Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity.

A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.

A long-acting formulation of the integrase inhibitor raltegravir protects humanized BLT mice from repeated high-dose vaginal HIV challenges.

OBJECTIVES: Pre-exposure prophylaxis (PrEP) using antiretroviral drugs (ARVs) has been shown to reduce HIV transmission in people at high risk of HIV infection. Adherence to PrEP strongly correlates with the level of HIV protection. Long-acting injectable ARVs provide sustained systemic drug exposures over many weeks and can improve adherence due to infrequent parenteral administration. Here, we evaluated a new long-acting formulation of raltegravir for prevention of vaginal HIV transmission. METHODS: Long-acting raltegravir was administered subcutaneously to BALB/c, NSG (NOD-scid-gamma) and humanized BLT (bone marrow-liver-thymus) mice and rhesus macaques. Raltegravir concentration in peripheral blood and tissue was analysed. Suppression of HIV replication was assessed in infected BLT mice. Two high-dose HIV vaginal challenges were used to evaluate protection from HIV transmission in BLT mice. RESULTS: Two weeks after a single subcutaneous injection of long-acting raltegravir in BLT mice (7.5 mg) and rhesus macaques (160 mg), the plasma concentration of raltegravir was comparable to 400 mg orally, twice daily in humans. Serum collected from mice 3 weeks post-administration of long-acting raltegravir efficiently blocked HIV infection of TZM-bl indicator cells in vitro. Administration of long-acting raltegravir suppressed viral RNA in plasma and cervico-vaginal fluids of infected BLT mice, demonstrating penetration of active raltegravir into the female reproductive tract. Using transmitted/founder HIV we observed that BLT mice administered a single subcutaneous dose of long-acting raltegravir were protected from two high-dose HIV vaginal challenges 1 week and 4 weeks after drug administration. CONCLUSIONS: These preclinical results demonstrated the efficacy of long-acting raltegravir in preventing vaginal HIV transmission.

Role of Semen on Vaginal HIV-1 Transmission and Maraviroc Protection.

We used bone marrow/liver/thymus (BLT) humanized mice to establish the effect of semen on vaginal HIV infection and on the efficacy of topically applied maraviroc. Our results demonstrate that vaginal transmission of cell-free HIV occurs efficiently in the presence of semen and that topically applied maraviroc efficiently prevents HIV transmission in the presence of semen. We also show that semen has no significant effect on the transmission of transmitted/founder viruses or cell-associated viruses.

HLA-B*57 elite suppressor and chronic progressor HIV-1 isolates replicate vigorously and cause CD4+ T cell depletion in humanized BLT mice.

UNLABELLED: Elite controllers or suppressors (ES) are HIV-1-infected patients who maintain undetectable viral loads without antiretroviral therapy. The mechanism of control remains unclear, but the HLA-B*57 allele is overrepresented in cohorts of these patients. However, many HLA-B*57 patients develop progressive disease, and some studies have suggested that infection with defective viruses may be the cause of the lack of high levels of virus replication and disease progression in ES. We therefore performed a comprehensive comparative in vivo and in vitro characterization of viruses isolated from well-defined ES. For this purpose, we first performed full-genome sequence analysis and in vitro fitness assays on replication-competent isolates from HLA-B*57 ES and HLA-B*57 chronic progressors (CPs). Under our experimental conditions, we found that isolates from ES and CPs can replicate in vitro. However, since inherently these assays involve the use of unnaturally in vitro-activated cells, we also investigated the replication competence and pathogenic potential of these HIV isolates in vivo using humanized BLT mice. The results from these analyses demonstrate that virus isolates from ES are fully replication competent in vivo and can induce peripheral and systemic CD4 T cell depletion. These results provide the first direct in vivo evidence that viral fitness does not likely determine clinical outcome in HLA-B*57 patients and that elite suppressors can control replication-competent, fully pathogenic viruses. A better understanding of the immunological bases of viral suppression in ES will serve to inform novel approaches to preventive and therapeutic HIV vaccine design. IMPORTANCE: Elite suppressors are HIV-1-infected patients who have undetectable levels of viremia despite not being on antiviral drugs. One of the most fundamental questions about this phenomenon involves the mechanism of control. To address this question, we isolated virus from elite suppressors and from HIV-1-infected patients who have the usual progressive disease course. We compared how well the isolates from the two groups of patients replicated in culture and in humanized mice. Our results suggest that elite suppressors are capable of controlling HIV-1 due to the possession of unique host factors rather than infection with defective virus.

