Weekly Oral Tenofovir Alafenamide Protects Macaques from Vaginal and Rectal Simian HIV Infection.

Pre-exposure prophylaxis (PrEP) with a weekly oral regimen of antiretroviral drugs could be a suitable preventative option for individuals who struggle with daily PrEP or prefer not to use long-acting injectables. We assessed in macaques the efficacy of weekly oral tenofovir alafenamide (TAF) at doses of 13.7 or 27.4 mg/kg. Macaques received weekly oral TAF for six weeks and were exposed twice-weekly to SHIV vaginally or rectally on day 3 and 6 after each dose. Median TFV-DP levels in PBMCs following the 13.7 mg/kg dose were 3110 and 1137 fmols/106 cells on day 3 and 6, respectively. With the 27.4 mg/kg dose, TFV-DP levels were increased (~2-fold) on day 3 and 6 (6095 and 3290 fmols/106 cells, respectively). Both TAF doses (13.7 and 27.4 mg/kg) conferred high efficacy (94.1% and 93.9%, respectively) against vaginal SHIV infection. Efficacy of the 27.4 mg/kg dose against rectal SHIV infection was 80.7%. We estimate that macaque doses of 13.7 and 27.4 mg/kg are equivalent to approximately 230 and 450 mg of TAF in humans, respectively. Our findings demonstrate the effectiveness of a weekly oral PrEP regimen and suggest that a clinically achievable oral TAF dose could be a promising option for non-daily PrEP.

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.

Characterisation of the bovine C-type lectin receptor Mincle and potential evidence for an endogenous ligand.

Innate immune receptors that form complexes with secondary receptors, activating multiple signalling pathways, modulate cellular activation and play essential roles in regulating homeostasis and immunity. We have previously identified a variety of bovine C-type lectin-like receptors that possess similar functionality than their human orthologues. Mincle (CLEC4E), a heavily glycosylated monomer, is involved in the recognition of the mycobacterial component Cord factor (trehalose 6,6'-dimycolate). Here we characterise the bovine homologue of Mincle (boMincle), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (huMincle), although there are some notable differences. In the absence of cross-reacting antibodies, boMincle-specific antibodies were created and used to demonstrate that, like the human receptor, boMincle is predominantly expressed by myeloid cells. BoMincle surface expression increases during the maturation of monocytes to macrophages. However, boMincle mRNA transcripts were also detected in granulocytes, B cells, and T cells. Finally, we show that boMincle binds to isolated bovine CD4+ T cells in a specific manner, indicating the potential to recognise endogenous ligands. This suggests that the receptor might also play a role in homeostasis in cattle.

HIV Pre-exposure Prophylaxis Persistence and Adherence Among Men Who Have Sex With Men in Four US Cities.

BACKGROUND: HIV pre-exposure prophylaxis (PrEP) persistence and adherence are critical to ending the HIV epidemic in the United States. SETTING: In 2017 National HIV Behavioral Surveillance, HIV-negative men who have sex with men (MSM) in 4 US cities completed a survey, HIV testing, and dried blood spots at recruitment. METHODS: We assessed 3 PrEP outcomes: persistence (self-reported PrEP use at any time in the past 12 months and had tenofovir, emtricitabine, or tenofovir diphosphate detected in dried blood spots), adherence at ≥4 doses/week (self-reported past-month PrEP use and tenofovir diphosphate concentration ≥700 fmol/punch), and adherence at 7 doses/week (self-reported past-month PrEP use and tenofovir diphosphate concentration ≥1250 fmol/punch). Associations with key characteristics were examined using log-linked Poisson regression models with generalized estimating equations. RESULTS: Among 391 MSM who took PrEP in the past year, persistence was 80% and was lower among MSM who were younger, had lower education, and had fewer sex partners. Of 302 MSM who took PrEP in the past month, adherence at ≥4 doses/week was 80% and adherence at 7 doses/week was 66%. Adherence was lower among MSM who were younger, were Black, and had fewer sex partners. CONCLUSIONS: Although persistence and adherence among MSM were high, 1 in 5 past-year PrEP users were not persistent and 1 in 5 past-month PrEP users were not adherent at levels that would effectively protect them from acquiring HIV (ie, ≥4 doses/week). Efforts to support PrEP persistence and adherence should include MSM who are young, are Black, and have less education.

