Effective and targeted latency reversal in CD4+ T cells from individuals on long term combined antiretroviral therapy initiated during chronic HIV-1 infection.

To date, an affordable, effective treatment for an HIV-1 cure remains only a concept with most "latency reversal" agents (LRAs) lacking specificity for the latent HIV-1 reservoir and failing in early clinical trials. We assessed HIV-1 latency reversal using a multivalent HIV-1-derived virus-like particle (HLP) to treat samples from 32 people living with HIV-1 (PLWH) in Uganda, US and Canada who initiated combined antiretroviral therapy (cART) during chronic infection. Even after 5-20 years on stable cART, HLP could target CD4+ T cells harbouring latent HIV-1 reservoir resulting in 100-fold more HIV-1 release into culture supernatant than by common recall antigens, and 1000-fold more than by chemotherapeutic LRAs. HLP induced release of a divergent and replication-competent HIV-1 population from PLWH on cART. These findings suggest HLP provides a targeted approach to reactivate the majority of latent HIV-1 proviruses among individuals infected with HIV-1.

The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses.

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on α-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of α-dystroglycan (α-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of α-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant α-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant α-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.

Enhanced SARS-CoV-2 entry via UPR-dependent AMPK-related kinase NUAK2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remodels the endoplasmic reticulum (ER) to form replication organelles, leading to ER stress and unfolded protein response (UPR). However, the role of specific UPR pathways in infection remains unclear. Here, we found that SARS-CoV-2 infection causes marginal activation of signaling sensor IRE1α leading to its phosphorylation, clustering in the form of dense ER-membrane rearrangements with embedded membrane openings, and XBP1 splicing. By investigating the factors regulated by IRE1α-XBP1 during SARS-CoV-2 infection, we identified stress-activated kinase NUAK2 as a novel host-dependency factor for SARS-CoV-2, HCoV-229E, and MERS-CoV entry. Reducing NUAK2 abundance or kinase activity impaired SARS-CoV-2 particle binding and internalization by decreasing cell surface levels of viral receptors and viral trafficking likely by modulating the actin cytoskeleton. IRE1α-dependent NUAK2 levels were elevated in SARS-CoV-2-infected and bystander non-infected cells, promoting viral spread by maintaining ACE2 cell surface levels and facilitating virion binding to bystander cells.

Does colonization with MRSA, ESBL - producing Enterobacteriaceae, and/or Acinetobacter baumannii - increase the risk for postoperative surgical site infection?

OBJECTIVE: Evaluation of the role of indicator pathogens in equine surgical site infection (SSI) and other infection-promoting factors. STUDY DESIGN: Cross-sectional study. ANIMALS: Horses presenting with an open injury or surgical colic during 1.5 years. METHODS: A nasal swab and a faecal sample were collected from every patient upon admission. Furthermore, a wound swab was collected from wounds of injured horses. Details on the wounds and procedures were documented. Laparotomy incisions and injuries were monitored for signs suggesting infection. RESULTS: In total, 156 horses presented because of a surgical colic (n = 48) or open injuries (n = 108). Thirteen surgical colic patients and three injured horses did not survive beyond 24 h, and four injured horses were discharged from the clinic at the day of admission. SSIs occurred in 31 (30.7%) injured horses and 11 (31.4%) horses after laparotomy. Regarding injuries, general anaesthesia increased the risk of developing a WI compared to sedation. Indicator pathogens were cultured from 29/42 SSI. In total, 10/11 infected laparotomy incisions and 19/31 injuries with SSI tested positive for multidrug-resistant pathogens (MDRPs) . Indicator pathogens were not detected at admission in any of the horses that developed incisional SSIs after laparotomy but were detected in two of the injured horses that developed SSIs. CONCLUSION: MDRPs were identified in almost 70% of the SSI. Less than 5% of the affected animals were colonized with the same pathogen before admission, indicating that colonization with MDR pathogens is only one of the crucial factors for the development of SSI. CLINICAL SIGNIFICANCE: Colonization with MDRP seems not to predispose horses to MDR SSIs.

ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

The scientific potential and technological challenges of the High-Luminosity Large Hadron Collider program.

We present an overview of the High-Luminosity (HL-LHC) program at the Large Hadron Collider (LHC), its scientific potential and technological challenges for both the accelerator and detectors. The HL-LHC program is expected to start circa 2027 and aims to increase the integrated luminosity delivered by the LHC by an order of magnitude at the collision energy of 14 TeV. This requires upgrades to the injector system, accelerator complex and luminosity levelling. The two experiments, ATLAS and CMS, require substantial upgrades to most of their systems in order to cope with the increased interaction rate, and much higher radiation levels than at the current LHC. We present selected examples based on novel ideas and technologies for applications at a hadron collider. Both experiments will replace their tracking systems. We describe the ATLAS pixel detector upgrade featuring novel tilted modules, and the CMS Outer Tracker upgrade with a new module design enabling use of tracks in the level-1 trigger system. CMS will also install state-of-the-art highly segmented calorimeter endcaps. Finally, we describe new picosecond precision timing detectors of both experiments. In addition, we discuss how the upgrades will enhance the physics performance of the experiments, and solve the computing challenges posed by the expected large data sets. The physics program of the HL-LHC is focused on precision measurements probing the limits of the Standard Model (SM) of particle physics and discovering new physics. We present a selection of studies that have been carried out to motivate the HL-LHC program. A central topic of exploration will be the characterization of the Higgs boson. The large HL-LHC data samples will extend the sensitivity of searches for new particles or new interactions whose existence has been hypothesized in order to explain shortcomings of the SM. Finally, we comment on the nature of large scientific collaborations.

Blocking T-cell egress with FTY720 extends DNA vaccine expression but reduces immunogenicity.

Optimal immunogenicity from nucleic acid vaccines requires a balance of antigen expression that effectively engages the host immune system without generating a cellular response that rapidly destroys cells producing the antigen and thereby limiting vaccine antigen expression. We investigated the role of the cellular response on the expression and antigenicity of DNA vaccines using a plasmid DNA construct expressing luciferase. Repeated intramuscular administration led to diminished luciferase expression, suggesting a role for immune-mediated clearance of expression. To investigate the role of cell trafficking, we used the sphingosine 1-phosphate receptor (S1PR) modulator, FTY720 (Fingolimod), which traps lymphocytes within the lymphoid tissues. When lymphocyte trafficking was blocked with FTY720, DNA transgene expression was maintained at a constant level for a significantly extended time period. Both continuous and staggered administration of FTY720 prolonged transgene expression. However, blocking lymphocyte egress during primary transgene administration did not result in an increase of transgene expression during secondary administration. Interestingly, there was a disconnect between transgene expression and immunogenicity, as increasing expression by this approach did not enhance the overall immune response. Furthermore, when FTY720 was administered alongside a DNA vaccine expressing the HIV gp140 envelope antigen, there was a significant reduction in both antigen-specific antibody and T-cell responses. This indicates that the developing antigen-specific cellular response clears DNA vaccine expression but requires access to the site of expression in order to develop an effective immune response.

Deep Gene Sequence Cluster Analyses of Multi-Virus-Infected Mucosal Tissue Reveal Enhanced Transmission of Acute HIV-1.

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue, while Envacute viruses penetrated the human tissue and established infection of CD4+ T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection.IMPORTANCE During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described (K. Klein et al., PLoS Pathog 14:e1006754, https://doi.org/10.1371/journal.ppat.1006754), and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance for understanding dynamics of infections and for designing focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early infection, compared to that from chronic infection, can more efficiently traverse the mucosal epithelium and be transmitted to T cells, suggesting higher transmission fitness. This study focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap.

Neuropilin-1 is a host factor for SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.

Investigation of magnetically driven passage of magnetic nanoparticles through eye tissues for magnetic drug targeting.

