Nanoparticle delivery of Tat synergizes with classical latency reversal agents to express HIV antigen targets.

Limited cellular levels of the HIV transcriptional activator Tat are one contributor to proviral latency that might be targeted in HIV cure strategies. We recently demonstrated that lipid nanoparticles containing HIV tat mRNA induce HIV expression in primary CD4 T cells. Here, we sought to further characterize tat mRNA in the context of several benchmark latency reversal agents (LRAs), including inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-Terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi). tat mRNA reversed latency across several different cell line models of HIV latency, an effect dependent on the TAR hairpin loop. Synergistic enhancement of tat mRNA activity was observed with IAPi, HDACi, and BETi, albeit to variable degrees. In primary CD4 T cells from durably suppressed people with HIV, tat mRNA profoundly increased the frequencies of elongated, multiply-spliced, and polyadenylated HIV transcripts, while having a lesser impact on TAR transcript frequencies. tat mRNAs alone resulted in variable HIV p24 protein induction across donors. However, tat mRNA in combination with IAPi, BETi, or HDACi markedly enhanced HIV RNA and protein expression without overt cytotoxicity or cellular activation. Notably, combination regimens approached or in some cases exceeded the latency reversal activity of maximal mitogenic T cell stimulation. Higher levels of tat mRNA-driven HIV p24 induction were observed in donors with larger mitogen-inducible HIV reservoirs, and expression increased with prolonged exposure time. Combination LRA strategies employing both small molecule inhibitors and Tat delivered to CD4 T cells are a promising approach to effectively target the HIV reservoir.

mRNA Therapeutic Vaccine for Hepatitis B Demonstrates Immunogenicity and Efficacy in the AAV-HBV Mouse Model.

Chronic infection with hepatitis B virus (HBV) develops in millions of patients per year, despite the availability of effective prophylactic vaccines. Patients who resolve acute HBV infection develop HBV-specific polyfunctional T cells accompanied by neutralizing antibodies, while in patients with chronic hepatitis B (CHB), immune cells are dysfunctional and impaired. We describe a lipid nanoparticle (LNP)-formulated mRNA vaccine, optimized for the expression of HBV core, polymerase, and surface (preS2-S) antigens with the aim of inducing an effective immune response in patients with CHB. Prime and prime/boost vaccination with LNP-formulated mRNA encoding for core, pol, and/or preS2-S dosing strategies were compared in naive C57BL/6 and BALB/c mice. Immune responses were assessed by IFN-γ ELISpot, intracellular cytokine staining (ICS), and ELISA for antibody production, whereas anti-viral efficacy was evaluated in the AAV-HBV mouse model. The mRNA vaccine induced strong antigen-specific polyfunctional T cell responses in these mouse models, accompanied by the emergence of anti-HBs and anti-HBe antibodies. After three immunizations, the antigen-specific immune stimulation resulted in up to 1.7 log10 IU/mL reduction in systemic HBV surface antigen (HBsAg), accompanied by a transient drop in systemic HBeAg, and this was observed in 50% of the AAV-HBV-transduced mice in the absence of additional modalities such as adjuvants, HBsAg reducing agents, or checkpoint inhibitors. However, no treatment-related effect on viremia was observed in the liver. These results warrant further optimization and evaluation of this mRNA vaccine as a candidate in a multimodal therapeutic regimen for the treatment of chronic HBV infection.

Potent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir.

The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells' transcriptome and proteome profiles.

A truncated HIV Tat demonstrates potent and specific latency reversal activity.

