Adeno-Associated Virus Serotype 2-hCHM Subretinal Delivery to the Macula in Choroideremia: Two-Year Interim Results of an Ongoing Phase I/II Gene Therapy Trial.

PURPOSE: To assess the safety of the subretinal delivery of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human choroideremia (CHM)-encoding cDNA in CHM. DESIGN: Prospective, open-label, nonrandomized, dose-escalation, phase I/II clinical trial. PARTICIPANTS: Fifteen CHM patients (ages 20-57 years at dosing). METHODS: Patients received uniocular subfoveal injections of low-dose (up to 5 × 1010 vector genome [vg] per eye, n = 5) or high-dose (up to 1 × 1011 vg per eye, n = 10) of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human CHM-encoding cDNA (AAV2-hCHM). Patients were evaluated preoperatively and postoperatively for 2 years with ophthalmic examinations, multimodal retinal imaging, and psychophysical testing. MAIN OUTCOME MEASURES: Visual acuity, perimetry (10-2 protocol), spectral-domain OCT (SD-OCT), and short-wavelength fundus autofluorescence (SW-FAF). RESULTS: We detected no vector-related or systemic toxicities. Visual acuity returned to within 15 letters of baseline in all but 2 patients (1 developed acute foveal thinning, and 1 developed a macular hole); the rest showed no gross changes in foveal structure at 2 years. There were no significant differences between intervention and control eyes in mean light-adapted sensitivity by perimetry or in the lateral extent of retinal pigment epithelium relative preservation by SD-OCT and SW-FAF. Microperimetry showed nonsignificant (< 3 standard deviations of the intervisit variability) gains in sensitivity in some locations and participants in the intervention eye. There were no obvious dose-dependent relationships. CONCLUSIONS: Visual acuity was within 15 letters of baseline after the subfoveal AAV2-hCHM injections in 13 of 15 patients. Acute foveal thinning with unchanged perifoveal function in 1 patient and macular hole in 1 patient suggest foveal vulnerability to the subretinal injections. Longer observation intervals will help establish the significance of the minor differences in sensitivities and rate of disease progression observed between intervention and control eyes.

T cell epitope engineering: an avian H7N9 influenza vaccine strategy for pandemic preparedness and response.

The delayed availability of vaccine during the 2009 H1N1 influenza pandemic created a sense of urgency to better prepare for the next influenza pandemic. Advancements in manufacturing technology, speed and capacity have been achieved but vaccine effectiveness remains a significant challenge. Here, we describe a novel vaccine design strategy called immune engineering in the context of H7N9 influenza vaccine development. The approach combines immunoinformatic and structure modeling methods to promote protective antibody responses against H7N9 hemagglutinin (HA) by engineering whole antigens to carry seasonal influenza HA memory CD4+ T cell epitopes - without perturbing native antigen structure - by galvanizing HA-specific memory helper T cells that support sustained antibody development against the native target HA. The premise for this vaccine concept rests on (i) the significance of CD4+ T cell memory to influenza immunity, (ii) the essential role CD4+ T cells play in development of neutralizing antibodies, (iii) linked specificity of HA-derived CD4+ T cell epitopes to antibody responses, (iv) the structural plasticity of HA and (v) an illustration of improved antibody response to a prototype engineered recombinant H7-HA vaccine. Immune engineering can be applied to development of vaccines against pandemic concerns, including avian influenza, as well as other difficult targets.

PAD4 Deficiency Leads to Decreased Organ Dysfunction and Improved Survival in a Dual Insult Model of Hemorrhagic Shock and Sepsis.