This paper elucidates the feasibility of magnetic drug targeting to the eye by using magnetic nanoparticles (MNPs) to which pharmaceutical drugs can be linked. Numerical simulations revealed that a magnetic field gradient of 20 T m-1 seems to be promising for dragging magnetic multicore nanoparticles of about 50 nm into the eye. Thus, a targeting magnet system made of superconducting magnets with a magnetic field gradient at the eye of about 20 T m-1 was simulated. For the proof-of-concept tissue experiments presented here the required magnetic field gradient of 20 T m-1 was realized by a permanent magnet array. MNPs with an optimized multicore structure were selected for this application by evaluating their stability against agglomeration of MNPs with different coatings in water for injections, physiological sodium chloride solution and biological media such as artificial tear fluid. From these investigations, starch turned out to be the most promising coating material because of its stability in saline fluids due to its steric stabilization mechanism. To evaluate the passage of MNPs through the sclera and cornea of the eye tissues of domestic pigs (Sus scrofa domesticus), a three-dimensionally printed setup consisting of two chambers (reservoir and target chamber) separated by the eye tissue was developed. With the permanent magnet array emulating the magnetic field gradient of the superconducting setup, experiments on magnetically driven transport of the MNPs from the reservoir chamber into the target chamber via the tissue were performed. The resulting concentration of MNPs in the target chamber was determined by means of quantitative magnetic particle spectroscopy. It was found that none of the tested particles passed the cornea, but starch-coated particles could pass the sclera at a rate of about 5 ng mm-2 within 24 h. These results open the door for future magnetic drug targeting to the eye.

A targeted reactivation of latent HIV-1 using an activator vector in patient samples from acute infection.

BACKGROUND: During combined anti-retroviral treatment, a latent HIV reservoir persists within resting memory CD4 T cells that initiates viral recrudescence upon treatment interruption. Strategies for HIV-1 cure have largely focused on latency reversing agents (LRAs) capable of reactivating and eliminating this viral reservoir. Previously investigated LRAs have largely failed to achieve a robust latency reversal sufficient for reduction of latent HIV pool or the potential of virus-free remission in the absence of treatment. METHODS: We utilize a polyvalent virus-like particle (VLP) formulation called Activator Vector (ACT-VEC) to 'shock' provirus into transcriptional activity. Ex vivo co-culture experiments were used to evaluate the efficacy of ACT-VEC in relation to other LRAs in individuals diagnosed and treated during the acute stage of infection. IFN-γ ELISpot, qRT-PCR and Illumina MiSeq were used to evaluate antigenicity, latency reversal, and diversity of induced virus respectively. FINDINGS: Using samples from HIV+ patients diagnosed and treated at acute/early infection, we demonstrate that ACT-VEC can reverse latency in HIV infected CD4 T cells to a greater extent than other major recall antigens as stimuli or even mitogens such as PMA/Iono. Furthermore, ACT-VEC activates more latent HIV-1 than clinically tested HDAC inhibitors or protein kinase C agonists. INTERPRETATION: Taken together, these results show that ACT-VEC can induce HIV reactivation from latently infected CD4 T cells collected from participants on first line combined antiretroviral therapy for at least two years after being diagnosed and treated at acute/early stage of infection. These findings could provide guidance to possible targeted cure strategies and treatments. FUNDING: NIH and CIHR.

Molecular Characterization of Equine Staphylococcus aureus Isolates Exhibiting Reduced Oxacillin Susceptibility.