Pharmacology of boosted and unboosted integrase strand transfer inhibitors for two-dose event-driven HIV prevention regimens among men.

BACKGROUND: Event-driven HIV prevention strategies are a priority for users who do not require daily pre-exposure prophylaxis (PrEP). Regimens containing integrase strand transfer inhibitors (INSTIs) are under evaluation as alternatives to daily PrEP. To better understand INSTI distribution and inform dosing selection we compared the pharmacology of two-dose boosted elvitegravir and unboosted bictegravir regimens in MSM. MATERIALS AND METHODS: Blood, rectal and penile secretions and rectal biopsies were collected from 63 HIV-negative MSM aged 18-49 years. Specimens were collected up to 96 h after two oral doses of tenofovir alafenamide and emtricitabine with elvitegravir boosted by cobicistat or unboosted bictegravir given 24 h apart. Antiretroviral drugs were measured by LC-MS. RESULTS: Mean bictegravir plasma concentrations remained above the 95% protein-adjusted effective concentration 96 h after dosing [273 (95% CI: 164-456) ng/mL] whereas elvitegravir plasma concentrations became undetectable 48 h after the second dose. Bictegravir and elvitegravir reached rectal tissues within 2 h after the first dose, and elvitegravir tissue concentrations [1.07 (0.38-13.51) ng/mg] were greater than bictegravir concentrations [0.27 (0.15-0.70) ng/mg]. Both INSTIs became undetectable in tissues within 96 h. Elvitegravir and bictegravir were not consistently detected in penile secretions. CONCLUSIONS: Whereas bictegravir plasma concentrations persist at least 4 days after a two-oral-dose HIV prophylaxis regimen, elvitegravir accumulates in mucosal tissues. Differing elvitegravir and bictegravir distribution may result in variable mucosal and systemic antiviral activity and can inform dosing strategies for event-driven HIV prevention.

Single dose topical inserts containing tenofovir alafenamide fumarate and elvitegravir provide pre- and post-exposure protection against vaginal SHIV infection in macaques.

BACKGROUND: Vaginal products for HIV prevention that can be used on-demand before or after sex may be a preferable option for women with low frequency or unplanned sexual activity or who prefer not to use daily or long-acting pre-exposure prophylaxis (PrEP). We performed dose ranging pharmacokinetics (PK) and efficacy studies of a vaginally applied insert containing tenofovir alafenamide fumarate (TAF) and elvitegravir (EVG) in macaques under PrEP or post-exposure prophylaxis (PEP) modalities. METHODS: PK studies were performed in 3 groups of pigtailed macaques receiving inserts with different fixed-dose combinations of TAF and EVG (10/8, 20/16 and 40/24 mg). PrEP and PEP efficacy of a selected insert was investigated in a repeat exposure vaginal SHIV transmission model. Inserts were administered 4 h before (n = 6) or after (n = 6) repeated weekly SHIV exposures. Infection outcome was compared with macaques receiving placebo inserts (n = 12). FINDINGS: Dose ranging studies showed rapid and sustained high drug concentrations in vaginal fluids and tissues across insert formulations with minimal dose proportionality. TAF/EVG (20/16 mg) inserts were selected for efficacy evaluation. Five of the 6 animals receiving these inserts 4 h before and 6/6 animals receiving inserts 4 h after SHIV exposure were protected after 13 challenges (p = 0.0088 and 0.0077 compared to placebo, respectively). The calculated PrEP and PEP efficacy was 91.0% (95% CI = 32.2%-98.8%) and 100% (95% CI = undefined), respectively. INTERPRETATION: Inserts containing TAF/EVG provided high protection against vaginal SHIV infection when administered within a 4 h window before or after SHIV exposure. Our results support the clinical development of TAF/EVG inserts for on-demand PrEP and PEP in women. FUNDING: Funded by CDC intramural funds, an interagency agreement between CDC and USAID (USAID/CDC IAA AID-GH-T-15-00002), and by the U.S. President's Emergency Plan for AIDS Relief (PEPFAR) through the U.S. Agency for International Development (USAID) under a Cooperative Agreement (AID-OAA-A-14-00010) with CONRAD/Eastern Virginia Medical School.

