Antibodies targeting the fusion peptide on the HIV envelope provide protection to rhesus macaques against mucosal SHIV challenge.

The fusion peptide (FP) on the HIV-1 envelope (Env) trimer can be targeted by broadly neutralizing antibodies (bNAbs). Here, we evaluated the ability of a human FP-directed bNAb, VRC34.01, along with two vaccine-elicited anti-FP rhesus macaque mAbs, DFPH-a.15 and DF1W-a.01, to protect against simian-HIV (SHIV)BG505 challenge. VRC34.01 neutralized SHIVBG505 with a 50% inhibitory concentration (IC50) of 0.58 µg/ml, whereas DF1W-a.01 and DFPH-a.15 were 4- or 30-fold less potent, respectively. VRC34.01 was infused into four rhesus macaques at a dose of 10 mg/kg and four rhesus macaques at a dose of 2.5 mg/kg. The animals were intrarectally challenged 5 days later with SHIVBG505. In comparison with all 12 control animals that became infected, all four animals infused with VRC34.01 (10 mg/kg) and three out of four animals infused with VRC34.01 (2.5 mg/kg) remained uninfected. Because of the lower potency of DF1W-a.01 and DFPH-a.15 against SHIVBG505, we infused both Abs at a higher dose of 100 mg/kg into four rhesus macaques each, followed by SHIVBG505 challenge 5 days later. Three of four animals that received DF1W-a.01 were protected against infection, whereas all animals that received DFPH-a.15 were protected. Overall, the protective serum neutralization titers observed in these animals were similar to what has been observed for other bNAbs in similar SHIV infection models and in human clinical trials. In conclusion, FP-directed mAbs can thus provide dose-dependent in vivo protection against mucosal SHIV challenges, supporting the development of prophylactic vaccines targeting the HIV-1 Env FP.

Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections.

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

Anti-viral efficacy of a next-generation CD4-binding site bNAb in SHIV-infected animals in the absence of anti-drug antibody responses.

Broadly neutralizing antibodies (bNAbs) against HIV-1 are promising immunotherapeutic agents for treatment of HIV-1 infection. bNAbs can be administered to SHIV-infected rhesus macaques to assess their anti-viral efficacy; however, their delivery into macaques often leads to rapid formation of anti-drug antibody (ADA) responses limiting such assessment. Here, we depleted B cells in five SHIV-infected rhesus macaques by pretreatment with a depleting anti-CD20 antibody prior to bNAb infusions to reduce ADA. Peripheral B cells were depleted following anti-CD20 infusions and remained depleted for at least 9 weeks after the 1st anti-CD20 infusion. Plasma viremia dropped by more than 100-fold in viremic animals after the initial bNAb treatment. No significant humoral ADA responses were detected for as long as B cells remained depleted. Our results indicate that transient B cell depletion successfully inhibited emergence of ADA and improved the assessment of anti-viral efficacy of a bNAb in a SHIV-infected rhesus macaque model.

Design and evaluation of a dynamic air cushion for pressure ulcers prevention.

Wheelchair users have a higher risk of developing pressure ulcers due to prolonged seated pressure. Pressure ulcers can be painful, may require surgical intervention, and even become life-threatening if infection occurs. To prevent pressure ulcers from forming the patient must either offload themselves or rely on a caregiver to move them allowing pressure redistribution over the seated area. In this work, we designed a dynamic air cushion to relieve pressure on loaded areas using sequences of inflation and deflation of the air cushion cells. The purpose of these sequences is to offload pressure from high-risk areas. To evaluate the effect of the alternating sequences on seated pressure and blood perfusion, we recorded interface pressure, skin blood flow, superficial tissue oxygen saturation, blood concentrations of oxygenated hemoglobin, and deoxygenated hemoglobin from twenty-one healthy volunteers who were asked to sit on the air cushion for static mode recording (3 min) and during the inflation/deflation sequences (up to 22 min). The alternating sequences consisted of ten combined inflation and deflation steps. Results showed that, after applying the alternating sequences, interface pressure reduced significantly (p=0.02) compared to the static mode. Moreover, the coefficient of variation of the seated pressure was higher (p<0.001) during the alternation sequence compared to the static mode. However, interface pressure under the right and left ischial tuberosities increased (p<0.001) during the alternation sequence compared to the static mode. In addition, during the alternating sequences, males had larger dispersion index values of both right and left ischial tuberosities pressure compared to females. Furthermore, the maximum value of oxygen saturation (p=0.04) and skin blood flow (p=0.001) increased during the pressure alternation sequences compared to the static mode. The study findings highlighted the positive effects of the designed dynamic air-cushion to relieve pressure on compressed areas and enhance blood perfusion similar to manual offloading approaches. The outcomes of this study are encouraging to evaluate the performance of the designed air cushion in studies involving wheelchair users.