Inefficient vaginal transmission of tenofovir-resistant HIV-1.

Transmission of drug-resistant HIV has been postulated to be a threat to current first-line antiretroviral therapy (ART) regimens and the efficacy of several antiretroviral-based preexposure prophylaxis (PrEP) strategies being tested. Here we evaluated the effect of the common tenofovir (TFV) resistance mutation K65R on vaginal HIV transmission. Our results demonstrate that despite no overt loss of overall replication competence in vivo, this mutation results in significantly reduced mucosal transmission. When transmitted, the mutant virus eventually reverted to the wild type in 2 of 3 animals examined.

Human breast milk and antiretrovirals dramatically reduce oral HIV-1 transmission in BLT humanized mice.

Currently, over 15% of new HIV infections occur in children. Breastfeeding is a major contributor to HIV infections in infants. This represents a major paradox in the field because in vitro, breast milk has been shown to have a strong inhibitory effect on HIV infectivity. However, this inhibitory effect has never been demonstrated in vivo. Here, we address this important paradox using the first humanized mouse model of oral HIV transmission. We established that reconstitution of the oral cavity and upper gastrointestinal (GI) tract of humanized bone marrow/liver/thymus (BLT) mice with human leukocytes, including the human cell types important for mucosal HIV transmission (i.e. dendritic cells, macrophages and CD4⁺ T cells), renders them susceptible to oral transmission of cell-free and cell-associated HIV. Oral transmission of HIV resulted in systemic infection of lymphoid and non-lymphoid tissues that is characterized by the presence of HIV RNA in plasma and a gradual decline of CD4⁺ T cells in peripheral blood. Consistent with infection of the oral cavity, we observed virus shedding into saliva. We then evaluated the role of human breast milk on oral HIV transmission. Our in vivo results demonstrate that breast milk has a strong inhibitory effect on oral transmission of both cell-free and cell-associated HIV. Finally, we evaluated the effect of antiretrovirals on oral transmission of HIV. Our results show that systemic antiretrovirals administered prior to exposure can efficiently prevent oral HIV transmission in BLT mice.

Expression of human endogenous retrovirus type K (HML-2) is activated by the Tat protein of HIV-1.

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The expression of HERV-K (HML-2), the family of HERVs that most recently entered the genome, is tightly regulated but becomes markedly increased after infection with HIV-1. To better understand the mechanisms involved in this activation, we explored the role of the HIV-1 Tat protein in inducing the expression of these endogenous retroviral genes. Administration of recombinant HIV-1 Tat protein caused a 13-fold increase in HERV-K (HML-2) gag RNA transcripts in Jurkat T cells and a 10-fold increase in primary lymphocytes, and the expression of the HERV-K (HML-2) rec and np9 oncogenes was also markedly increased. This activation was seen especially in lymphocytes and monocytic cells, the natural hosts for HIV-1 infection. Luciferase reporter gene assays demonstrated that the effect of Tat on HERV-K (HML-2) expression occurred at the level of the transcriptional promoter. The transcription factors NF-κB and NF-AT contribute to the Tat-induced activation of the promoter, as shown by chromatin immunoprecipitation assays, mutational analysis of the HERV-K (HML-2) long terminal repeat, and treatments with agents that inhibit NF-κB or NF-AT activation. These studies demonstrate that HIV-1 Tat plays an important role in activating expression of HERV-K (HML-2) in the setting of HIV-1 infection.

Generation of HIV latency in humanized BLT mice.

Here we demonstrate that a combination of tenofovir, emtricitabine, and raltegravir effectively suppresses peripheral and systemic HIV replication in humanized BLT mice. We also demonstrate that antiretroviral therapy (ART)-treated humanized BLT mice harbor latently infected resting human CD4+ T cells that can be induced ex vivo to produce HIV. We observed that the levels of infected resting human CD4+ T cells present in BLT mice are within the range of those observed circulating in patients undergoing suppressive ART. These results demonstrate the potential of humanized BLT mice as an attractive model for testing the in vivo efficacy of novel HIV eradication strategies.

Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble.

Current approaches used to identify protein-binding small molecules are not suited for identifying small molecules that can bind emerging RNA drug targets. By docking small molecules onto an RNA dynamic ensemble constructed by combining NMR spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from HIV type 1 (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with high affinity and inhibit its interaction with a Tat peptide in vitro (K(i) values of 710 nM-169 µM). One compound binds HIV-1 TAR with marked selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T-cell lines and HIV replication in an HIV-1 indicator cell line (IC(50) ∼23.1 µM).

