Extended Postexposure Protection Against Vaginal Simian/Human Immunodeficiency Virus Infection With Tenofovir Alafenamide Fumarate/Elvitegravir Inserts in Macaques.

Vaginal inserts that can be used on demand before or after sex may be a desirable human immunodeficiency virus (HIV) prevention option for women. We recently showed that inserts containing tenofovir alafenamide fumarate (TAF, 20 mg) and elvitegravir (EVG, 16 mg) were highly protective against repeated simian/human immunodeficiency virus (SHIV) vaginal exposures when administered to macaques 4 hours before or after virus exposure (93% and 100%, respectively). Here, we show in the same macaque model that insert application 8 hours or 24 hours after exposure maintains high efficacy (94.4% and 77.2%, respectively). These data extend the protective window by TAF/EVG inserts and inform their clinical development for on-demand prophylaxis in women.

Pharmacokinetics of vaginally applied integrase inhibitors in macaques.

OBJECTIVES: We conducted a detailed pharmacokinetic assessment in macaques treated with vaginal gels formulated with HIV integrase strand transfer inhibitors (INSTIs) to better understand drug distribution and identify INSTI concentrations associated with previously demonstrated in vivo protection against vaginal simian HIV challenge. METHODS: Six macaques received vaginal gel containing 1% raltegravir (30 mg) once-weekly over 6 weeks. Following a washout period, five macaques received once-weekly gel containing 0.23% L-870,812 (7 mg). Drug concentrations were measured in plasma, mucosal fluids and vaginal tissues at baseline and 2, 5 and 24 h post-dosing. RESULTS: The median maximum concentration (Cmax) for raltegravir and L-870,812 in plasma was below the limit of quantification and 41.1 ng/mL, respectively. The Cmax in vaginal fluids (1441 and 1250 µg/mL) and tissues (266.7 and 368.4 µg/g) was achieved 2-5 h after dosing, respectively. A similar half-life was observed for raltegravir and L-870,812 in vaginal fluids (8-10 h) and remained 3-4 orders of magnitude above the protein-adjusted IC95 (0.016 and 0.106 µg/mL, respectively) at 24 h. Drug concentrations in vaginal fluids correlated well with those in vaginal tissues (Pearson r ≥ 0.788). Both drugs were consistently detected in rectal fluids 2 h after vaginal dosing, albeit at much lower levels (31-92-fold) than those in vaginal fluids. CONCLUSIONS: To the best of our knowledge, this study provides the first data on INSTI levels in vaginal tissues associated with in vivo protection and demonstrates rectal drug distribution of INSTIs after vaginal dosing. These findings may inform dose selection for topical products with INSTIs for HIV prevention.

Long-Acting Cabotegravir Protects Macaques Against Repeated Penile Simian-Human Immunodeficiency Virus Exposures.

We used a novel penile simian-human immunodeficiency virus (SHIV) transmission model to investigate whether long-acting cabotegravir (CAB LA) prevents penile SHIV acquisition in macaques. Twenty-two macaques were exposed to SHIV via the foreskin and urethra once weekly for 12 weeks. Of these, 6 received human-equivalent doses of CAB LA, 6 received oral emtricitabine/tenofovir disoproxil fumarate, and 10 were untreated. The efficacy of CAB LA was high (94.4%; 95% confidence interval, 58.2%-99.3%) and similar to that seen with oral emtricitabine/tenofovir disoproxil fumarate (94.0%; 55.1%-99.2%). The high efficacy of CAB LA in the penile transmission model supports extending the clinical advancement of CAB LA preexposure prophylaxis to heterosexual men.

Efficacy of Vaginally Administered Gel Containing Emtricitabine and Tenofovir Against Repeated Rectal Simian Human Immunodeficiency Virus Exposures in Macaques.

