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.

Screening for Viral Nucleic Acids in the Cerebrospinal Fluid of Dogs With Central Nervous System Inflammation.

Central nervous system (CNS) inflammation is a common cause of neurological dysfunction in dogs. Most dogs with CNS inflammation are diagnosed with presumptive autoimmune disease. A smaller number are diagnosed with an infectious etiology. Additionally, at necropsy, a subset of dogs with CNS inflammation do not fit previously described patterns of autoimmune disease and an infectious cause is not readily identifiable. Because viral infection is a common cause of meningoencephalitis in people, we hypothesize that a subset of dogs presented with CNS inflammation have an occult viral infection either as a direct cause of CNS inflammation or a trigger for autoimmunity. The goal of this research was to screen cerebrospinal fluid from a large number dogs with CNS inflammation for occult viral infection. One hundred seventy-two dogs with neurological dysfunction and cerebrospinal fluid (CSF) pleocytosis were identified. Of these, 42 had meningoencephalitis of unknown origin, six had steroid-responsive meningitis-arteritis, one had eosinophilic meningoencephalitis, five had documented infection, 21 had and undetermined diagnosis, and 97 had a diagnosis not consistent with primary inflammatory disease of the CNS (e.g., neoplasia). CSF samples were subsequently screened with broadly reactive PCR for eight viral groups: adenovirus, bunyavirus, coronavirus, enterovirus, flavivirus, herpesvirus, paramyxovirus, and parechovirus. No viral nucleic acids were detected from 168 cases screened for eight viral groups, which does not support occult viral infection as a cause of CNS inflammation in dogs. La Crosse virus (LACV) nucleic acids were detected from four cases in Georgia. Subclinical infection was supported in two of these cases but LACV could not be ruled-out as a cause of infection in the other two cases, suggesting further research is warranted to determine if LACV is an occult cause of CNS inflammation in dogs.

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).

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.

HIV-1 genetic diversity to estimate time of infection and infer adherence to preexposure prophylaxis.

OBJECTIVE: To estimate time of HIV infection in participants from the Bangkok Tenofovir Study (BTS) with daily oral tenofovir disoproxil fumarate (TDF) for preexposure prophylaxis (PrEP) and relate infection with adherence patterns. DESIGN: We used the diversity structure of the virus population at the first HIV RNA-positive sample to estimate the date of infection, and mapped these estimates to medication diaries obtained under daily directly observed therapy (DOT). METHODS: HIV genetic diversity was investigated in all 17 PrEP breakthrough infections and in 16 placebo recipients. We generated 10-25 HIV env sequences from each participant by single genome amplification, and calculated time since infection (and 95% confidence interval) using Poisson models of early virus evolution. Study medication diaries obtained under daily DOT were then used to compute the number of missed TDF doses at the approximate date of infection. RESULTS: Fifteen of the 17 PrEP breakthrough infections were successfully amplified. Of these, 13 were initiated by a single genetic variant and generated reliable estimates of time since infection (median = 47 [IQR = 35] days). Eleven of these 13 were under daily DOT at the estimated time of infection. Analysis of medication diaries in these 11 participants showed 100% adherence in five, 90-95% adherence in two, 55% adherence in one, and nonadherence in three. CONCLUSION: We estimated time of infection in participants from BTS and found several infections when high levels of adherence to TDF were reported. Our results suggest that the biological efficacy of daily TDF against parenteral HIV exposure is not 100%.

Efficacy of Oral Tenofovir Alafenamide/Emtricitabine Combination or Single-Agent Tenofovir Alafenamide Against Vaginal Simian Human Immunodeficiency Virus Infection in Macaques.

BACKGROUND: Tenofovir alafenamide (TAF)-based regimens are being evaluated for pre-exposure prophylaxis (PrEP). We used a macaque model of repeated exposures to simian human immunodeficiency virus (SHIV) to investigate whether TAF alone or the combination of TAF and emtricitabine (FTC) can prevent vaginal infection. METHODS: Pigtail macaques were exposed vaginally to SHIV162p3 once a week for up to 15 weeks. Animals received clinical doses of FTC/TAF (n = 6) or TAF (n = 9) orally 24 hours before and 2 hours after each weekly virus exposure. Infection was compared with 21 untreated controls. RESULTS: Five of the 6 animals in the FTC/TAF and 4 of the 9 animals in the TAF alone group were protected against infection (P = .001 and P = .049, respectively). The calculated efficacy of FTC/TAF and TAF was 91% (95% confidence interval [CI], 34.9%-98.8%) and 57.8% (95% CI, -8.7% to 83.6%), respectively. Infection in FTC/TAF but not TAF-treated macaques was delayed relative to controls (P = .005 and P = .114). Median tenofovir diphosphate (TFV-DP) levels in peripheral blood mononuclear cells (PBMCs) were similar among infected and uninfected macaques receiving TAF PrEP (351 and 143 fmols/106 cells, respectively; P = .921). CONCLUSIONS: Emtricitabine/TAF provided a level of protection against vaginal challenge similar to FTC/TFV disoproxil fumarate combination in the macaque model. Our results support the clinical evaluation of FTC/TAF for PrEP in women.