Interactions of human mesenchymal stromal cells with peripheral blood mononuclear cells in a Mitogenic proliferation assay.

BACKGROUND: Immunomodulation by mesenchymal stromal cells (MSCs) is a potentially important therapeutic modality. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, suggesting a mechanism for suppressing inflammatory responses in vivo. This study details the interactions of PBMCs and MSCs. METHODS: Pooled human PBMCs and MSCs were co-cultured at different MSC:PBMC ratios and harvested from 0 to 120 h, with and without phytohaemagglutin A (PHA) stimulation. Proliferation of adherent MSCs and non-adherent PBMCs was assessed by quantitation of ATP levels. PBMC surface marker expression was analyzed by flow cytometry. Indoleamine 2,3-dioxygenase (IDO) activity was determined by kynurenine assay and IDO mRNA by RT-PCR. Cytokine release was measured by ELISA. Immunofluorescent microscopy detected MSC, PBMC, monocyte (CD14+) and apoptotic events. RESULTS: PBMC proliferation in response to PHA gave a robust ATP signal by 72 h, which was suppressed by co-culture with densely plated MSCs. Very low level MSC seeding densities relative to PBMC number reproducibly stimulated PBMC proliferation. The CD4+/CD3+ population significantly decreased over time while the CD8+/CD3+ population significantly increased. No change in CD4+/CD8+ ratio is seen with high density MSC co-culture; very low density MSCs augment the changes seen in PHA stimulated PBMCs alone. IDO activity in MSCs co-cultured with PBMCs correlated with PBMC suppression. MSCs increased the secretion of IL-10 and IL-6 from stimulated co-cultures and decreased TNF-α secretion. In stimulated co-culture, low density MSCs decreased in number; fluorescence immunomicroscopy detected association of PBMC with MSC and phosphatidyl serine externalization in both cell populations. CONCLUSIONS: A bidirectional interaction between MSCs and PBMCs occurs during co-culture. High numbers of MSCs inhibit PHA-stimulated PBMC proliferation and the PBMC response to stimulation; low numbers of MSCs augment these responses. Low density MSCs are susceptible to attrition, apparently by PBMC-induced apoptosis. These results may have direct application when considering therapeutic dosing of patients; low MSC doses may have unintended detrimental consequences.

Use of multiple potency assays to evaluate human mesenchymal stromal cells.

BACKGROUND: There is broad interest in the use of cell therapies and cell products for treatment of a variety of diseases and problems. Of interest to the military, cellular therapies have the potential to confer tremendous benefit for treatment of both acute and chronic injuries. Although many different cell therapy products are currently under investigation, mesenchymal stromal cells (MSCs) are good candidates, based on their ability to respond to inflammation, limit vascular permeability, and modulate immune responses to injury. Although a large number of clinical trials utilize MSCs or their products, there is no firm consensus defining the characteristics and activities of a good MSC product. Here, we test multiple human MSCs in several assays designed to test potency, to determine if functionally relevant differences between MSCs can be defined using in vitro assays, allowing identification of superior MSC products for preclinical or clinical testing. METHODS: Human MSCs derived from several tissue sources (adipose, bone marrow, umbilical cord) were evaluated for their ability to respond to inflammatory signaling by upregulating indoleamine-2,3-dioxygenase and TSG6, suppress lymphocyte proliferation, alter the polarization of macrophages, and affect tube formation by endothelial cells. RESULTS: All MSCs tested displayed activity in the functional assays utilized, but differences in potency were observed in each assay. CONCLUSION: The indoleamine-2,3-dioxygenase enzyme activity assay represents a simple way to screen multiple samples. The mixed lymphocyte reaction and monocyte assays used to test interactions between MSCs and immune cells are more involved but give direct information on immunomodulation potential. The endothelial cell tube formation assay is relatively simple to perform but a large number of images must be generated and analyzed. However, it tests a functional activity other than immunomodulation and, therefore, adds another facet to MSC evaluation.