A major barrier to HIV-1 cure is caused by the pool of latently infected CD4 T-cells that persist under combination antiretroviral therapy (cART). This latent reservoir is capable of producing replication-competent infectious viruses once prolonged suppressive cART is withdrawn. Inducing the reactivation of HIV-1 gene expression in T-cells harboring a latent provirus in people living with HIV-1 under cART may result in depletion of this latent reservoir due to cytopathic effects or immune clearance. Studies have investigated molecules that reactivate HIV-1 gene expression, but to date, no latency reversal agent has been identified to eliminate latently infected cells harboring replication-competent HIV in cART-treated individuals. Stochastic fluctuations in HIV-1 tat gene expression have been described and hypothesized to allow the progression into proviral latency. We hypothesized that exposing latently infected CD4+ T-cells to Tat would result in effective latency reversal. Our results indicate the capacity of a truncated Tat protein and mRNA to reactivate HIV-1 in latently infected T-cells ex vivo to a similar degree as the protein kinase C agonist: phorbol 12-myristate 13-acetate, without T-cell activation or any significant transcriptome perturbation.

A Therapeutic Hepatitis B Virus DNA Vaccine Induces Specific Immune Responses in Mice and Non-Human Primates.

Despite the availability of an effective prophylactic vaccine for more than 30 years, nearly 300 million people worldwide are chronically infected with the hepatitis B virus (HBV), leading to 1 death every 30 s mainly from viral hepatitis-related cirrhosis and liver cancer. Chronic HBV patients exhibit weak, transient, or dysfunctional CD8+ T-cell responses to HBV, which contrasts with high CD8+ T-cell responses seen for resolvers of acute HBV infection. Therefore, a therapeutic DNA vaccine was designed, expressing both HBV core and polymerase proteins, and was sequence optimized to ensure high protein expression and secretion. Although the vaccine, administered intramuscularly via electroporation, had no effect on plasma viral parameters in a mouse model of persistent HBV infection, it did induce robust HBV-specific immune responses in healthy and adeno-associated hepatitis B virus (AAV-HBV) infected mice as well as in healthy non-human primates.

Humanized mice recapitulate key features of HIV-1 infection: a novel concept using long-acting anti-retroviral drugs for treating HIV-1.

BACKGROUND: Humanized mice generate a lymphoid system of human origin subsequent to transplantation of human CD34+ cells and thus are highly susceptible to HIV infection. Here we examined the efficacy of antiretroviral treatment (ART) when added to food pellets, and of long-acting (LA) antiretroviral compounds, either as monotherapy or in combination. These studies shall be inspiring for establishing a gold standard of ART, which is easy to administer and well supported by the mice, and for subsequent studies such as latency. Furthermore, they should disclose whether viral breakthrough and emergence of resistance occurs similar as in HIV-infected patients when ART is insufficient. METHODS/PRINCIPAL FINDINGS: NOD/shi-scid/γ(c)null (NOG) mice were used in all experimentations. We first performed pharmacokinetic studies of the drugs used, either added to food pellets (AZT, TDF, 3TC, RTV) or in a LA formulation that permitted once weekly subcutaneous administration (TMC278: non-nucleoside reverse transcriptase inhibitor, TMC181: protease inhibitor). A combination of 3TC, TDF and TMC278-LA or 3TC, TDF, TMC278-LA and TMC181-LA suppressed the viral load to undetectable levels in 15/19 (79%) and 14/14 (100%) mice, respectively. In successfully treated mice, subsequent monotherapy with TMC278-LA resulted in viral breakthrough; in contrast, the two LA compounds together prevented viral breakthrough. Resistance mutations matched the mutations most commonly observed in HIV patients failing therapy. Importantly, viral rebound after interruption of ART, presence of HIV DNA in successfully treated mice and in vitro reactivation of early HIV transcripts point to an existing latent HIV reservoir. CONCLUSIONS/SIGNIFICANCE: This report is a unique description of multiple aspects of HIV infection in humanized mice that comprised efficacy testing of various treatment regimens, including LA compounds, resistance mutation analysis as well as viral rebound after treatment interruption. Humanized mice will be highly valuable for exploring the antiviral potency of new compounds or compounds targeting the latent HIV reservoir.

Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli.