Quantification of clesrovimab, an investigational, half-life extended, anti-respiratory syncytial virus protein F human monoclonal antibody in the nasal epithelial lining fluid of healthy adults.

BACKGROUND: Clesrovimab (MK-1654) is an investigational, half-life extended human monoclonal antibody (mAb) against RSV F glycoprotein in clinical trials as a prophylactic agent against RSV infection for infants. METHODS: This adult study measured clesrovimab concentrations in the serum and nasal epithelial lining fluid (ELF) to establish the partitioning of the antibody after dosing. Clesrovimab concentrations in the nasal ELF were normalized for sampling dilution using urea concentrations from ELF and serum. Furthermore, in vitro RSV neutralization of human nasal ELF following dosing was also measured to examine the activity of clesrovimab in the nasal compartment. FINDINGS: mAbs with YTE mutations are reported in literature to partition ∼1-2 % of serum antibodies into nasal mucosa. Nasal: serum ratios of 1:69-1:30 were observed for clesrovimab in two separate adult human trials after urea normalization, translating to 1.4-3.3 % of serum concentrations. The nasal PK and estimates of peripheral volume of distribution correlated with higher extravascular distribution of clesrovimab. These higher concentration of the antibody in the nasal ELF corroborated with the nasal sample's ability to neutralize RSV ex vivo. An overall trend of decreased viral plaque AUC was also noted with increasing availability of clesrovimab in the nasal ELF from a human RSV challenge study. INTERPRETATION: Along with its extended half-life, the higher penetration of clesrovimab into the nasal epithelial lining fluid and the associated local increase in RSV neutralization activity could offer infants better protection against RSV infection.

Immunogenicity Risk Assessment of Spontaneously Occurring Therapeutic Monoclonal Antibody Aggregates.

Aggregates of therapeutic proteins have been associated with increased immunogenicity in pre-clinical models as well as in human patients. Recent studies to understand aggregates and their immunogenicity risks use artificial stress methods to induce high levels of aggregation. These methods may be less biologically relevant in terms of their quantity than those that occur spontaneously during processing and storage. Here we describe the immunogenicity risk due to spontaneously occurring therapeutic antibody aggregates using peripheral blood mononuclear cells (PBMC) and a cell line with a reporter gene for immune activation: THP-1 BLUE NFκB. The spontaneously occurring therapeutic protein aggregates were obtained from process intermediates and final formulated drug substance from stability retains. Spontaneously occurring aggregates elicited innate immune responses for several donors in a PBMC assay with cytokine and chemokine production as a readout for immune activation. Meanwhile, no significant adaptive phase responses to spontaneously occurring aggregate samples were detected. While the THP-1 BLUE NFκB cell line and PBMC assays both responded to high stress induced aggregates, only the PBMC from a limited subset of donors responded to processing-induced aggregates. In this case study, levels of antibody aggregation occurring at process relevant levels are lower than those induced by stirring and may pose lower risk in vivo. Our methodologies can further inform additional immunogenicity risk assessments using a pre-clinical in vitro risk assessment approach utilizing human derived immune cells.

A lectin isolated from bananas is a potent inhibitor of HIV replication.

BanLec is a jacalin-related lectin isolated from the fruit of bananas, Musa acuminata. This lectin binds to high mannose carbohydrate structures, including those found on viruses containing glycosylated envelope proteins such as human immunodeficiency virus type-1 (HIV-1). Therefore, we hypothesized that BanLec might inhibit HIV-1 through binding of the glycosylated HIV-1 envelope protein, gp120. We determined that BanLec inhibits primary and laboratory-adapted HIV-1 isolates of different tropisms and subtypes. BanLec possesses potent anti-HIV activity, with IC(50) values in the low nanomolar to picomolar range. The mechanism for BanLec-mediated antiviral activity was investigated by determining if this lectin can directly bind the HIV-1 envelope protein and block entry of the virus into the cell. An enzyme-linked immunosorbent assay confirmed direct binding of BanLec to gp120 and indicated that BanLec can recognize the high mannose structures that are recognized by the monoclonal antibody 2G12. Furthermore, BanLec is able to block HIV-1 cellular entry as indicated by temperature-sensitive viral entry studies and by the decreased levels of the strong-stop product of early reverse transcription seen in the presence of BanLec. Thus, our data indicate that BanLec inhibits HIV-1 infection by binding to the glycosylated viral envelope and blocking cellular entry. The relative anti-HIV activity of BanLec compared favorably to other anti-HIV lectins, such as snowdrop lectin and Griffithsin, and to T-20 and maraviroc, two anti-HIV drugs currently in clinical use. Based on these results, BanLec is a potential component for an anti-viral microbicide that could be used to prevent the sexual transmission of HIV-1.