Vaginal microbicides containing antiretrovirals (ARVs) have shown to prevent vaginally acquired human immunodeficiency virus (HIV), but these products may not protect women who engage in anal sex. Intravaginal dosing with ARVs has shown to result in drug exposures in rectal tissues, thus raising the possibility of dual compartment protection. To test this concept, we investigated whether intravaginal dosing with emtricitabine (FTC)/tenofovir (TFV) gel, which fully protected macaques against repeated vaginal exposures to simian human immunodeficiency virus (SHIV), protects against rectal SHIV exposures. Pharmacokinetic studies revealed rapid distribution of FTC and TFV to rectal tissues and luminal fluids, albeit at concentrations 1-2 log10 lower than those in the vaginal compartment. Efficacy measurements against repeated rectal SHIV challenges demonstrated a 4.5-fold reduction in risk of infection in macaques that received intravaginal FTC/TFV compared to placebo gel (P = .047; log-rank test). These data support the concept of dual compartment protection by vaginal dosing and warrants developing ARV-based vaginal products with improved bidirectional dosing.

A Recombinant Chimeric Ad5/3 Vector Expressing a Multistage Plasmodium Antigen Induces Protective Immunity in Mice Using Heterologous Prime-Boost Immunization Regimens.

An ideal malaria vaccine should target several stages of the parasite life cycle and induce antiparasite and antidisease immunity. We have reported a Plasmodium yoelii chimeric multistage recombinant protein (P. yoelii linear peptide chimera/recombinant modular chimera), engineered to express several autologous T cell epitopes and sequences derived from the circumsporozoite protein and the merozoite surface protein 1. This chimeric protein elicits protective immunity, mediated by CD4(+) T cells and neutralizing Abs. However, experimental evidence, from pre-erythrocytic vaccine candidates and irradiated sporozoites, has shown that CD8(+) T cells play a significant role in protection. Recombinant viral vectors have been used as a vaccine platform to elicit effective CD8(+) T cell responses. The human adenovirus (Ad) serotype 5 has been tested in malaria vaccine clinical trials with excellent safety profile. Nevertheless, a major concern for the use of Ad5 is the high prevalence of anti-vector neutralizing Abs in humans, hampering its immunogenicity. To minimize the impact of anti-vector pre-existing immunity, we developed a chimeric Ad5/3 vector in which the knob region of Ad5 was replaced with that of Ad3, conferring partial resistance to anti-Ad5 neutralizing Abs. Furthermore, we implemented heterologous Ad/protein immunization regimens that include a single immunization with recombinant Ad vectors. Our data show that immunization with the recombinant Ad5/3 vector induces protective efficacy indistinguishable from that elicited by Ad5. Our study also demonstrates that the dose of the Ad vectors has an impact on the memory profile and protective efficacy. The results support further studies with Ad5/3 for malaria vaccine development.

Aluminum exclusion from root zone and maintenance of nutrient uptake are principal mechanisms of Al tolerance in Pisum sativum L.

Our study aimed to evaluate intraspecific variability of pea (Pisum sativum L.) in Al tolerance and to reveal mechanisms underlying genotypic differences in this trait. At the first stage, 106 pea genotypes were screened for Al tolerance using root re-elongation assay based on staining with eriochrome cyanine R. The root re-elongation zone varied from 0.5 mm to 14 mm and relationships between Al tolerance and provenance or phenotypic traits of genotypes were found. Tolerance index (TI), calculated as a biomass ratio of Al-treated and non-treated contrasting genotypes grown in hydroponics for 10 days, varied from 30% to 92% for roots and from 38% to 90% for shoots. TI did not correlate with root or shoot Al content, but correlated positively with increasing pH and negatively with residual Al concentration in nutrient solution in the end of experiments. Root exudation of organic acid anions (mostly acetate, citrate, lactate, pyroglutamate, pyruvate and succinate) significantly increased in several Al-treated genotypes, but did not correlate with TI. Al-treatment decreased Ca, Co, Cu, K, Mg, Mn, Mo, Ni, S and Zn contents in roots and/or shoots, whereas contents of several elements (P, B, Fe and Mo in roots and B and Fe in shoots) increased, suggesting that Al toxicity induced substantial disturbances in uptake and translocation of nutrients. Nutritional disturbances were more pronounced in Al sensitive genotypes. In conclusion, pea has a high intraspecific variability in Al tolerance and this trait is associated with provenance and phenotypic properties of plants. Transformation of Al to unavailable (insoluble) forms in the root zone and the ability to maintain nutrient uptake are considered to be important mechanisms of Al tolerance in this plant species.

