A Novel Prime and Boost Regimen of HIV Virus-Like Particles with TLR4 Adjuvant MPLA Induces Th1 Oriented Immune Responses against HIV

HIV virus-like particles (VLPs) present the HIV envelope protein in its native conformation, providing an ideal vaccine antigen. To enhance the immunogenicity of the VLP vaccine, we sought to improve upon two components; the route of administration and the additional adjuvant. Using HIV VLPs, we evaluated sub-cheek as a novel route of vaccine administration when combined with other conventional routes of immunization. Of five combinations of distinct prime and boost sequences, which included sub-cheek, intranasal, and intradermal routes of administration, intranasal prime and sub-cheek boost (IN+SC) resulted in the highest HIV-specific IgG titers among the groups tested. Using the IN+SC regimen we tested the adjuvant VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) + monophosphoryl lipid A (MPLA) at MPLA concentrations of 0, 7.5, 12.5, and 25 μg/dose in combination with our VLPs. Mice that received 12.5 or 25 μg/dose MPLA had the highest concentrations of Env-specific IgG2c (20.7 and 18.4 μg/ml respectively), which represents a Th1 type of immune response in C57BL/6 mice. This was in sharp contrast to mice which received 0 or 7.5 μg MPLA adjuvant (6.05 and 5.68 μg/ml of IgG2c respectively). In contrast to IgG2c, MPLA had minor effects on Env-specific IgG1; therefore, 12.5 and 25 μg/dose of MPLA induced the optimal IgG1/IgG2c ratio of 1.3. Additionally, the percentage of germinal center B cells increased significantly from 15.4% in the control group to 31.9% in the CALV + 25 μg MPLA group. These mice also had significantly more IL-2 and less IL-4 Env-specific CD8⁺ T cells than controls, correlating with an increased percentage of Env-specific central memory CD4⁺ and CD8⁺ T cells. Our study shows the strong potential of IN+SC as an efficacious route of administration and the effectiveness of VLPs combined with MPLA adjuvant to induce Env specific Th1-oriented HIV-specific immune responses.     

PrEP uptake,persistence, adherence,and effect of retrospective drug level feedback on PrEP adherence among young women in southern Africa: Results from HPTN 082, a randomized controlled trial

BackgroundPre-exposure prophylaxis (PrEP) is highly effective and an important prevention tool for African adolescent girls and young women (AGYW), but adherence and persistence are challenging. PrEP adherence support strategies for African AGYW were studied in an implementation study.Methods and findingsHIV Prevention Trials Network (HPTN) 082 was conducted in Cape Town, Johannesburg (South Africa) and Harare (Zimbabwe) from October 2016 to October 2018 to evaluate PrEP uptake, persistence, and the effect of drug level feedback on adherence. Sexually active HIV–negative women ages 16–25 were offered PrEP and followed for 12 months; women who accepted PrEP were randomized to standard adherence support (counseling, 2-way SMS, and adherence clubs) or enhanced adherence support with adherence feedback from intracellular tenofovir-diphosphate (TFV-DP) levels in dried blood spots (DBS). PrEP uptake, persistence through 12 months (no PrEP hold or missed visits), and adherence were assessed. The primary outcome was high adherence (TFV-DP ≥700 fmol/punch) at 6 months, compared by study arm. Of 451 women enrolled, median age was 21 years, and 39% had curable sexually transmitted infections (STIs). Most (95%) started PrEP, of whom 55% had uninterrupted PrEP refills through 12 months. Of those with DBS, 84% had detectable TFV-DP levels at month 3, 57% at month 6, and 31% at month 12. At 6 months, 36/179 (21%) of AGYW in the enhanced arm had high adherence and 40/184 (22%) in the standard adherence support arm (adjusted odds ratio [OR] of 0.92; 95% confidence interval [CI] 0.55, 1.34; p = 0.76). Four women acquired HIV (incidence 1.0/100 person-years), with low or undetectable TFV-DP levels at or prior to seroconversion, and none of whom had tenofovir or emtricitabine resistance mutations. The study had limited power to detect a modest effect of drug level feedback on adherence, and there was limited awareness of PrEP at the time the study was conducted.ConclusionsIn this study, PrEP initiation was high, over half of study participants persisted with PrEP through month 12, and the majority of young African women had detectable TFV-DP levels through month 6 with one-fifth having high adherence. Drug level feedback in the first 3 months of PrEP use did not increase the proportion with high adherence at month 6. HIV incidence was 1% in this cohort with 39% prevalence of curable STIs and moderate PrEP adherence. Strategies to support PrEP use and less adherence-dependent formulations are needed for this population.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02732730","term_id":"NCT02732730"}}NCT02732730.

Connie Celum and co-workers report on use of pre-exposure prophylaxis by young women in Southern Africa.     