Targeted disruption of pi-pi stacking in Malaysian banana lectin reduces mitogenicity while preserving antiviral activity.

Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi-pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi-pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi-pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi-pi stacking without disrupting the overall lectin structure. These findings show that pi-pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes.

Ultra-long-acting tunable biodegradable and removable controlled release implants for drug delivery.

Here we report an ultra-long-acting tunable, biodegradable, and removable polymer-based delivery system that offers sustained drug delivery for up to one year for HIV treatment or prophylaxis. This robust formulation offers the ability to integrate multiple drugs in a single injection, which is particularly important to address the potential for drug resistance with monotherapy. Six antiretroviral drugs were selected based on their solubility in N-methyl-2-pyrrolidone and relevance as a combination therapy for HIV treatment or prevention. All drugs released with concentrations above their protein-adjusted inhibitory concentration and retained their physical and chemical properties within the formulation and upon release. The versatility of this formulation to integrate multiple drugs and provide sustained plasma concentrations from several weeks to up to one year, combined with its ability to be removed to terminate the treatment if necessary, makes it attractive as a drug delivery platform technology for a wide range of applications.

T cells establish and maintain CNS viral infection in HIV-infected humanized mice.

The human brain is an important site of HIV replication and persistence during antiretroviral therapy (ART). Direct evaluation of HIV infection in the brains of otherwise healthy individuals is not feasible; therefore, we performed a large-scale study of bone marrow/liver/thymus (BLT) humanized mice as an in vivo model to study HIV infection in the brain. Human immune cells, including CD4+ T cells and macrophages, were present throughout the BLT mouse brain. HIV DNA, HIV RNA, and/or p24+ cells were observed in the brains of HIV-infected animals, regardless of the HIV isolate used. HIV infection resulted in decreased numbers of CD4+ T cells, increased numbers of CD8+ T cells, and a decreased CD4+/CD8+ T cell ratio in the brain. Using humanized T cell-only mice (ToM), we demonstrated that T cells establish and maintain HIV infection of the brain in the complete absence of human myeloid cells. HIV infection of ToM resulted in CD4+ T cell depletion and a reduced CD4+/CD8+ T cell ratio. ART significantly reduced HIV levels in the BLT mouse brain, and the immune cell populations present were indistinguishable from those of uninfected controls, which demonstrated the effectiveness of ART in controlling HIV replication in the CNS and returning cellular homeostasis to a pre-HIV state.

ART influences HIV persistence in the female reproductive tract and cervicovaginal secretions.

The recently completed HIV prevention trials network study 052 is a landmark collaboration demonstrating that HIV transmission in discordant couples can be dramatically reduced by treating the infected individual with antiretroviral therapy (ART). However, the cellular and virological events that occur in the female reproductive tract (FRT) during ART that result in such a drastic decrease in transmission were not studied and remain unknown. Here, we implemented an in vivo model of ART in BM/liver/thymus (BLT) humanized mice in order to better understand the ability of ART to prevent secondary HIV transmission. We demonstrated that the entire FRT of BLT mice is reconstituted with human CD4+ cells that are shed into cervicovaginal secretions (CVS). A high percentage of the CD4+ T cells in the FRT and CVS expressed CCR5 and therefore are potential HIV target cells. Infection with HIV increased the numbers of CD4+ and CD8+ T cells in CVS of BLT mice. Furthermore, HIV was present in CVS during infection. Finally, we evaluated the effect of ART on HIV levels in the FRT and CVS and demonstrated that ART can efficiently suppress cell-free HIV-RNA in CVS, despite residual levels of HIV-RNA+ cells in both the FRT and CVS.

Rectal transmission of transmitted/founder HIV-1 is efficiently prevented by topical 1% tenofovir in BLT humanized mice.