Efficacy of topical tenofovir against transmission of a tenofovir-resistant SHIV in macaques.

BACKGROUND: Topically delivered tenofovir (TFV) from intravaginal rings, tablets, or gels is being evaluated for HIV prevention. We previously demonstrated that TFV delivered vaginally by gel protected macaques from vaginal infection with SHIV. Here we investigated efficacy of the TFV gel against vaginal transmission of a TFV-resistant SHIV containing the K65R mutation (SHIV162P3K65R) and its relationship to drug levels in vaginal tissues. RESULTS: SHIV162P3K65R shows approximately a 5-fold reduction in susceptibility to TFV compared to wild-type SHIV. Efficacy was evaluated in pig-tailed macaques exposed vaginally twice-weekly (up to 10 weeks) to SHIV162P3K65R 30 min after receiving placebo (n = 6) or 1% TFV (n = 6) gel. Four of the six controls were infected after a median of 5 exposures. In contrast, five of six macaques that received TFV gel remained uninfected after 20 vaginal SHIV162P3K65R exposures, resulting in an estimated efficacy of 75%. The mean intracellular TFV-diphosphate (TFV-DP) concentrations in vaginal lymphocytes 4 h after a single gel dose were found to be high (1,631 fmol/10(6) cells, range 492-3,847) and within the in vitro IC75 range (1,206 fmol/10(6) cells) for SHIV162P3K65R. CONCLUSION: Both the modest resistance conferred by K65R and the high TFV-DP exposure in vaginal lymphocytes, likely explain the observed protection. The findings in this model do not predict complete loss of protection by topical TFV against vaginal exposure to HIV-1K65R viruses and provide a tissue drug target for high efficacy. These data will facilitate the development of TFV delivery platforms that have high activity on both wild-type and TFV-resistant viruses.

Development and optimization of a non-enzymatic method of leukocyte isolation from macaque tissues.

BACKGROUND AND METHODS: Cell isolation from macaque tissues involves laborious enzymatic digestion. The Medimachine provides a simpler, quicker non-enzymatic method, yielding 1.5­5 million cells/g of vaginal or rectal tissue from pigtailed macaques. RESULTS AND CONCLUSIONS: Flow cytometry analysis of the two methods revealed similar levels of cell viability and most major cell lineage and activation markers.

Pharmacokinetics and efficacy of topical inserts containing tenofovir alafenamide fumarate and elvitegravir administered rectally in macaques.

BACKGROUND: Topical on-demand forms for HIV pre-exposure prophylaxis (PrEP) may be a desirable alternative for people that prefer not to use daily PrEP. CONRAD has developed inserts containing tenofovir alafenamide (TAF) and elvitegravir (EVG) for on-demand vaginal or rectal pericoital use. We assessed the pharmacokinetics (PK) and pre-exposure efficacy of rectally applied TAF/EVG inserts in macaques. METHODS: PK was assessed in 12 pigtailed macaques. Tenofovir (TFV) and EVG levels were assayed in rectal biopsies and secretions, and tenofovir-diphosphate (TFV-DP) levels in biopsies and peripheral blood mononuclear cells (PBMC). Drug biodistribution was evaluated in 10 animals at necropsy 4 h post-dosing. For efficacy assessments, one or two TAF/EVG inserts were administered to macaques (n = 6) 4 h before repeated rectal SHIV162p3 challenges. FINDINGS: One TAF/EVG insert resulted in rapid and high EVG and TFV-DP in rectal tissue 4 h after application. Adding a second insert led to a 10-fold increase in EVG and TFV-DP in rectal tissue. Efficacy of one and two TAF/EVG inserts were 72.6% (CI 24.5%-92.6%) and 93.1% (CI 73.3%-99.2%), respectively. INTERPRETATION: Although high TFV-DP and EVG levels were observed with one rectal TAF/EVG insert, it only conferred partial protection from rectal SHIV challenges. Adding a second insert led to an increase in TFV and EVG in rectal tissues resulting in higher (>90%) efficacy. These results highlight the high efficacy of TAF/EVG inserts as topical on-demand rectal PrEP, as well as the need for appropriate drug coverage in the deep rectum and colon to achieve high protection. FUNDING: The work related to animal studies was funded by CDC intramural funds and an interagency agreement between CDC and USAID (USAID/CDC IAA AID-GH-T-15-00002). The work related to the insert formulation was funded by U.S. PEPFAR through USAID under a Cooperative Agreement (AID-OAA-A-14-00010) with CONRAD/Eastern Virginia Medical School. The findings and conclusions of this manuscript are those of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention (CDC), USAID, President's Emergency Plan for AIDS Relief (PEPFAR), Eastern Virginia Medical School (EVMS), or the US government.

Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1-based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.

Evaluation of cellular determinants required for in vitro xenotropic murine leukemia virus-related virus entry into human prostate cancer and noncancerous cells.

The newly identified retrovirus-the xenotropic murine leukemia virus-related virus (XMRV)-has recently been shown to be strongly associated with familial prostate cancer in humans (A. Urisman et al., PLoS Pathog. 2:e25, 2006). While that study showed evidence of XMRV infection exclusively in the prostatic stromal fibroblasts, a recent study found XMRV protein antigens mainly in malignant prostate epithelial cells (R. Schlaberg et al., Proc. Natl. Acad. Sci. U. S. A. 106:16351-16356, 2009). To help elucidate the mechanisms behind XMRV infection, we show that prostatic fibroblast cells express Xpr1, a known receptor of XMRV, but its expression is absent in other cell lines of the prostate (i.e., epithelial and stromal smooth muscle cells). We also show that certain amino acid residues located within the predicted extracellular loop (ECL3 and ECL4) sequences of Xpr1 are required for efficient XMRV entry. Although we found strong evidence to support XMRV infection of prostatic fibroblast cell lines via Xpr1, we learned that XMRV was indeed capable of infecting cells that did not necessarily express Xpr1, such as those of the prostatic epithelial and smooth muscle origins. Further studies suggest that the expression of Xpr1 and certain genotypes of the RNASEL gene, which could restrict XMRV infection, may play important roles in defining XMRV tropisms in certain cell types. Collectively, our data reveal important cellular determinants required for XMRV entry into different human prostate cells in vitro, which may provide important insights into the possible role of XMRV as an etiologic agent in human prostate cancer.

Development, Characterization and In Vivo Pharmacokinetic Assessment of Rectal Suppositories Containing Combination Antiretroviral Drugs for HIV Prevention.

Receptive anal intercourse (RAI) contributes significantly to HIV acquisition underscoring the need to develop HIV prevention options for populations engaging in RAI practices. We explored the feasibility of formulating rectal suppositories with potent antiviral drugs for on-demand use. A fixed-dose combination of tenofovir (TFV) and elvitegravir (EVG) (40 mg each) was co-formulated in six different suppository bases (three fat- and three water-soluble). Fat-soluble witepsol H15 and water-soluble polyethylene glycol (PEG) based suppositories demonstrated favorable in vitro release and were advanced to assess in vivo pharmacokinetics following rectal administration in macaques. In vivo drug release profiles were similar for both suppository bases. Median concentrations of TFV and EVG detected in rectal fluids at 2 h were 1- and 2-logs higher than the in vitro IC50, respectively; TFV-diphosphate levels in rectal tissues met or exceeded those associated with high efficacy against rectal simian HIV (SHIV) exposure in macaques. Leveraging on these findings, a PEG-based suppository with a lower dose combination of tenofovir alafenamide (TAF) and EVG (8 mg each) was developed and found to achieve similar rectal drug exposures in macaques. This study establishes the utility of rectal suppositories as a promising on-demand strategy for HIV PrEP and supports their clinical development.