Drug resistance emergence in macaques administered cabotegravir long-acting for pre-exposure prophylaxis during acute SHIV infection.

A long-acting injectable formulation of the HIV integrase inhibitor cabotegravir (CAB-LA) is currently in clinical development for PrEP. Although the long plasma half-life of CAB-LA is an important attribute for PrEP, it also raises concerns about drug resistance emergence if someone becomes infected with HIV, or if PrEP is initiated during undiagnosed acute infection. Here we use a macaque model of SHIV infection to model risks of drug resistance to CAB-LA PrEP. Six macaques infected with SHIV received CAB-LA before seroconversion. We show integrase mutations G118R, E92G/Q, or G140R in plasma from 3/6 macaques as early as day 57, and identify G118R and E92Q in viruses from vaginal and rectal fluids. G118R and G140R confer > 800-fold resistance to CAB and cross-resistance to all licensed integrase inhibitors. Our results emphasize the need for appropriate HIV testing strategies before and possibly shortly after initiating CAB LA PrEP to exclude acute infection.

Brief Report: HIV-1 Evolution in Breakthrough Infections in a Human Trial of Oral Pre-exposure Prophylaxis With Emtricitabine and Tenofovir Disoproxil Fumarate.

We describe HIV-1 evolutionary dynamics in the 4 participants from the TDF2-PrEP trial who became HIV-1 infected while prescribed emtricitabine and tenofovir disoproxil fumarate (FTC/TDF). At seroconversion, virus diversity in the 2 participants with detectable drug was only 0.05% (95% confidence intervals: 0.04 to 0.06) and 0.07% (0.06 to 0.08) compared with 2.25% (1.95 to 2.6) and 0.42% (0.36 to 0.49) in those with no detectable drug and 0.07%-0.69% in 5 placebo recipients (P > 0.5). At 10 months, diversity in adherent participants was only 0.37% (0.31 to 0.41) and 0.86% (0.82 to 0.90) compared with 0.5%-1.7% among participants who did not take FTC/TDF (P > 0.5). Although limited by the small number of infections that reduced the power to detect differences, we found that sequences from seroconverters with detectable drug were more homogeneous than those from placebo or nonadherent seroconverters.

Limited SHIV env diversification in macaques failing oral antiretroviral pre-exposure prophylaxis.

BACKGROUND: Pre-exposure prophylaxis (PrEP) with daily Truvada [a combination of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF)] is a novel HIV prevention strategy recently found to prevent HIV transmission among men who have sex with men and heterosexual couples. Acute infection in adherent persons who fail PrEP will inevitably occur under concurrent antiretroviral therapy, thus raising questions regarding the potential impact of PrEP on early viral dynamics. We investigated viral evolution dynamics in a macaque model of PrEP consisting of repeated rectal exposures to SHIV162P3 in the presence of PrEP. RESULTS: Four macaques were infected during daily or intermittent PrEP with FTC or FTC/TDF, and five were untreated controls. SHIV env sequence evolution was monitored by single genome amplification with phylogenetic and sequence analysis. Mean nucleotide divergence from transmitted founder viruses calculated 17 weeks (range = 12-20) post peak viremia was significantly lower in PrEP failures than in control animals (7.2 × 10-3 compared to 1.6 × 10-2 nucleotide substitutions per site per year, respectively, p < 0.0001). Mean virus diversity was also lower in PrEP failures after 17 weeks (0.13% vs. 0.53% in controls, p < 0.0001). CONCLUSIONS: Our results in a macaque model of acute HIV infection suggest that infection during PrEP limits early virus evolution likely because of a direct antiviral effect of PrEP and/or reduced target cell availability. Reduced virus diversification during early infection might enhance immune control by slowing the selection of escape mutants.

Identification of adenoviruses in fecal specimens from wild chimpanzees (Pan trogylodytes schweinfurthii) in western Tanzania.

DNA of two distinctive adenoviruses was detected in wild chimpanzees in western Tanzania that showed clinical signs of acute, upper respiratory disease, notably coughing. The amplified sequences from part of the capsid hexon gene suggests that one virus is a novel adenovirus serotype candidate and the other virus is a species C adenovirus most closely related to recent isolates from captive chimpanzees in the United States, Simian AdV 37 with 86% nucleic acid identity and Simian AdV 40 with 95% nucleic acid identity, respectively. The species C adenovirus sequences suggest possible recombination with a human adenovirus. The source of these viruses and disease association is not known.