Rectal microbicides are being developed to prevent new HIV infections in both men and women. We focused our in vivo preclinical efficacy study on rectally-applied tenofovir. BLT humanized mice (n = 43) were rectally inoculated with either the primary isolate HIV-1JRCSF or the MSM-derived transmitted/founder (T/F) virus HIV-1THRO within 30 minutes following treatment with topical 1% tenofovir or vehicle. Under our experimental conditions, in the absence of drug treatment we observed 50% and 60% rectal transmission by HIV-1JRCSF and HIV-1THRO, respectively. Topical tenofovir reduced rectal transmission to 8% (1/12; log rank p = 0.03) for HIV-1JRCSF and 0% (0/6; log rank p = 0.02) for HIV-1THRO. This is the first demonstration that any human T/F HIV-1 rectally infects humanized mice and that transmission of the T/F virus can be efficiently blocked by rectally applied 1% tenofovir. These results obtained in BLT mice, along with recent ex vivo, Phase 1 trial and non-human primate reports, provide a critically important step forward in the development of tenofovir-based rectal microbicides.

Murine colitis is mediated by vimentin.

Vimentin, an abundant intermediate filament protein, presumably has an important role in stabilizing intracellular architecture, but its function is otherwise poorly understood. In a vimentin knockout (Vim KO) mouse model, we note that Vim KO mice challenged with intraperitoneal Escherichia coli control bacterial infection better than do wild-type (WT) mice. In vitro, Vim KO phagocytes show significantly increased capacity to mediate bacterial killing by abundant production of reactive oxygen species (ROS) and nitric oxides, likely due to interactions with the p47phox active subunit of NADPH oxidase. In acute colitis induced by dextran sodium sulfate (DSS), Vim KO mice develop significantly less gut inflammation than do WT mice. Further, Vim KO mice have markedly decreased bacterial extravasation in the setting of DSS-induced acute colitis, consistent with decreased intestinal disease. Our results suggest that vimentin impedes bacterial killing and production of ROS, thereby contributing to the pathogenesis of acute colitis.

Combinations of griffithsin with other carbohydrate-binding agents demonstrate superior activity against HIV Type 1, HIV Type 2, and selected carbohydrate-binding agent-resistant HIV Type 1 strains.

Carbohydrate-binding agents (CBAs) are potential HIV microbicidal agents with a high genetic barrier to resistance. We wanted to evaluate whether two mannose-specific CBAs, recognizing multiple and often distinct glycan structures on the HIV envelope gp120, can interact synergistically against HIV-1, HIV-2, and HIV-1 strains that were selected for resistance against particular CBAs [i.e., 2G12 mAb and microvirin (MVN)]. Paired CBA/CBA combinations mainly showed synergistic activity against both wild-type HIV-1 and HIV-2 but also 2G12 mAb- and MVN-resistant HIV-1 strains as based on the median effect principle with combination indices (CIs) ranging between 0.29 and 0.97. Upon combination, an increase in antiviral potency of griffithsin (GRFT) up to ∼12-fold (against HIV-1), ∼8-fold (against HIV-2), and ∼6-fold (against CBA-resistant HIV-1) was observed. In contrast, HHA/GNA combinations showed additive activity against wild-type HIV-1 and HIV-2 strains, but remarkable synergy with HHA and GNA was observed against 2G12 mAb- and MVN-resistant HIV-1 strains (CI, 0.64 and 0.49, respectively). Overall, combinations of GRFT and other CBAs showed synergistic activity against HIV-1, HIV-2, and even against certain CBA-resistant HIV-1 strains. The CBAs tested appear to have distinct binding patterns on the gp120 envelope and therefore do not necessarily compete with each other's glycan binding sites on gp120. As a result, there might be no steric hindrance between two different CBAs in their competition for glycan binding (except for the HHA/GNA combination). These data are encouraging for the use of paired CBA combinations in topical microbicide applications (e.g., creams, gels, or intravaginal rings) to prevent HIV transmission.

Synergistic in vitro anti-HIV type 1 activity of tenofovir with carbohydrate-binding agents (CBAs).

Tenofovir, a well-known and highly prescribed anti-HIV-1 drug for the treatment of HIV/AIDS infections, has recently also shown its effectiveness as a potential microbicide drug in the prevention of HIV transmission. Here, we evaluated the combination of tenofovir with various members of the class of carbohydrate-binding agents (CBAs) targeting the glycans on the viral envelope gp120 for their anti-HIV efficacy. The tenofovir/CBA combinations predominantly showed synergistic antiviral activity using the median effect principle. These findings illustrate that combination of tenofovir with CBAs may increase the antiviral potency of the individual drugs and reducing the risk on potential side-effects.