Antiretroviral therapy (ART) suppresses HIV replication but is unable to eliminate latent viral reservoirs (VR) comprising of CD4+ T cells and macrophages. These cell populations have elevated expression of Inhibitors of Apoptosis Proteins (IAPs), enabling prolonged cell survival. The most effective LRA, PKC agonists, reactivate VR by activating the NF-kB pathway. However, since they are likely to dramatically increase inflammation in vivo, they are difficult to evaluate clinically. Consequently, there is a need for novel therapeutic interventions for VR eradication. Herein, we demonstrate that a bivalent SMAC Mimetic (SM) reactivates up to 60% CD4+ T cells in models of latency (J-Lat 10.6, J-Lat 5A8 and 2D10) by activating the non-canonical NF-kB pathway. As well, the SM induces apoptosis in up to 30% and 50% CD4+T and myeloid cell models of latency (OM-10.1, U1 and THP89GFP) respectively, via degradation of IAPs and upregulation of caspases. The results were validated in primary CD4+ T cells and differentiated THP-1 cells infected with a dual reporter virus-HIV-CRMZ. The ability of the SM to reactivate and reduce VR was then tested in virally-suppressed humanised bone-marrow–liver–thymus (hu-mice). SM was non-cytotoxic in hu-mice for up to 4 weeks and induced viral reactivation in at least 70% of ART-suppressed animals as exemplified by the detection of HIV viral load in sera and increased HIV-RNA and p24-antigen expression in almost all analysed tissues. Furthermore, SM reduced the VR by at least 10-fold as measured by the frequency of cells harboring integrated and total HIV-DNA in lymphoid and non-lymphoid tissues. In conclusion, this SM is an effective LRA with a capacity for preferential elimination of VR (both CD4+ T cells and Macrophages) that has not been studied in the previously published articles. This promising approach for systemic clearance of VR might increases opportunities for HIV eradication.

In 2018, reports from the Tsepamo study in Botswana highlighted a concerning interaction between exposure from the time of conception to the HIV integrase strand transfer inhibitor dolutegravir (DTG), and neural-tube defects (NTDs) in the offspring of women accessing antiretroviral therapy. Due to the importance of folates in fetal development, and the established association of folate-deficiency with NTDs, it is critical to investigate potential interactions between DTG and folate pathways in the developing fetus. In this study, we investigated the effect of in utero DTG exposure on the functional expression of placental proteins important for folate delivery to the fetus, including reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT), as well as the folate receptor-α (FRα).
We first characterized the expression of RFC, PCFT and FRα in a panel of human placenta cell-lines and in human placental explants by qPCR, immunoblotting and immunohistochemistry analyses. In the first-trimester human placental cell line HTR-8/SVneo, exposure to clinically-relevant concentrations of DTG was associated with a modest but significant reduction in the expression of RFC and PCFT both at the mRNA and protein levels, following 3h and 6h of exposure. DTG exposure was also associated with reduced uptake of 50 nM [3H]-folic acid, a PCFT substrate, in HTR-8/SVneo cells at 3h and 6h exposure. To investigate the interaction of DTG with placental folate pathways in vivo, pregnant mice were treated daily with clinically relevant doses of DTG, and placentas were isolated on gestational day 15.5. In these mouse placentas, we identified a very modest induction of both RFC and PCFT at the mRNA level associated with in utero DTG exposure.
Together, these findings suggest a potential interaction between DTG and folate pathways in the placenta, which could potentially impact folate delivery to the fetus in the context of antiretroviral therapy during pregnancy.

Antiretroviral therapy (ART) controls HIV-1; it however does not eliminate the virus, and virus re-emerges upon ART interruption. Thus, new approaches aimed at eradicating or functionally curing HIV are needed. A promising approach to eliminate latently infected cells after viral reactivation relies on the ability of immune cells to mediate antibody-dependent cellular cytotoxicity (ADCC). Through ADCC, effector cells such as NK cells and monocytes can kill infected cells expressing envelope glycoproteins (Env) through recognition by HIV-specific antibodies (Abs). Small CD4-mimetic compounds (CD4mc) sensitize infected cells to ADCC mediated by CD4-induced (CD4i) Abs present in HIV+ sera. Two families of CD4i Abs are required to sensitize infected cells to ADCC in the presence of CD4mc: anti-cluster A and anti-coreceptor binding site Abs. These CD4i Abs in combination with CD4mc stabilize a new Env conformation, State 2A, which is highly vulnerable to ADCC. We employed new-generation SRG-15 humanized mice that supports NK cell and Fc-effector functions in vivo, to demonstrate that brief treatment with CD4mc and these two families of Abs significantly decreases HIV-1 replication, reduces the virus reservoir and substantially delays virus rebound after ART interruption. The decrease in the size of the reservoir was significantly less pronounced when using Fc-impaired Abs or hu-mice depleted of NK cells. Thus, indicating a role for Fc-mediated effector functions in viral reservoir elimination. Viral rebound was also suppressed in HIV-1+-donor cell-derived humanized mice supplemented with autologous plasma and CD4mc. These results indicate that CD4mc could have therapeutic utility in HIV-1-infected individuals for decreasing the size of the virus reservoir and/or achieving a functional cure.