Indirect acute respiratory distress syndrome (iARDS) is caused by a nonpulmonary inflammatory process resulting from insults such as nonpulmonary sepsis. Neutrophils are thought to play a significant role in mediating ARDS, with the development of iARDS being characterized by dysregulation and recruitment of activated neutrophils into the lung. Recently, a novel mechanism of microbial killing by neutrophils was identified through the formation of neutrophil extracellular traps (NETs). NETs are composed of large webs of decondensed chromatin released from activated neutrophils into the extracellular space; they are regulated by the enzyme peptidylarginine deiminase 4 (PAD4) through mediation of chromatin decondensation via citrullination of target histones. Components of NETs have been implicated in ARDS. However, it is unknown whether there is any pathological significance of NET formation in ARDS caused indirectly by nonpulmonary insult. We subjected PAD4-/- mice and wild-type mice to a "two-hit" model of hypovolemic shock (fixed-pressure hemorrhage [Hem]) followed by septic cecal ligation and puncture (CLP) insult (Hem/CLP). Mice were hemorrhaged and resuscitated; 24 h after Hem, mice were then subjected to CLP. Overall, PAD4 deletion led to an improved survival as compared with wild-type mice. PAD4-/- mice displayed a marked decrease in neutrophil influx into the lung, as well decreased presence of proinflammatory mediators. PAD4-/- mice were also able to maintain baseline kidney function after Hem/CLP. These data taken together suggest PAD4-mediated NET formation contributes to the mortality associated with shock/sepsis and may play a role in the pathobiology of end organ injury in response to combined hemorrhage plus sepsis.

Cl-Amidine Prevents Histone 3 Citrullination and Neutrophil Extracellular Trap Formation, and Improves Survival in a Murine Sepsis Model.

Sepsis refers to the presence of a serious infection that correlates with systemic and uncontrolled immune activation. Posttranslational histone modification plays an important role in chromatin decondensation, which is regulated by citrullination. Citrullinated histone H3 (H3cit) has been identified as a component of neutrophil extracellular traps (NETs), which are released into the extracellular space as part of the neutrophil response to infection. The conversion of arginine to citrulline residues on histones is catalyzed by peptidylarginine deiminase 4 (PAD4). This study's goals were to characterize the presence of PAD4-catalyzed H3cit and NET formation during the onset of sepsis and elucidate the effects on the immune response when this mechanism of action is blocked. Adult C57BL/6 male mice were treated with Cl-amidine, an inhibitor of PAD4, 1 h prior to sepsis induced by cecal ligation and puncture (CLP). Twenty-four hours after CLP, cytokine levels, H3cit protein expression, neutrophil counts, and NET production were evaluated in the peritoneal cavity. Survival studies were also performed. Here we demonstrate that Cl-amidine treatment prior to CLP improves overall survival in sepsis and the abrogation of PAD4 has minimal effects on the proinflammatory immune response to sepsis, while it has no effect on overall neutrophil migration to the peritoneum.

Consequences of extracellular trap formation in sepsis.

PURPOSE OF REVIEW: This review will focus on in-vivo findings derived from animal models of sepsis regarding the trapping role of neutrophil extracellular traps (NETs) which is difficult to assess ex vivo. The NETotic response of neutrophils at sites of sterile injury or autoimmune disease is destructive as no antimicrobial advantage to the host is realized and dampening NETosis is largely beneficial. In early stages of local infection or in sepsis, the trapping function of NETs may help abscess formation and limit microbial dissemination. RECENT FINDINGS: The trapping function of NETs limits bacterial dissemination keeping an abscess from becoming bacteremic or confining tissue infection to local sites. Once containment is lost and disease has progressed, the best therapeutic approach suggested by animal studies to date is to inhibit protein arginine deiminase 4 and prevent NETosis rather than attempting to neutralize caustic NET components. Prognostic value may best be realized by taking cell free DNA, citrulllinated histones, neutrophil function and counts of immature granulocytes into consideration rather than rely on any one measure alone. SUMMARY: The trapping function of NETs may supercede the value of antimicrobial function in the early phases of sepsis such that degradation of the DNA backbone is contraindicated.

Enhanced Innate Inflammation Induced by Anti-BTLA Antibody in Dual Insult Model of Hemorrhagic Shock/Sepsis.