The detection of borderline oxacillin-resistant Staphylococcus aureus (BORSA) represents a challenge to both, veterinary and human laboratories. Between 2015 and 2017, 19 equine S. aureus with elevated minimal inhibitory concentrations for oxacillin were detected in routine diagnostics. The aim of this study was to characterize these isolates to identify factors possibly associated with the BORSA phenotype. All S. aureus were subjected to antimicrobial susceptibility testing and whole genome sequencing (WGS). A quantifiable ß-lactamase activity assay was performed for a representative subset of 13 isolates. The WGS data analysis of the 19 BORSA isolates identified two different genomic lineages, sequence type (ST) 1 and ST1660. The core genome multilocus sequence typing (cgMLST) revealed a close relatedness of all isolates belonging to either ST1 or ST1660. The WGS analysis identified the resistance genes aadD, dfrG, tet(L), and/or blaZ and aacA-aphD. Phenotypic resistance to penicillins, aminoglycosides, tetracyclines, fluoroquinolones and sulfamethoxazole/trimethoprim was observed in the respective isolates. For the penicillin-binding proteins 1-4, amino acid substitutions were predicted using WGS data. Since neither transglycosylase nor transpeptidase domains were affected, these alterations might not explain the BORSA phenotype. Moreover, ß-lactamase activity was found to be associated with an inducible blaZ gene. The lineage-specific differences regarding the expression profiles were noted.

Equine Methicillin-Resistant Sequence Type 398 Staphylococcus aureus (MRSA) Harbor Mobile Genetic Elements Promoting Host Adaptation.

Continuing introduction of multi-drug resistant, zoonotic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) in horse clinics challenges the biosafety of employees and animal patients. This study was aimed to determine the occurrence of mobile genetic elements facilitating survival in the early stages of invasive infection in different host species, including humans and horses, in MRSA carried by equine patients admitted to a large horse clinic. A total of 341 equine patients were investigated for carriage of MRSA by hygiene screening directly at hospital admission. MRSA were further investigated by antimicrobial susceptibility testing, whole-genome sequencing and genomic composition, including virulence factors involved in immune evasion and host adaption. From a total of 340 validated specimens from equine nostrils, 3.5% yielded positive results for MRSA. All MRSA were found to be closely related belonging to sequence type (ST) 398_t011 with up to four additional antimicrobial resistances. All MRSA harbored a specific Staphylococcal Pathogenicity Island (SaPIbov5) involved in facilitating survival in ruminant and equine plasma. Moreover, a ß-hemolysin (hlb) converting ΦSa3 phage encoding the human-specific Immune Evasion Cluster (IEC) was present in 72% of the isolates. An equid-specific leukotoxin encoded by a further temperate phage (Saeq1) was only rarely detected (22%). Despite the absence of ß-hemolysin production for all IEC-positive ST398, a prominent hemolysis zone was demonstrable on sheep blood agar. Thus, IEC might remain undetected among the ST398 lineage, since the presence of IEC is commonly associated with reduction of hemolysis in S. aureus belonging to other genetic backgrounds. Here we describe MRSA-ST398 harboring different mobile genetic elements encoding variants of immune evasion factors and toxins previously shown to contribute to S. aureus invasive diseases in specific host species or ecologic niches. We suggest these combinations contribute to the adaptation of MRSA belonging to ST398 with respect to epidemic spread across different habitats and hosts, and may therefore confer a host "generalist" phenotype.

Fast Access to Amphiphilic Multiblock Architectures by the Anionic Copolymerization of Aziridines and Ethylene Oxide.

An ideal system for stimuli-responsive and amphiphilic (block) polymers would be the copolymerization of aziridines with epoxides. However, to date, no copolymerization of these two highly strained three-membered heterocycles had been achieved. Herein, we report the combination of the living oxy- and azaanionic ring-opening polymerization of ethylene oxide (EO) and sulfonamide-activated aziridines. In a single step, well-defined amphiphilic block copolymers are obtained by a one-pot copolymerization. Real-time 1H NMR spectroscopy revealed the highest difference in reactivity ratios ever reported for an anionic copolymerization (with r1 = 265 and r2 = 0.004 for 2-methyl- N-tosylaziridine/EO and r1 = 151 and r2 = 0.013 for 2-methyl- N-mesylaziridine/EO), leading to the formation of block copolymers with monomodal and moderate molecular weight distributions ( Mw/ Mn mostly ≤1.3). The amphiphilic diblock copolymers were used to stabilize emulsions and to prepare polymeric nanoparticles by miniemulsion polymerization, representing a novel class of nonionic and responsive surfactants. In addition, this unique comonomer reactivity of activated-Az/EO allows fast access to multiblock copolymers, and we prepared the first amphiphilic penta- or tetrablock copolymers containing aziridines in only one or two steps, respectively. These examples render the combination of epoxide and aziridine copolymerizations via a powerful strategy for producing sophisticated macromolecular architectures and nanostructures.

