unable to eradicate HIV-1 from infected individuals because of the establishment of proviral latency in long-lived cellular reservoirs. The shock-and-kill approach aims to reactivate viral replication from the latent state (shock) using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells (kill) by specific therapeutics. The NF-κB RelA/p50 heterodimer has been characterized as an essential component of reactivation of the latent HIV-1 long terminal repeat (LTR). Nevertheless, prolonged NF-κB activation contributes to the development of various autoimmune, inflammatory, and malignant disorders. In the present study, we established a cellular model of HIV-1 latency in J-Lat CD4+ T cells that stably expressed the NF-κB superrepressor IκB-α 2NΔ4 and demonstrate that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivated HIV-1 from latency, even under conditions where NF-κB activation was repressed. Using specific calcineurin phosphatase, p38, and MEK1/MEK2 kinase inhibitors or specific short hairpin RNAs, c-Jun was identified to be an essential factor binding to the LTR enhancer κB sites and mediating the combined synergistic reactivation effect. Furthermore, acetylsalicylic acid (ASA), a potent inhibitor of the NF-κB activator kinase IκB kinase ß (IKK-ß), did not significantly diminish reactivation in a primary CD4+ T central memory (TCM) cell latency model. The present work demonstrates that the shock phase of the shock-and-kill approach to reverse HIV-1 latency may be achieved in the absence of NF-κB, with the potential to avoid unwanted autoimmune- and or inflammation-related side effects associated with latency-reversing strategies.IMPORTANCE The shock-and-kill approach consists of the reactivation of HIV-1 replication from latency using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells. The cellular transcription factor NF-κB is considered a master mediator of HIV-1 escape from latency induced by LRAs. Nevertheless, a systemic activation of NF-κB in HIV-1-infected patients resulting from the combined administration of different LRAs could represent a potential risk, especially in the case of a prolonged treatment. We demonstrate here that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivate HIV-1 from latency, even under conditions where NF-κB activation is repressed. Our study provides a molecular proof of concept for the use of anti-inflammatory drugs, like aspirin, capable of inhibiting NF-κB in patients under combination antiretroviral therapy during the shock-and-kill approach, to avoid potential autoimmune and inflammatory disorders that can be elicited by combinations of LRAs.

Genetic diversity in the env V1-V2 region of proviral quasispecies from long-term controller MHC-typed cynomolgus macaques infected with SHIVSF162P4cy.

Intra-host evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) has been shown by viral RNA analysis in subjects who naturally suppress plasma viremia to low levels, known as controllers. However, little is known about the variability of proviral DNA and the inter-relationships among contained systemic viremia, rate of reservoir reseeding and specific major histocompatibility complex (MHC) genotypes, in controllers. Here, we analysed the proviral DNA quasispecies of the env V1-V2 region, in PBMCs and in anatomical compartments of 13 long-term controller monkeys after 3.2 years of infection with simian/human immunodeficiency virus (SHIV)SF162P4cy. A considerable variation in the genetic diversity of proviral quasispecies was present among animals. Seven monkeys exhibited env V1-V2 proviral populations composed of both clusters of identical ancestral sequences and new variants, whereas the other six monkeys displayed relatively high env V1-V2 genetic diversity with a large proportion of diverse novel sequences. Our results demonstrate that in SHIVSF162P4cy-infected monkeys there exists a disparate pattern of intra-host viral diversity and that reseeding of the proviral reservoir occurs in some animals. Moreover, even though no particular association has been observed between MHC haplotypes and the long-term control of infection, a remarkably similar pattern of intra-host viral diversity and divergence was found within animals carrying the M3 haplotype. This suggests that in animals bearing the same MHC haplotype and infected with the same virus, viral diversity follows a similar pattern with similar outcomes and control of infection.

Effect of MHC haplotype on immune response upon experimental SHIVSF162P4cy infection of Mauritian cynomolgus macaques.

Little is known about the effects of Major Histocompatibility Complex (MHC) haplotypes on immunity to primate lentiviruses involving both acquired and innate immune responses. We present statistical evidence of the influence of MHC polymorphism on antiviral immunity of Mauritian cynomolgus macaques (MCM) following simian/human immunodeficiency virus SHIVSF162P4cy infection, involving the production of pro- and anti-inflammatory cytokines and α-defensins, which may modulate acquired immune responses. During the acute phase of infection, IL-10 correlated positively with viral load and negatively with CD4+T cell counts. Furthermore, α-defensins production was directly correlated with plasma viral RNA, particularly at peak of viral load. When the effects of the MHC were analyzed, a significant association between lower anti-Env binding and neutralizing antibody levels with class IB M4 haplotype and with class IA, IB M4 haplotype, respectively, was observed in the post-acute phase. Lower antibody responses may have resulted into a poor control of infection thus explaining the previously reported lower CD4 T cell counts in these monkeys. Class II M3 haplotype displayed significantly lower acute and post-acute IL-10 levels. In addition, significantly lower levels of α-defensins were detected in class IA M3 haplotype monkeys than in non-M3 macaques, in the post-acute phase of infection. These data indicate that the MHC could contribute to the delicate balance of pro-inflammatory mechanisms, particularly with regard to the association between IL-10 and α-defensins in lentivirus infection. Our results show that host genetic background, virological and immunological parameters should be considered for the design and interpretation of HIV-1 vaccine efficacy studies.

Containment of infection in tat vaccinated monkeys after rechallenge with a higher dose of SHIV89.6P(cy243).

We previously reported that cynomolgus monkeys vaccinated with the human immunodeficiency virus (HIV)-1 Tat protein controlled infection after challenge with the simian human immunodeficiency virus (SHIV) 89.6P(cy243) for up to 2 y of follow-up. To evaluate the breadth of the protective immunity elicited by the Tat protein, the vaccines along with the naïve monkeys were intravenously rechallenged with a fivefold higher dose (50 MID(50)) of the same SHIV-89.6P(cy243). The vaccinated monkeys exhibited a statistically significant and long-lasting reduction of viral replication compared to control monkeys. This effect was associated with a strong anamnestic response to Tat, while responses to Gag and Env were nearly undetectable. Taken together, these data provide further evidence for the usefulness of Tat-based vaccines.