Fluorescent reporter genes are valuable tools for real-time monitoring of gene expression in living cells. In this study we describe the construction of novel promoter-probe vectors containing a synthetic mCherry fluorescent protein gene, codon-optimized for lactic acid bacteria, divergently linked, or not, to a gene encoding the S65T and F64L variant of the green fluorescent protein. The utility of the transcriptional fusion vectors was demonstrated by the cloning of a single or two divergent promoter regions and by the quantitative evaluation of fluorescence during growth of Lactococcus lactis, Enterococcus faecalis, and Escherichia coli.

The virologic and immunologic effects of cyclosporine as an adjunct to antiretroviral therapy in patients treated during acute and early HIV-1 infection.

Acute human immunodeficiency virus type 1 (HIV-1) infection is characterized by high levels of immune activation. Immunomodulation with cyclosporine combined with antiretroviral therapy (ART) in the setting of acute and early HIV-1 infection has been reported to result in enhanced immune reconstitution. Fifty-four individuals with acute and early infection were randomized to receive ART with 4 weeks of cyclosporine versus ART alone. In 48 subjects who completed the study, there were no significant differences between treatment arms in levels of proviral DNA or CD4(+) T cell counts. Adjunctive therapy with cyclosporine in this setting does not provide apparent virologic or immunologic benefit.

Adjunctive passive immunotherapy in human immunodeficiency virus type 1-infected individuals treated with antiviral therapy during acute and early infection.

Three neutralizing monoclonal antibodies (MAbs), 2G12, 2F5, and 4E10, with activity in vitro and in vivo were administered in an open-label, nonrandomized, proof-of-concept study to attempt to prevent viral rebound after interruption of antiretroviral therapy (ART). Ten human immunodeficiency virus type 1-infected individuals identified and treated with ART during acute and early infection were enrolled. The first six patients were administered 1.0 g of each of the three MAbs per infusion. The remaining four patients received 2G12 at 1.0 g/infusion and 2.0 g/infusion of 2F5 and 4E10. The MAbs were well tolerated. Grade I post-partial thromboplastin time prolongations were noted. Viral rebound was observed in 8/10 subjects (28 to 73 days post-ART interruption), and 2/10 subjects remained aviremic over the course of the study. In seven of eight subjects with viral rebound, clear resistance to 2G12 emerged, whereas reductions in the susceptibilities of plasma-derived recombinant viruses to 2F5 and 4E10 were neither sustained nor consistently measured. Viral rebound was associated with a preferential depletion of CD4(+) T cells within the gastrointestinal tract. Though safe, the use of MAbs generally delayed, but did not prevent, virologic rebound. Consideration should be given to further pilot studies with alternative combinations of MAbs and perhaps additional novel treatment modalities.

Coreceptor switch in R5-tropic simian/human immunodeficiency virus-infected macaques.

The basis for the switch from CCR5 to CXCR4 coreceptor usage seen in approximately 50% of human immunodeficiency virus type 1 (HIV-1) subtype B-infected individuals as disease advances is not well understood. Among the reasons proposed are target cell limitation and better immune recognition of the CXCR4 (X4)-tropic compared to the CCR5 (R5)-tropic virus. We document here X4 virus emergence in a rhesus macaque (RM) infected with R5-tropic simian/human immunodeficiency virus, demonstrating that coreceptor switch can happen in a nonhuman primate model of HIV/AIDS. The switch to CXCR4 usage in RM requires envelope sequence changes in the V3 loop that are similar to those found in humans, suggesting that the R5-to-X4 evolution pathways in the two hosts overlap. Interestingly, compared to the inoculating R5 virus, the emerging CXCR4-using virus is highly neutralization sensitive. This finding, coupled with the observation of X4 evolution and appearance in an animal with undetectable circulating virus-specific antibody and low cellular immune responses, lends further support to an inhibitory role of antiviral immunity in HIV-1 coreceptor switch.

Overcoming HIV-1 resistance to RNA interference.