Hydrophilicity Regulates the Stealth Properties of Polyphosphoester-Coated Nanocarriers.

Increasing the plasma half-life is an important goal in the development of drug carriers, and can be effectively achieved through the attachment of polymers, in particular poly(ethylene glycol) (PEG). While the increased plasma half-life has been suggested to be a result of decreased overall protein adsorption on the hydrophilic surface in combination with the adsorption of specific proteins, the molecular reasons for the success of PEG and other hydrophilic polymers are still widely unknown. We prepared polyphosphoester-coated nanocarriers with defined hydrophilicity to control the stealth properties of the polymer shell. We found that the log P value of the copolymer controls the composition of the protein corona and the cell interaction. Upon a significant change in hydrophilicity, the overall amount of blood proteins adsorbed on the nanocarrier remained unchanged, while the protein composition varied. This result underlines the importance of the protein type for the protein corona and cellular uptake.

A heterogeneous human immunodeficiency virus-like particle (VLP) formulation produced by a novel vector system.

First identified as the etiological agent behind Acquired Immunodeficiency Syndrome (AIDS) in the early 1980s, HIV-1 has continued to spread into a global pandemic and major public health concern. Despite the success of antiretroviral therapy at reducing HIV-1 viremia and preventing the dramatic CD4+ T-cell collapse, infected individuals remain HIV positive for life. Unfortunately, it is increasingly clear that natural immunity is not, and may never be, protective against this pathogen. Therefore, efficacious vaccine interventions, which can either prevent infection or eradicate the latent viral reservoir and effect cure, are a major medical priority. Here we describe the development of a safe vaccine platform, currently being utilized in on-going prophylactic and therapeutic preclinical studies and consisting of highly heterogeneous virus-like particle formulations that represent the virus diversity within infected individuals. These VLPs contain no 5'LTR, no functional integrase, and have a severely mutated stem loop 1-thereby preventing any potential reverse transcription, integration, and RNA packaging. Furthermore, we demonstrate that these VLPs are morphologically identical to wild-type virus with polyvalent Env in a functional form. Finally, we show that the VLPs are antigenic and capable of generating strong immune recall responses.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Acinetobacter baumannii among horses entering a veterinary teaching hospital: The contemporary "Trojan Horse".

Pathogens frequently associated with multi-drug resistant (MDR) phenotypes, including extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) and Acinetobacter baumannii isolated from horses admitted to horse clinics, pose a risk for animal patients and personnel in horse clinics. To estimate current rates of colonization, a total of 341 equine patients were screened for carriage of zoonotic indicator pathogens at hospital admission. Horses showing clinical signs associated with colic (n = 233) or open wounds (n = 108) were selected for microbiological examination of nostril swabs, faecal samples and wound swabs taken from the open wound group. The results showed alarming carriage rates of Gram-negative MDR pathogens in equine patients: 10.7% (34 of 318) of validated faecal specimens were positive for ESBL-E (94%: ESBL-producing Escherichia coli), with recorded rates of 10.5% for the colic and 11% for the open wound group. 92.7% of the ESBL-producing E. coli were phenotypically resistant to three or more classes of antimicrobials. A. baumannii was rarely detected (0.9%), and all faecal samples investigated were negative for Salmonella, both directly and after two enrichment steps. Screening results for the equine nostril swabs showed detection rates for ESBL-E of 3.4% among colic patients and 0.9% in the open wound group, with an average rate of 2.6% (9/340) for both indications. For all 41 ESBL-producing E. coli isolated, a broad heterogeneity was revealed using pulsed-field gel electrophoresis (PFGE) patterns and whole genome sequencing (WGS) -analysis. However, a predominance of sequence type complex (STC)10 and STC1250 was observed, including several novel STs. The most common genes associated with ESBL-production were identified as blaCTX-M-1 (31/41; 75.6%) and blaSHV-12 (24.4%). The results of this study reveal a disturbingly large fraction of multi-drug resistant and ESBL-producing E. coli among equine patients, posing a clear threat to established hygiene management systems and work-place safety of veterinary staff in horse clinics.