Broadly reactive pan-paramyxovirus reverse transcription polymerase chain reaction and sequence analysis for the detection of Canine distemper virus in a case of canine meningoencephalitis of unknown etiology.

Despite the immunologic protection associated with routine vaccination protocols, Canine distemper virus (CDV) remains an important pathogen of dogs. Antemortem diagnosis of systemic CDV infection may be made by reverse transcription polymerase chain reaction (RT-PCR) and/or immunohistochemical testing for CDV antigen; central nervous system infection often requires postmortem confirmation via histopathology and immunohistochemistry. An 8-month-old intact male French Bulldog previously vaccinated for CDV presented with multifocal neurologic signs. Based on clinical and postmortem findings, the dog's disease was categorized as a meningoencephalitis of unknown etiology. Broadly reactive, pan-paramyxovirus RT-PCR using consensus-degenerate hybrid oligonucleotide primers, combined with sequence analysis, identified CDV amplicons in the dog's brain. Immunohistochemistry confirmed the presence of CDV antigens, and a specific CDV RT-PCR based on the phosphoprotein gene identified a wild-type versus vaccinal virus strain. This case illustrates the utility of broadly reactive PCR and sequence analysis for the identification of pathogens in diseases with unknown etiology.

Identification of a novel astrovirus (astrovirus VA1) associated with an outbreak of acute gastroenteritis.

The etiology of a large proportion of gastrointestinal illness is unknown. In this study, random Sanger sequencing and pyrosequencing approaches were used to analyze fecal specimens from a gastroenteritis outbreak of unknown etiology in a child care center. Multiple sequences with limited identity to known astroviruses were identified. Assembly of the sequences and subsequent reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends generated a complete genome of 6,586 nucleotides. Phylogenetic analysis demonstrated that this virus, named astrovirus VA1 (AstV-VA1), is highly divergent from all previously described astroviruses. Based on RT-PCR, specimens from multiple patients in this outbreak were unequivocally positive for Ast-VA1.

Detection of novel SARS-like and other coronaviruses in bats from Kenya.

Diverse coronaviruses have been identified in bats from several continents but not from Africa. We identified group 1 and 2 coronaviruses in bats in Kenya, including SARS-related coronaviruses. The sequence diversity suggests that bats are well-established reservoirs for and likely sources of coronaviruses for many species, including humans.

Severe postvaccinia encephalitis with acute disseminated encephalomyelitis: recovery with early intravenous immunoglobulin, high-dose steroids, and vaccinia immunoglobulin.

We report the second case of severe postvaccinial encephalitis with acute disseminated encephalomyelitis since smallpox vaccination was reintroduced in 2002. Both affected patients responded dramatically with early intervention of intravenous immunoglobulin. Our patient, who also received concurrent vaccinia immunoglobulin and corticosteroids, demonstrated full recovery.

Role of the human T-cell leukemia virus type 1 PTAP motif in Gag targeting and particle release.

Human T-cell leukemia virus type 1 (HTLV-1) Gag is targeted to the plasma membrane for particle assembly and release. How HTLV-1 Gag targeting occurs is not well understood. The PPPY and PTAP motifs were previously shown to be involved in HTLV-1 particle release with PTAP playing a more subtle role in virus budding. These L domains function through the interaction with host cellular proteins normally involved in multivesicular body (MVB) morphogenesis. The plasma membrane pathway rather than the MVB pathway was found to be the primary pathway for HTLV-1 particle release in HeLa cells. Intriguingly, disruption of the PTAP motif led to a defect in the targeting of Gag from the plasma membrane to CD63-positive MVBs. Particles or particle buds were observed to be associated with MVBs by electron microscopy, implying that Gag targeting to the MVB resulted in particle budding. Blocking clathrin-dependent endocytosis was found not to influence localization of the HTLV-1 Gag PTAP mutant, indicating that Gag did not reach the MVBs through clathrin-dependent endocytosis. Our observations imply that the interaction between Gag and TSG101 is not required for Gag targeting to the MVB. Overexpression of dynamitin p50 increased particle release, suggesting that there was an increase in the intracellular transport of MVBs to the cell periphery by the utilization of the dynein-dynactin motor complex. Intriguingly, virus particle release with this mutant was reduced by 20-fold compared to that of wild type in HeLa cells, which is in marked contrast to the less-than-twofold defect observed for particle production of the HTLV-1 Gag PTAP mutant from 293T cells. These results indicate that the role of the PTAP motif in L domain function is cell type dependent.

Structural features of nucleosomes reorganized by yeast FACT and its HMG box component, Nhp6.