RNAi refers to the sequence-specific degradation of RNA that follows the cellular introduction of homologous short interfering (si) RNA. RNAi has emerged as a powerful tool to probe the function of genes of known sequence in vitro and in vivo. Advances in vector design permit the effective expression of siRNA in human cells. Numerous recent investigations have described the ability of RNAi to decrease the replication of human immunodeficiency virus type 1 (HIV-1) in lymphocytic cells using siRNA targeting viral (e.g. tat, gag, rev) and host (e.g. CCR5, CD4) proteins. Can RNAi be used as a form of genetic therapy for HIV-1 infection? Recent data indicate that the dynamic replication kinetics of HIV-1 pose a considerable barrier to achieving durable virus suppression by RNAi with the rapid emergence of HIV-1 mutants resistant to siRNA. This review summarizes recent work on HIV-1 specific RNAi with a focus on potential strategies to overcome HIV-1 resistance to RNAi.

Mechanisms of gastrointestinal CD4+ T-cell depletion during acute and early human immunodeficiency virus type 1 infection.

During acute and early human immunodeficiency virus type 1 (HIV-1) infection (AEI) more than 50% of CD4+ T cells are preferentially depleted from the gastrointestinal (GI) lamina propria. To better understand the underlying mechanisms, we studied virological and immunological events within the peripheral blood (PB) and GI tract during AEI. A total of 32 AEI subjects and 18 uninfected controls underwent colonic biopsy. HIV-1 viral DNA and RNA levels were quantified in CD4+ T cells derived from the GI tract and PB by using real-time PCR. The phenotype of infected cells was characterized by using combinations of immunohistochemistry and in situ hybridization. Markers of immunological memory, activation, and proliferation were examined by flow cytometry and immunohistochemistry, and the host-derived cytotoxic cellular response was examined by using immunohistochemistry. GI CD4+ T cells harbored, on average, 13-fold higher HIV-1 viral DNA levels and 10-fold higher HIV-1 RNA levels than PB CD4+ T cells during AEI. HIV-1 RNA was detected in both "activated" and "nonactivated" mucosal CD4+ T cells. A significantly higher number of activated and proliferating T cells were detected in the GI tract compared to the PB, and a robust cytotoxic response (HIV-1 specificity not determined) was detected in the GI tract as early as 18 days postinfection. Mucosal CD4+ T-cell depletion is multifactorial. Direct viral infection likely accounts for the earliest loss of CD4+ T cells. Subsequently, ongoing infection of susceptible CD4+ T cells, along with activation-induced cellular death and host cytotoxic cellular response, are responsible for the persistence of the lesion.

Lack of mucosal immune reconstitution during prolonged treatment of acute and early HIV-1 infection.

BACKGROUND: During acute and early HIV-1 infection (AEI), up to 60% of CD4(+) T cells in the lamina propria of the lower gastrointestinal (GI) tract are lost as early as 2-4 wk after infection. Reconstitution in the peripheral blood during therapy with highly active antiretroviral therapy (HAART) is well established. However, the extent of immune reconstitution in the GI tract is unknown. METHODS AND FINDINGS: Fifty-four AEI patients and 18 uninfected control participants underwent colonic biopsy. Forty of the 54 AEI patients were followed after initiation of antiretroviral therapy (18 were studied longitudinally with sequential biopsies over a 3-y period after beginning HAART, and 22 were studied cross sectionally after 1-7 y of uninterrupted therapy). Lymphocyte subsets, markers of immune activation and memory in the peripheral blood and GI tract were determined by flow cytometry and immunohistochemistry. In situ hybridization was performed in order to identify persistent HIV-1 RNA expression. Of the patients studied, 70% maintained, on average, a 50%-60% depletion of lamina propria lymphocytes despite 1-7 y of HAART. Lymphocytes expressing CCR5 and both CCR5 and CXCR4 were persistently and preferentially depleted. Levels of immune activation in the memory cell population, CD45RO+ HLA-DR+, returned to levels seen in the uninfected control participants in the peripheral blood, but were elevated in the GI tract of patients with persistent CD4+ T cell depletion despite therapy. Rare HIV-1 RNA-expressing cells were detected by in situ hybridization. CONCLUSIONS: Apparently suppressive treatment with HAART during acute and early infection does not lead to complete immune reconstitution in the GI mucosa in the majority of patients studied, despite immune reconstitution in the peripheral blood. Though the mechanism remains obscure, the data suggest that there is either viral or immune-mediated accelerated T cell destruction or, possibly, alterations in T cell homing to the GI tract. Although clinically silent over the short term, the long-term consequences of the persistence of this lesion may emerge as the HIV-1-infected population survives longer owing to the benefits of HAART.