Higher sequence diversity in the vaginal tract than in blood at early HIV-1 infection.

In the majority of cases, human immunodeficiency virus type 1 (HIV-1) infection is transmitted through sexual intercourse. A single founder virus in the blood of the newly infected donor emerges from a genetic bottleneck, while in rarer instances multiple viruses are responsible for systemic infection. We sought to characterize the sequence diversity at early infection, between two distinct anatomical sites; the female reproductive tract vs. systemic compartment. We recruited 72 women from Uganda and Zimbabwe within seven months of HIV-1 infection. Using next generation deep sequencing, we analyzed the total genetic diversity within the C2-V3-C3 envelope region of HIV-1 isolated from the female genital tract at early infection and compared this to the diversity of HIV-1 in plasma. We then compared intra-patient viral diversity in matched cervical and blood samples with three or seven months post infection. Genetic analysis of the C2-V3-C3 region of HIV-1 env revealed that early HIV-1 isolates within blood displayed a more homogeneous genotype (mean 1.67 clones, range 1-5 clones) than clones in the female genital tract (mean 5.7 clones, range 3-10 clones) (p<0.0001). The higher env diversity observed within the genital tract compared to plasma was independent of HIV-1 subtype (A, C and D). Our analysis of early mucosal infections in women revealed high HIV-1 diversity in the vaginal tract but few transmitted clones in the blood. These novel in vivo finding suggest a possible mucosal sieve effect, leading to the establishment of a homogenous systemic infection.

Eradication of HIV-1 latent reservoirs through therapeutic vaccination.

Despite the significant success of combination anti-retroviral therapy to reduce HIV viremia and save lives, HIV-1 infection remains a lifelong infection that must be appropriately managed. Advances in the understanding of the HIV infection process and insights from vaccine development in other biomedical fields such as cancer, imaging, and genetic engineering have fueled rapid advancements in HIV cure research. In the last few years, several studies have focused on the development of "Kick and Kill" therapies to reverse HIV latency and kick start viral translational activity. This has been done with the aim that concomitant anti-retroviral treatment and the elicited immune responses will prevent de novo infections while eradicating productively infected cells. In this review, we describe our perspective on HIV cure and the new approaches we are undertaking to eradicate the established pro-viral reservoir.

An in vitro Model to Mimic Selection of Replication-Competent HIV-1 Intersubtype Recombination in Dual or Superinfected Patients.

The low frequency of HIV-1 recombinants within entire viral populations in both individual patients and culture-based infection models impedes investigation of the underlying factors contributing to either the occurrence of recombinants or the survival of recombinants once they are formed. So far, most of the related studies have no consideration of recombinants' functionality. Here, we established a functional recombinant production (FRP) system to produce pure and functional HIV-1 intersubtype Env recombinants and utilized 454 pyrosequencing to investigate the distribution of over 4000 functional and non-functional recombination breakpoints from either the FRP system or dual infection cultures. The results revealed that most of the breakpoints converged in gp41 (62%) and C1 (25.3%) domains of gp120, which has strong correlation with the similarity between the two recombining sequences. Yet, the breakpoints also appeared in C2 (5.2%) and C5 (4.6%) domains not correlated with the recombining sequence similarity. Interestingly, none of the intersubtype gp120 recombinants recombined between C1 and gp41 regions either from the FRP system or from the dual infection culture, and very few from the HIV epidemic were functional. The present study suggests that the selection of functional Env recombinants is one of the reasons for the predominance of C1 and gp41 Env recombinants in the HIV epidemic, and it provides an in vitro model to mimic the selection of replication-competent HIV-1 intersubtype recombination in dual or superinfected patients.