Background: Women with HIV-subtype-B infection are understudied in persistence research. Towards addressing this, we reconstructed within-host HIV evolution in three seroconverters from the Women's Interagency HIV Study to infer proviral ages and temporal stability during-cART, and to compare ages of rebound viruses to the overall proviral pool.

Methods: Participants 1, 2 and 3 (P1, P2, P3) initiated cART in chronic infection; total follow-up was 22, 14 and 15yrs. Pre-cART longitudinal single-genome-amplified (SGA) plasma HIV RNA env-gp120 sequences were previously published. We now used SGA (env-gp120) to characterize plasma HIV emerging after initial suppression (P1); proviruses sampled on-cART (P1:4 times over 5yrs; P2:thrice over 4yrs; P3:once). Within-host phylogenies were inferred from intact and unique sequences, from which rebound virus and proviral ages were estimated by root-to-tip regression.

Results: P1, P2 and P3's intact-env dataset sizes were 340, 176 and 231, where proviruses were 80%, 80% and 55% unique. For P1, unique proviruses initially sampled-on-cART were an estimated 8yrs-old on average (oldest 16yrs) with most dating to chronic phase. Proviral integration dates remained stable over 5yrs on-cART (p=0.68) but rebound viruses emerging after 1yr of suppressive-cART were younger than the persisting proviral pool (p<0.0001). P2's unique proviruses were 5yrs-old on average (oldest 8yrs) at initial sampling, with most dating to chronic phase; proviral integration dates remained stable over 9yrs (p=0.55). P3's unique proviruses were an estimated 7yrs-old on average (oldest 13yrs) with many dating to early infection.

Conclusion: Proviral age distributions support ongoing archiving and persistence of diverse proviral lineages pre-cART. Modest skewing towards chronic-phase deposition in 2 participants is consistent with reports in HIV-subtype-C and among men. Stability of proviral integration dates on-cART supports negligible decay of the proviral pool. Younger ages of rebound viruses suggest these emerged from a recently established replication-competent reservoir, as compared to the older proviral pool.

HIV-1 infection is subject to restriction by host factors. In turn, HIV-1 often has countering mechanisms to antagonize and evade these host restriction factors. The interferon-inducible myxovirus resistance B (MxB) protein has been shown to inhibit HIV-1 infection by blocking the nuclear import of viral DNA. HIV-1 is able to escape from MxB restriction by mutating viral capsid protein. In this study, we report a new anti-HIV-1 mechanism of MxB by inhibiting HIV-1 Rev protein. Rev is a nuclear protein, exports full-length and singly spliced viral RNA into the cytoplasm for translation, thus plays an essential role in viral gene expression. We observed that MxB sequesters Rev within the cytoplasm, impairs Rev-dependent viral RNA export, and suppresses viral Gag protein expression. Specifically, MxB disrupts the association of Rev with its nuclear transport receptor transportin 1 (TNPO1), thus causes Rev cytoplasm retention. By testing a group of Rev variants in HIV-1 strains isolated from HIV patients, we identified MxB-resistant Rev mutations which allow Rev to enter the nucleus independent of TNPO1. Also, HIV-1 is able to overcome MxB inhibition of Rev by increasing Rev protein expression thanks to the feedback mechanism that regulates HIV-1 gene expression. Overall, these results have extended our understanding of the anti-HIV-1 mechanisms of MxB from blocking the nuclear import of viral DNA to interfering with the nuclear import of essential viral proteins.

The human APOBEC3 (A3) family of cytidine deaminases includes five host restriction factors that inhibit HIV-1 in the absence of its Vif protein that causes their degradation. The deamination of cytidine in HIV-1 (-)DNA forms uracil that causes inactivating mutations when uracil is used in the (+)DNA synthesis template. The A3 family exhibits considerable variation in their antiviral activity. For example, A3G is the most restrictive and A3C is the least restrictive. In most cases this activity in human A3 enzymes is overall less than chimpanzee A3 enzymes, suggesting that activity has been decreasing through evolution. This is thought to have occurred since A3 enzymes can erroneously cause mutations in the human genome and contribute to cancer. However, for A3C, although we have previously found that chimpanzee and gorilla A3C are more active than human A3C, we also observed the surprising result that old world monkey A3C from rhesus macaque (rh) was not active against HIV-1. The activity in hominid A3C is determined by an I188 or a K115 that enable the A3C to dimerize which increases its deamination activity and processivity on (-)DNA. The rhA3C has an I188, but this does not enable dimerization due to the steric hindrance imparted by the residue M115. Mutational analysis showed that although rhA3C shares a similar dimer interface with hominid A3C, the key amino acids needed to promote activity were at positions 44, 45, and 144. Overall, the data show that A3C was less active in old world monkeys and gained activity in the hominid lineage, which is the opposite to evolution of other A3 enzymes. A3s were thought to be more active in old world monkeys due to higher lentivirus burdens, but these data demonstrate that there are more things to learn about the evolutionary pressures that formed the A3 family.