An anti-HIV microbicide engineered in commensal bacteria: secretion of HIV-1 fusion inhibitors by lactobacilli.

OBJECTIVES: To engineer Lactobacillus spp. to secrete HIV-1 fusion inhibitors with potent neutralizing activity against primary HIV-1 isolates. METHODS: HIV-1 fusion inhibitors (FI-1, FI-2, and FI-3) were introduced into the previously developed shuttle vector pTSV2 and transformed in L. plantarum and L. gasseri. The signal peptide Usp45 from L. lactis was used to achieve high secretion efficiency of peptides into the bacterial supernatant. The antiviral activity of lactobacillus-derived HIV-1 fusion inhibitors was tested against a panel of primary HIV-1 isolates and a chimeric simian/HIV (SHIV-162P3) using the TZM infection assay. TZM-bl cells are engineered HeLa cells that express CD4, CCR5, and CXCR4 and contain integrated reporter genes for firefly luciferase and beta-galactosidase under the control of an HIV-1 long terminal repeat. The amount of secreted fusion inhibitor FI-3 was determined by Western blot analysis and the antiviral specificity verified by antibody-mediated depletion of peptide FI-3 and HIV-1 infection with VSV-G envelope pseudotyped virions. RESULTS: Viral infectivity of primary HIV-1 isolates and SHIV-162P3 was neutralized by up to 98% and 72%, respectively, by 10% (v/v) lactobacillus supernatant containing fusion inhibitor FI-3. The antiviral activity of the lactobacillus-derived fusion inhibitor FI-3 was clearly shown to be attributable to the secreted fusion inhibitor peptide. CONCLUSION: The development of recombinant lactobacilli expressing HIV-1 fusion inhibitors with potent neutralizing activity represents an important step toward the development of a live microbial (topical) microbicide against HIV-1 transmission.

Engineering mucosal RNA interference in vivo.

Mucosal surfaces serve as a gateway to disease. Here, we demonstrate that RNA interference can be used to manipulate mucosal gene expression in vivo. Using a murine model, we show that direct application of liposome-complexed siRNA mediates gene-specific silencing in cervicovaginal and rectal mucosa. A single vaginal or rectal administration of siRNA targeting hematopoietic or somatic cell gene products reduced corresponding mRNA levels by up to 90%. Using a murine model of inflammatory bowel disease, we found that the rectal application of siRNA targeting TNF-alpha led to relative mucosal resistance to experimental colitis. Liposomal siRNA formulations proved nontoxic, did not elicit a nonspecific interferon response, and provide a means for genetic engineering of mucosal surfaces in vivo.

Tracking the prevalence of transmitted antiretroviral drug-resistant HIV-1: a decade of experience.