Lysophosphatidic acid induces neointima formation through PPARgamma activation.

Neointimal lesions are characterized by accumulation of cells within the arterial wall and are a prelude to atherosclerotic disease. Here we report that a brief exposure to either alkyl ether analogs of the growth factor-like phospholipid lysophosphatidic acid (LPA), products generated during the oxidative modification of low density lipoprotein, or to unsaturated acyl forms of LPA induce progressive formation of neointima in vivo in a rat carotid artery model. This effect is completely inhibited by the peroxisome proliferator-activated receptor (PPAR)gamma antagonist GW9662 and mimicked by PPARgamma agonists Rosiglitazone and 1-O-hexadecyl-2-azeleoyl-phosphatidylcholine. In contrast, stearoyl-oxovaleryl phosphatidylcholine, a PPARalpha agonist and polypeptide epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor failed to elicit neointima. The structure-activity relationship for neointima induction by LPA analogs in vivo is identical to that of PPARgamma activation in vitro and disparate from that of LPA G protein-coupled receptor activation. Neointima-inducing LPA analogs up-regulated the CD36 scavenger receptor in vitro and in vivo and elicited dedifferentiation of cultured vascular smooth muscle cells that was prevented by GW9662. These results suggest that selected LPA analogs are important novel endogenous PPARgamma ligands capable of mediating vascular remodeling and that activation of the nuclear transcription factor PPARgamma is both necessary and sufficient for neointima formation by components of oxidized low density lipoprotein.

Microbial Consortium of PGPR, Rhizobia and Arbuscular Mycorrhizal Fungus Makes Pea Mutant SGECdt Comparable with Indian Mustard in Cadmium Tolerance and Accumulation.

Cadmium (Cd) is one of the most widespread and toxic soil pollutants that inhibits plant growth and microbial activity. Polluted soils can be remediated using plants that either accumulate metals (phytoextraction) or convert them to biologically inaccessible forms (phytostabilization). The phytoremediation potential of a symbiotic system comprising the Cd-tolerant pea (Pisum sativum L.) mutant SGECdt and selected Cd-tolerant microorganisms, such as plant growth-promoting rhizobacterium Variovorax paradoxus 5C-2, nodule bacterium Rhizobium leguminosarum bv. viciae RCAM1066, and arbuscular mycorrhizal fungus Glomus sp. 1Fo, was evaluated in comparison with wild-type pea SGE and the Cd-accumulating plant Indian mustard (Brassica juncea L. Czern.) VIR263. Plants were grown in pots in sterilized uncontaminated or Cd-supplemented (15 mg Cd kg-1) soil and inoculated or not with the microbial consortium. Cadmium significantly inhibited growth of uninoculated and particularly inoculated SGE plants, but had no effect on SGECdt and decreased shoot biomass of B. juncea. Inoculation with the microbial consortium more than doubled pea biomass (both genotypes) irrespective of Cd contamination, but had little effect on B. juncea biomass. Cadmium decreased nodule number and acetylene reduction activity of SGE by 5.6 and 10.8 times, whereas this decrease in SGECdt was 2.1 and 2.8 times only, and the frequency of mycorrhizal structures decreased only in SGE roots. Inoculation decreased shoot Cd concentration and increased seed Cd concentration of both pea genotypes, but had little effect on Cd concentration of B. juncea. Inoculation also significantly increased concentration and/or accumulation of nutrients (Ca, Fe, K, Mg, Mn, N, P, S, and Zn) by Cd-treated pea plants, particularly by the SGECdt mutant. Shoot Cd concentration of SGECdt was twice that of SGE, and the inoculated SGECdt had approximately similar Cd accumulation capacity as compared with B. juncea. Thus, plant-microbe systems based on Cd-tolerant micro-symbionts and plant genotypes offer considerable opportunities to increase plant HM tolerance and accumulation.

The early- and late stages in phenotypic modulation of vascular smooth muscle cells: differential roles for lysophosphatidic acid.