The Saccharomyces cerevisiae Spt16/Cdc68, Pob3, and Nhp6 proteins (SPN or yFACT) bind to and alter nucleosomes in vitro, providing a potential explanation for their importance in both transcription and replication in vivo. We show that nucleosomes bound by either Nhp6 alone or the yFACT complex remain largely intact and immobile but are significantly reorganized, as indicated by changes in the pattern of sensitivity to DNase I and enhanced digestion by some restriction endonucleases. In contrast, yFACT enhanced access to exonuclease III only at very high levels of enzyme, suggesting that the DNA near the entry and exit sites of nucleosomes is largely unperturbed and that the position of the histone octamers relative to the DNA is not altered during reorganization. DNase I sensitivity was enhanced at sites clustered near the center of the nucleosomal DNA, away from the entry and exit points, and the pattern of nuclease sensitivity was only mildly affected by the configuration of linker extensions, further indicating that linkers play only a minor role in the reorganization of nucleosomes by yFACT. The DNA in contact with H2A-H2B dimers is therefore the region whose nuclease sensitivity was the least affected by yFACT reorganization. The most dramatic changes in nucleosome structure occurred when Spt16-Pob3 and the HMG box protein Nhp6 were both present, but Nhp6 alone altered DNase I sensitivity at some specific sites, supporting an independent role for this class of proteins in the general management of chromatin properties. yFACT activity does not require ATP hydrolysis and does not alter the position of nucleosomes, indicating that it acts through a mechanism distinct from chromatin remodeling. The results presented here suggest instead that yFACT promotes polymerase progression by reorganizing nucleosome cores into a less inhibitory conformation in which the properties of DNA sequences near the center of the nucleosomes are altered.

Multiple Nhp6 molecules are required to recruit Spt16-Pob3 to form yFACT complexes and to reorganize nucleosomes.

The Saccharomyces cerevisiae Nhp6 protein contains a DNA-binding motif that is similar to those found in the high mobility group B family of chromatin proteins. Nhp6 bound to nucleosomes and made at least two changes in them: the nucleosomal DNA became more sensitive to DNase I at specific sites, and the nucleosomes became competent to bind Spt16-Pob3 to form yFACT.nucleosome complexes. Both changes occurred at similar concentrations of Nhp6, suggesting that they reflect the same structural reorganization of the nucleosome. Nucleosomes have multiple binding sites for Nhp6, and structural reorganization was associated with a concentration of Nhp6 about 10-fold higher than that needed for simple binding. We propose that the coordinated action of multiple Nhp6 molecules is required to convert nucleosomes to an alternative form as the first step in a two-step reorganization of nucleosomes with the second step being dependent on Spt16-Pob3. The presence of linker DNA had only subtle effects on these processes, indicating that both Nhp6 and yFACT act on core nucleosome structure rather than on the interaction between nucleosomes and adjacent DNA. These results suggest that Nhp6 and the related high mobility group B proteins may have a general role in promoting rearrangements of chromatin by initiating the destabilization of core nucleosomal structure.

Defects in SPT16 or POB3 (yFACT) in Saccharomyces cerevisiae cause dependence on the Hir/Hpc pathway: polymerase passage may degrade chromatin structure.

Spt16/Cdc68, Pob3, and Nhp6 collaborate in vitro and in vivo as the yeast factor SPN, which is homologous to human FACT. SPN/FACT complexes mediate passage of polymerases through nucleosomes and are important for both transcription and replication. An spt16 mutation was found to be intolerable when combined with a mutation in any member of the set of functionally related genes HIR1, HIR2/SPT1, HIR3/HPC1, or HPC2. Mutations in POB3, but not in NHP6A/B, also display strong synthetic defects with hir/hpc mutations. A screen for other mutations that cause dependence on HIR/HPC genes revealed genes encoding members of the Paf1 complex, which also promotes transcriptional elongation. The Hir/Hpc proteins affect the expression of histone genes and also promote normal deposition of nucleosomes; either role could explain an interaction with elongation factors. We show that both spt16 and pob3 mutants respond to changes in histone gene numbers, but in opposite ways, suggesting that Spt16 and Pob3 each interact with histones but perhaps with different subsets of these proteins. Supporting this, spt16 and pob3 mutants also display different sensitivities to mutations in the N-terminal tails of histones H3 and H4 and to mutations in enzymes that modulate acetylation of these tails. Our results support a model in which SPN/FACT has two functions: it disrupts nucleosomes to allow polymerases to access DNA, and it reassembles the nucleosomes afterward. Mutations that impair the reassembly activity cause chromatin to accumulate in an abnormally disrupted state, imposing a requirement for a nucleosome reassembly function that we propose is provided by Hir/Hpc proteins.