Transmitted resistance to antiretroviral drugs in acute and early HIV-1 infection has been well documented, although overall trends vary depending on geography and cohort characteristics. To describe the changing pattern of transmitted drug-resistant HIV-1 in a well-defined cohort in New York City, a total of 361 patients with acute or recent HIV-1 infection were prospectively studied over a decade (1995-2004) with respect to HIV-1 genotypes and longitudinal T-cell subsets and HIV-1 RNA levels. The prevalence of overall transmitted resistance changed from 13.2% to 24.1% (P = 0.11) during the periods 1995 to 1998 and 2003 to 2004. Nonnucleoside reverse transcriptase inhibitor resistance prevalence increased significantly from 2.6% to 13.4% (P = 0.007) during the same periods, whereas prevalence of multidrug-resistant virus shifted from 2.6% to 9.8% (P = 0.07) but did not achieve statistical significance. A comparable immunologic and virologic response of appropriately treated individuals was observed regardless of viral drug susceptibility status, suggesting that initial combination therapy guided by baseline resistance testing in the case of acute and early infection may result in a favorable treatment response even in the case of a drug-resistant virus. These data have important implications for selection of empiric first-line regimens for treatment of acutely infected antiretroviral-naive individuals and reinforce the need for baseline resistance testing in acute and early HIV-1 infection.

Bioengineering lactic acid bacteria to secrete the HIV-1 virucide cyanovirin.

An urgent need exists to prevent the sexual transmission of HIV-1. With prevalence rates exceeding 35% in parts of sub-Saharan Africa, increasing attention has been placed on developing and testing microbicidal agents capable of preventing virus transmission at mucosal sites. HIV-1 microbicides must meet several requirements before their widespread use. The drugs must be able to neutralize a diversity of HIV-1 strains, not induce mucosal inflammation, be associated with minimal side effects, and be effective for a prolonged period after a single application. Recent work has demonstrated the utility of recombinant lactic acid bacteria (LAB) as agents of mucosal drug delivery. Here, we describe the bioengineering of strains of LAB to secrete the prototypic virucidal compound cyanovirin (CV-N) and demonstrate the anti-HIV-1 activity of secreted CV-N. Our results suggest that recombinant LAB may serve as effective microbicidal compounds and deserve in vivo testing in simian immunodeficiency virus models of mucosal virus transmission.

Infection with multidrug resistant, dual-tropic HIV-1 and rapid progression to AIDS: a case report.

BACKGROUND: Rapid progression to AIDS after acute HIV-1 infection, though uncommon, has been noted, as has the transmission of multidrug resistant viruses. Here, we describe a patient in whom these two factors arose concomitantly and assess the effects. METHODS: We did a case study of a patient with HIV-1 seroconversion. We genotyped the virus and host genetic markers by PCR and nucleotide sequencing. To ascertain the drug susceptibility of our patient's HIV-1 we did phenotypic studies with the PhenoSense assay. We assessed viral coreceptor use via syncytium formation in vitro and with a modified PhenoSense assay. FINDINGS: Our patient seems to have been recently infected by a viral variant of HIV-1 resistant to multiple classes of antiretroviral drugs. Furthermore, his virus population is dual tropic for cells that express CCR5 or CXCR4 coreceptor. The infection has resulted in progression to symptomatic AIDS in 4-20 months. INTERPRETATION: The intersection of multidrug resistance and rapid development of AIDS in this patient is of concern, especially in view of his case history, which includes high-risk sexual contacts and use of metamfetamine. The public health ramifications of such a case are great.

HIV-1-specific RNA interference.

The preclinical development of RNA interference (RNAi) as a novel therapeutic agent for HIV-1 infection is reviewed. RNAi refers to the sequence-specific degradation of RNA that follows the cellular introduction of homologous, short-interfering RNA (siRNA). RNAi has emerged as a powerful tool to probe the function of genes of known sequence in vitro and in vivo. Advances in vector design permit the effective expression of siRNA in human cells by transfer of short hairpin RNA expression cassettes. Recent investigations have described the ability of RNAi to decrease the replication of HIV-1 in lymphocytic cells using siRNA targeting viral (eg, Tat, Gag and Rev) and host (eg, CCR5 and CD4) proteins. Can RNAi be used as a form of genetic therapy for HIV-1 and associated infections? There are numerous challenges associated with converting RNAi from a laboratory technique to an antiviral therapeutic. Recent research on the cellular delivery, antiviral durability and gene-silencing specificity of HIV-1-specific RNAi is reviewed.