Tracking and forecasting ecosystem interactions in real time

Evidence shows that species interactions are not constant but change as the ecosystem shifts to new states. Although controlled experiments and model investigations demonstrate how nonlinear interactions can arise in principle, empirical tools to track and predict them in nature are lacking. Here we present a practical method, using available time-series data, to measure and forecast changing interactions in real systems, and identify the underlying mechanisms. The method is illustrated with model data from a marine mesocosm experiment and limnologic field data from Sparkling Lake, WI, USA. From simple to complex, these examples demonstrate the feasibility of quantifying, predicting and understanding state-dependent, nonlinear interactions as they occur in situ and in real time—a requirement for managing resources in a nonlinear, non-equilibrium world.     

Anti-CD45 Radioimmunotherapy with 90Y but Not 177Lu Is Effective Treatment in a Syngeneic Murine Leukemia Model

Radioimmunotherapy (RIT) for treatment of hematologic malignancies has primarily employed monoclonal antibodies (Ab) labeled with ¹³¹I or ⁹⁰Y which have limitations, and alternative radionuclides are needed to facilitate wider adoption of RIT. We therefore compared the relative therapeutic efficacy and toxicity of anti-CD45 RIT employing ⁹⁰Y and ¹⁷⁷Lu in a syngeneic, disseminated murine myeloid leukemia (B6SJLF1/J) model. Biodistribution studies showed that both ⁹⁰Y- and ¹⁷⁷Lu-anti-murine CD45 Ab conjugates (DOTA-30F11) targeted hematologic tissues, as at 24 hours 48.8±21.2 and 156±14.6% injected dose per gram of tissue (% ID/g) of ⁹⁰Y-DOTA-30F11 and 54.2±9.5 and 199±11.7% ID/g of ¹⁷⁷Lu-DOTA-30F11 accumulated in bone marrow (BM) and spleen, respectively. However, ⁹⁰Y-DOTA-30F11 RIT demonstrated a dose-dependent survival benefit: 60% of mice treated with 300 µCi ⁹⁰Y-DOTA-30F11 lived over 180 days after therapy, and mice treated with 100 µCi ⁹⁰Y-DOTA-30F11 had a median survival 66 days. ⁹⁰Y-anti-CD45 RIT was associated with transient, mild myelotoxicity without hepatic or renal toxicity. Conversely, ¹⁷⁷Lu- anti-CD45 RIT yielded no long-term survivors. Thus, ⁹⁰Y was more effective than ¹⁷⁷Lu for anti-CD45 RIT of AML in this murine leukemia model.     

Mycobacterium tuberculosis Hip1 Modulates Macrophage Responses through Proteolysis of GroEL2

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.     

Structure of herpes simplex virus glycoprotein D bound to the human receptor nectin-1

Binding of herpes simplex virus (HSV) glycoprotein D (gD) to a cell surface receptor is required to trigger membrane fusion during entry into host cells. Nectin-1 is a cell adhesion molecule and the main HSV receptor in neurons and epithelial cells. We report the structure of gD bound to nectin-1 determined by x-ray crystallography to 4.0 Å resolution. The structure reveals that the nectin-1 binding site on gD differs from the binding site of the HVEM receptor. A surface on the first Ig-domain of nectin-1, which mediates homophilic interactions of Ig-like cell adhesion molecules, buries an area composed by residues from both the gD N- and C-terminal extensions. Phenylalanine 129, at the tip of the loop connecting β-strands F and G of nectin-1, protrudes into a groove on gD, which is otherwise occupied by C-terminal residues in the unliganded gD and by N-terminal residues in the gD/HVEM complex. Notably, mutation of Phe129 to alanine prevents nectin-1 binding to gD and HSV entry. Together these data are consistent with previous studies showing that gD disrupts the normal nectin-1 homophilic interactions. Furthermore, the structure of the complex supports a model in which gD-receptor binding triggers HSV entry through receptor-mediated displacement of the gD C-terminal region.

Authors Summary

Herpes simplex virus (HSV) is a widespread human pathogen. Four viral glycoproteins (gD, gB, gH/gL) are required for HSV entry into host cells. gD binding to a cell surface receptor triggers conformational changes in the other viral glycoproteins leading to membrane fusion and viral entry. Two structurally unrelated cellular protein receptors, nectin-1 and HVEM, can mediate HSV entry upon binding to gD. The structure presented here reveals the molecular basis for the stable interaction between HSV-1 gD and the receptor nectin-1. Comparison with the previously determined structures of the gD/HVEM complex and unliganded gD shows that, despite the fact that the two receptors interact with different binding sites, they both cause a similar conformational change in gD. Therefore, our data point to a conserved mechanism for receptor mediated activation of the HSV entry process. In addition, the gD/Nectin-1 structure reveals that the gD-binding site overlaps with a surface involved in nectin-1 homo-dimerization. This observation explains how gD interferes with the cell adhesion function of nectin-1. Finally, the gD/Nectin-1 complex displays similarities with other viral ligands bound to immunoglobulin-like receptors suggesting a convergent mechanism for receptors selection and usage.     

Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo

The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.