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.

Genital Immune Cell Activation and Tenofovir Gel Efficacy: A Case-Control Study.

Genital inflammation (GI) undermines topical human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) efficacy through unknown mechanisms. Here, associations between activated endocervical CD4 + T-cell numbers and higher deoxyadenosine triphosphate (dATP) concentrations suggest that competition for intracellular metabolites within HIV target cells may reduce the efficacy of antiretroviral-based PrEP in women with GI.

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.

Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called "Infection and Treatment Method" (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

Bovine Neutrophils Release Extracellular Traps and Cooperate With Macrophages in Mycobacterium avium subsp. paratuberculosis clearance In Vitro.

Mycobacterium avium subsp. paratuberculosis (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an in vitro mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Mycobacterium bovis Bacillus-Calmette-Guérin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1ß and IL-8 release were assessed. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1ß and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection site.

Studying the heterogeneous pathogenesis of canine diabetes: Observational characterization of an island population.

BACKGROUND: Canine diabetes mellitus has mostly been studied in northern European, Australian and American populations, whereas other regions have received less attention. OBJECTIVES: We evaluated the epidemiological, clinical and histopathological features of diabetic dogs in Gran Canaria, Spain. METHODS: Prevalence and incidence were estimated. Clinical features were analysed, and serum and genomic DNA were obtained. Dogs with presumed idiopathic or immune-mediated diabetes, were DLA-typed and antibodies against GAD65 and IA-2 were assessed. Pancreases from ten diabetic dogs were examined and compared with pancreases from non-diabetic dogs. RESULTS AND CONCLUSIONS: Twenty-nine diabetic dogs were identified in a population of 5,213 (prevalence: 0.56%; incidence: 0.37%). Most were female (79%) and sexually intact (87% of females, 83% of males). Diabetes secondary to dioestrus (55.2%) and insulin-deficient diabetes (20.7%) were the most frequent types. Antibodies against GAD65 and IA-2 were identified in two out of five cases and DLA-genotyping revealed novel haplotypes. Breed distribution differed between diabetic and non-diabetic dogs. Reduced number of pancreatic islets and ß-cell mass were observed, with vacuolation of islet cells and ductal epithelium. In this population, where neutering is not standard practice, diabetes secondary to dioestrus is the most frequent diabetes subtype. Genetic susceptibility also differed from previous studies. These results support the heterogeneous pathogenesis of canine diabetes.

The effect of depot medroxyprogesterone acetate on tenofovir alafenamide in rhesus macaques.

Prevention of HIV infection and unintended pregnancies are public health priorities. In sub-Saharan Africa, where HIV prevalence is highest, depot medroxyprogesterone acetate (DMPA) is widely used as contraception. Therefore, understanding potential interactions between DMPA and antiretrovirals is critical. Here, we use a macaque model to investigate the effect of DMPA on the pharmacology of the antiretroviral tenofovir alafenamide (TAF). Female rhesus macaques received 30 mg of DMPA (n = 9) or were untreated (n = 9). Macaques received a human equivalent dose of TAF (1.5 mg/kg) orally by gavage. Tenofovir (TFV) and TFV-diphosphate (TFV-DP) were measured in blood, secretions, and tissues over 72 h. The median area under the curve (AUC0-72h) values for TFV-DP in peripheral blood mononuclear cells were similar in DMPA-treated (6991 fmol*h/106 cells) and untreated controls (5256 fmol*h/106 cells) (P = 0.174). Rectal tissue TFV-DP concentrations from DMPA+ animals [median: 20.23 fmol/mg of tissue (range: 4.94-107.95)] were higher than the DMPA- group [median: below the limit of quantification (BLOQ-11.92)], (P = 0.019). TFV-DP was not detectable in vaginal tissue from either group. A high-dose DMPA treatment in macaques was associated with increased rectal TFV-DP levels, indicating a potential tissue-specific drug-drug interaction. The lack of detectable TFV-DP in the vaginal tissue warrants further investigation of PrEP efficacy with single-agent TAF products. DMPA did not affect systemic TAF metabolism, with similar PBMC TFV-DP in both groups, suggesting that DMPA use should not alter the antiviral activity of TAF.

Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality.

Recent evidence suggests that several cattle breeds may be more resistant to infection with the zoonotic pathogen Mycobacterium bovis. Our data presented here suggests that the response to mycobacterial antigens varies in macrophages generated from Brown Swiss (BS) and Holstein Friesian (HF) cattle, two breeds belonging to the Bos taurus family. Whole genome sequencing of the Brown Swiss genome identified several potential candidate genes, in particular Toll-like Receptor-2 (TLR2), a pattern recognition receptor (PRR) that has previously been described to be involved in mycobacterial recognition. Further investigation revealed single nucleotide polymorphisms (SNP) in TLR2 that were identified between DNA isolated from cells of BS and HF cows. Interestingly, one specific SNP, H326Q, showed a different genotype frequency in two cattle subspecies, Bos (B.) taurus and Bos indicus. Cloning of the TLR2 gene and subsequent gene-reporter and chemokine assays revealed that this SNP, present in BS and Bos indicus breeds, resulted in a significantly higher response to mycobacterial antigens as well as tri-acylated lipopeptide ligands in general. Comparing wild-type and H326Q containing TLR2 responses, wild-type bovine TLR2 response showed clear, diminished mycobacterial antigen responses compared to human TLR2, however bovine TLR2 responses containing H326Q were found to be partially recovered compared to human TLR2. The creation of human:bovine TLR2 chimeras increased the response to mycobacterial antigens compared to the full-length bovine TLR2, but significantly reduced the response compared to the full-length human TLR2. Thus, our data, not only present evidence that TLR2 is a major PRR in the mammalian species-specific response to mycobacterial antigens, but furthermore, that there are clear differences between the response seen in different cattle breeds, which may contribute to their enhanced or reduced susceptibility to mycobacterial infection.

Single oral dose for HIV pre or post-exposure prophylaxis: user desirability and biological efficacy in macaques.

BACKGROUND: Daily oral pre- or post-exposure prophylaxis (PrEP or PEP) is highly effective in preventing HIV infection. However, many people find it challenging to adhere to a daily oral regimen. Chemoprophylaxis with single oral doses of antiretroviral drugs taken before or after sex may better adapt to changing or unanticipated sexual practices and be a desirable alternative to daily PrEP or PEP. We investigated willingness to use a single oral pill before or after sex among men who have sex with men (MSM) and assessed the biological efficacy of a potent antiretroviral combination containing elvitegravir (EVG), emtricitabine (FTC), and tenofovir alafenamide (TAF). METHODS: Data on willingness to use single-dose PrEP or PEP were obtained from the 2017 cycle of the American Men's Internet Survey (AMIS), an annual online behavioral surveillance survey of MSM in the United States. Antiretroviral drug levels were measured in humans and macaques to define drug distribution in rectal tissue and identify clinically relevant doses for macaque modeling studies. The biological efficacy of a single dose of FTC/TAF/EVG as PrEP or PEP was investigated using a repeat-challenge macaque model of rectal HIV infection. FINDINGS: Through pharmacokinetic assessment in humans and macaques we found that EVG penetrates and concentrates in rectal tissues supporting its addition to FTC/TAF to boost and extend chemoprophylactic activity. Efficacy estimates for a single oral dose given to macaques 4h before or 2h after SHIV exposure was 91•7%[35•7%-98•9%] and 100%, respectively, compared to 80•1%[13•9%-95•4%] and 64•6%[-19•4%-89•5%] when single doses were given 6 and 24h post challenge, respectively. A two-dose regimen at 24h and 48h after exposure was also protective [77•1%[1•7%-94•7%]. INTERPRETATION: Informed by user willingness, human and macaque pharmacokinetic data, and preclinical efficacy we show that single-dose prophylaxis before or after sex is a promising HIV prevention strategy. Carefully designed clinical trials are needed to determine if any of these strategies will be effective in humans. FUNDING: Funded by CDC intramural funds, CDC contract HCVJCG2-2016-03948 (to CFK), and a grant from the MAC AIDS Fund and by the National Institutes of Health [P30AI050409] - the Emory Center for AIDS Research (to MZ and TS).