Sepsis following hemorrhagic shock is a common clinical condition, in which innate immune system suffers from severe suppression. B and T lymphocyte attenuator (BTLA) is an immune-regulatory coinhibitory receptor expressed not only on adaptive, but also on innate immune cells. Our previous data showed that BTLA gene deficient mice were protected from septic mortality when compared with wild-type control C57BL/6 mice. Here, we extended our study by treating C57BL/6 mice with an anti-BTLA monoclonal antibody (clone 6A6; reported to have the ability to neutralize or agonize/potentiate BTLA signaling) in a mouse model of hemorrhagic shock (Hem) followed by sepsis induced by cecal ligation and puncture (CLP); positing initially that if BTLA engagement was neutralized, like gene deficiency, an anti-BTLA mAb would have the similar effects on the inflammatory response/morbidity in these mice after such insults. Here, we report that BTLA expression is elevated on innate immune cells after Hem/CLP. However, anti-BTLA antibody treatment increased cytokine (TNF-α, IL-12, IL-10)/chemokine (KC, MIP-2, MCP-1) levels and inflammatory cells (neutrophils, macrophages, dendritic cells) recruitment in the peritoneal cavity, which in turn aggravated organ injury and elevated these animals' mortality in Hem/CLP. When compared with the protective effects of our previous study using BTLA gene deficient mice in a model of lethal septic challenge, we further confirmed BTLA's contribution to enhanced innate cell recruitment, elevated IL-10 levels, and reduced survival, and that engagement of antibody with BTLA potentiates/exacerbates the pathophysiology in Hem/sepsis.

Biomarkers for Sepsis: What Is and What Might Be?

Every year numerous individuals develop the morbid condition of sepsis. Therefore, novel biomarkers that might better inform clinicians treating such patients are sorely needed. Difficulty in identifying such markers is in part due to the complex heterogeneity of sepsis, resulting from the broad and vague definition of this state/condition based on numerous possible clinical signs and symptoms as well as an incomplete understanding of the underlying pathobiology of this complex condition. This review considers some of the attempts that have been made so far, looking at both the pro- and anti-inflammatory response to sepsis, as well as genomic analysis, as sources of potential biomarkers. Irrespective, for functional biomarker(s) of sepsis to successfully translate from the laboratory to a clinical setting, the biomarker must be target specific and sensitive as well as easy to implement/interpret, and be cost effective, such that they can be utilized routinely in patient diagnosis and treatment.

Efficacy of ETI-204 monoclonal antibody as an adjunct therapy in a New Zealand white rabbit partial survival model for inhalational anthrax.

Inhalational anthrax is characterized by extensive bacteremia and toxemia as well as nonspecific to mild flu-like symptoms, until the onset of hypotension, shock, and mortality. Without treatment, the mortality rate approaches 100%. Antibiotic treatment is not always effective, and alternative treatments are needed, such as monotherapy for antibiotic-resistant inhalational anthrax or as an adjunct therapy in combination with antibiotics. The Bacillus anthracis antitoxin monoclonal antibody (MAb) ETI-204 is a high-affinity chimeric deimmunized antibody which targets the anthrax toxin protective antigen (PA). In this study, a partial protection New Zealand White (NZW) rabbit model was used to evaluate the protective efficacy of the adjunct therapy with the MAb. Following detection of PA in the blood, NZW rabbits were administered either an antibiotic (doxycycline) alone or the antibiotic in conjunction with ETI-204. Survival was evaluated to compare the efficacy of the combination adjunct therapy with that of an antibiotic alone in treating inhalational anthrax. Overall, the results from this study indicate that a subtherapeutic regimen consisting of an antibiotic in combination with an anti-PA MAb results in increased survival compared to the antibiotic alone and would provide an effective therapeutic strategy against symptomatic anthrax in nonvaccinated individuals.