Lysophosphatidic acid (LPA) has been implicated as causative in phenotypic modulation (PM) of cultured vascular smooth muscle cells (VSMC) in their transition to the dedifferentiated phenotype. We evaluated the contribution of the three major LPA receptors, LPA1 and LPA2 GPCR and PPARgamma, on PM of VSMC. Expression of differentiated VSMC-specific marker genes, including smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM-22alpha, and h-caldesmon, was measured by quantitative real-time PCR in VSMC cultures and aortic rings kept in serum-free chemically defined medium or serum- or LPA-containing medium using wild-type C57BL/6, LPA1, LPA2, and LPA1&2 receptor knockout mice. Within hours after cells were deprived of physiological cues, the expression of VSMC marker genes, regardless of genotype, rapidly decreased. This early PM was neither prevented by IGF-I, inhibitors of p38, ERK1/2, or PPARgamma nor significantly accelerated by LPA or serum. To elucidate the mechanism of PM in vivo, carotid artery ligation with/without replacement of blood with Krebs solution was used to evaluate contributions of blood flow and pressure. Early PM in the common carotid was induced by depressurization regardless of the presence/absence of blood, but eliminating blood flow while maintaining blood pressure or after sham surgery elicited no early PM. The present results indicate that LPA, serum, dissociation of VSMC, IGF-I, p38, ERK1/2, LPA1, and LPA2 are not causative factors of early PM of VSMC. Tensile stress generated by blood pressure may be the fundamental signal maintaining the fully differentiated phenotype of VSMC.

Serine phosphorylation differentially affects RhoA binding to effectors: implications to NGF-induced neurite outgrowth.

Activation of RhoA prevents NGF-induced outgrowth and causes retraction of neurites in neuronal cells, including PC12 cells. Despite its inhibitory effect on neurite outgrowth, NGF activates GTP loading of and effector binding to RhoA, setting up an apparent contradiction. According to the molecular switch hypothesis of GTPase function GTP-loading of RhoA should be sufficient to activate its effectors uniformly. However, when monitoring NGF-induced binding of GTP-RhoA to multiple targets, we noted differential interactions with its effectors. We found that NGF elicits a protein kinase A-mediated phosphorylation of RhoA on serine(188), which renders it unable to bind to Rho-associated kinase (ROK), whereas it retains the ability to interact with other RhoA targets including rhotekin, mDia-1 and PKN. We show in vitro and in vivo that phosphorylation of serine(188) represents an additional switch, capable of directing signals among effector pathways. In the context of PC12 cell differentiation, NGF-induced phosphorylation of RhoA on serine(188) prevents it from interacting with ROK, which would otherwise block neurite outgrowth. Transfection of RhoA(S188A) mutant into PC12 cells prevents NGF-induced neurite outgrowth, just like constitutively activated RhoA(14V) does, indicating the requirement of this phosphorylation site. Replacement of serine(188) with the phosphomimetic glutamate residue in RhoA(V14/S188E) selectively impairs interaction with ROK and when transfected into PC12 cells restores NGF-induced neurite outgrowth. Therefore, phosphorylation of serine(188) may serve as a novel secondary switch of RhoA capable of overriding GTP-binding-elicited effector activation to a subset of targets such as ROK, which interact with the C-terminus of RhoA.

Topical tenofovir protects against vaginal simian HIV infection in macaques coinfected with Chlamydia trachomatis and Trichomonas vaginalis.