Analysis of Genetic Variation in the Bovine SLC11A1 Gene, Its Influence on the Expression of NRAMP1 and Potential Association With Resistance to Bovine Tuberculosis.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic zoonotic disease where host genetics is thought to contribute to susceptibility or resistance. One of the genes implicated is the SLC11A1 gene, that encodes for the natural resistance-associated macrophage protein 1 (NRAMP1). The aim of this study was to identify SLC11A1 polymorphisms and to investigate any resulting functional differences in NRAMP1 expression that might be correlated with resistance/susceptibility to M. bovis infection. Sequencing of the SLC11A1 gene in cDNA isolated from Brown Swiss, Holstein Friesian, and Sahiwal cattle identified five single nucleotide polymorphisms (SNPs) in the coding region, but only one of these (SNP4, c.1066C>G, rs109453173) was present in all three cattle breeds and therefore warranted further investigation. Additionally, variations of 10, 11, and 12 GT repeats were identified in a microsatellite (MS1) in the SLC11A1 3'UTR. Measurement of NRAMP1 expression in bovine macrophages by ELISA showed no differences between cells generated from the different breeds. Furthermore, variations in the length of the MS1 microsatellite did not impact on NRAMP1 protein expression as analyzed by luciferase reporter assay. However, further analysis of the ELISA data identified that the presence of the alternative G allele at SNP4 was associated with increased expression of NRAMP1 in bovine macrophages. Since NRAMP1 has been shown to influence the survival of intracellular pathogens such as M. bovis through the sequestering of iron, it is possible that cattle expressing the alternative G allele might have an increased resistance to bTB through increased NRAMP1 expression in their macrophages.

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.

Mammalian lectin arrays for screening host-microbe interactions.

Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen-receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.

Training rhesus macaques to take daily oral antiretroviral therapy for preclinical evaluation of HIV prevention and treatment strategies.

BACKGROUND: Macaque models of simian or simian/human immunodeficiency virus (SIV or SHIV) infection are critical for the evaluation of antiretroviral (ARV)-based HIV treatment and prevention strategies. However, modelling human oral ARV administration is logistically challenging and fraught by limited adherence. Here, we developed a protocol for administering daily oral doses of ARVs to macaques with a high rate of compliance. METHODS: Parameters of positive reinforcement training (PRT), behavioral responses and optimal drug delivery foods were defined in 7 male rhesus macaques (Macaca mulatta). Animals were trained to sit in a specified cage location prior to receiving ARVs, emtricitabine (FTC) and tenofovir alafenamide (TAF), in a blended food mixture, which was followed immediately with a juice chaser. Consistency of daily oral adherence was evaluated in 4 trained macaques receiving clinically equivalent doses of FTC and TAF (20 and 1.5 mg/kg, respectively) in a short-term (1 month) and an extended (6 month) trial. Adherence was monitored using medication diaries and by quantifying intracellular FTC-triphosphate (FTC-TP) and tenofovir-diphosphate (TFV-DP) concentrations in peripheral mononuclear blood cells (PBMCs). RESULTS: Trained macaques quickly and consistently took daily oral ARVs for 1 month with an average 99.8% observed adherence. Intracellular concentrations of TFV-DP (median = 845.8 fmol/million cells [range, 620.8-1031.3]) and FTC-TP (median = 367.0 fmol/million cells [range, 289.5-413.5) in PBMCs were consistent with high adherence. Extended treatment with select subjects yielded similar observations for three months (99.5% adherence, 352/356 complete doses taken), although a sudden drop in adherence was observed after splenic biopsy surgery. CONCLUSIONS: We demonstrate that trained macaques reliably adhere to a daily oral ARV regimen, although unexpected adherence issues are possible. Our approach, using clinical doses of oral FTC and TAF daily, further refines macaque models of HIV treatment and prevention by mimicking the human route and timing of ARV administration.