BACKGROUND: Chlamydia trachomatis and Trichomonas vaginalis, two prevalent sexually transmitted infections, are known to increase HIV risk in women and could potentially diminish preexposure prophylaxis efficacy, particularly for topical interventions that rely on local protection. We investigated in macaques whether coinfection with Chlamydia trachomatis/Trichomonas vaginalis reduces protection by vaginal tenofovir (TFV) gel. METHODS: Vaginal TFV gel dosing previously shown to provide 100 or 74% protection when applied either 30 min or 3 days before simian HIV(SHIV) challenge was assessed in pigtailed macaques coinfected with Chlamydia trachomatis/Trichomonas vaginalis and challenged twice weekly with SHIV162p3 for up to 10 weeks (two menstrual cycles). Three groups of six macaques received either placebo or 1% TFV gel 30 min or 3 days before each SHIV challenge. We additionally assessed TFV and TFV diphosphate concentrations in plasma and vaginal tissues in Chlamydia trachomatis/Trichomonas vaginalis coinfected (n = 4) and uninfected (n = 4) macaques. RESULTS: Chlamydia trachomatis/Trichomonas vaginalis coinfections were maintained during the SHIV challenge period. All macaques that received placebo gel were SHIV infected after a median of seven challenges (one menstrual cycle). In contrast, no infections were observed in macaques treated with TFV gel 30 min before SHIV challenge (P < 0.001). Efficacy was reduced to 60% when TFV gel was applied 3 days before SHIV challenge (P = 0.07). Plasma TFV and TFV diphosphate concentrations in tissues and vaginal lymphocytes were significantly higher in Chlamydia trachomatis/Trichomonas vaginalis coinfected compared with Chlamydia trachomatis/Trichomonas vaginalis uninfected macaques. CONCLUSION: Our findings in this model suggest that Chlamydia trachomatis/Trichomonas vaginalis coinfection may have little or no impact on the efficacy of highly effective topical TFV modalities and highlight a significant modulation of TFV pharmacokinetics.

Stable knock-down of the sphingosine 1-phosphate receptor S1P1 influences multiple functions of human endothelial cells.

OBJECTIVES: Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein-coupled receptors S1P1-5 and as a second messenger. Because S1P1 knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P1 in endothelial cells is needed. METHODS AND RESULTS: All human endothelial cells analyzed expressed abundant S1P1 transcripts. We permanently silenced (by RNA interference) the expression of S1P1 in the human endothelial cell lines AS-M.5 and ISO-HAS.1. The S1P1 knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress-mediated injury. New S1P1-dependent gene targets were identified in human endothelial cells. S1P1 silencing decreased the expression of platelet-endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-alpha. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P1 silencing. CONCLUSIONS: Long-term silencing of S1P1 enabled us for the first time to demonstrate the involvement of S1P1 in key functions of endothelial cells and to identify new S1P1-dependent gene targets.

CryoEM visualization of an adenovirus capsid-incorporated HIV antigen.

Adenoviral (Ad) vectors show promise as platforms for vaccine applications against infectious diseases including HIV. However, the requirements for eliciting protective neutralizing antibody and cellular immune responses against HIV remain a major challenge. In a novel approach to generate 2F5- and 4E10-like antibodies, we engineered an Ad vector with the HIV membrane proximal ectodomain region (MPER) epitope displayed on the hypervariable region 2 (HVR2) of the viral hexon capsid, instead of expressed as a transgene. The structure and flexibility of MPER epitopes, and the structural context of these epitopes within viral vectors, play important roles in the induced host immune responses. In this regard, understanding the critical factors for epitope presentation would facilitate optimization strategies for developing viral vaccine vectors. Therefore we undertook a cryoEM structural study of this Ad vector, which was previously shown to elicit MPER-specific humoral immune responses. A subnanometer resolution cryoEM structure was analyzed with guided molecular dynamics simulations. Due to the arrangement of hexons within the Ad capsid, there are twelve unique environments for the inserted peptide that lead to a variety of conformations for MPER, including individual α-helices, interacting α-helices, and partially extended forms. This finding is consistent with the known conformational flexibility of MPER. The presence of an extended form, or an induced extended form, is supported by interaction of this vector with the human HIV monoclonal antibody 2F5, which recognizes 14 extended amino acids within MPER. These results demonstrate that the Ad capsid influences epitope structure, flexibility and accessibility, all of which affect the host immune response. In summary, this cryoEM structural study provided a means to visualize an epitope presented on an engineered viral vector and suggested modifications for the next generation of Ad vectors with capsid-incorporated HIV epitopes.

Antibody responses against xenotropic murine leukemia virus-related virus envelope in a murine model.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV. RESULTS: Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations. CONCLUSIONS: Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.