Antibodies from Rabbits Immunized with HIV-1 Clade B SOSIP Trimers Can Neutralize Multiple Clade B Viruses by Destabilizing the Envelope Glycoprotein.

The high viral diversity of HIV-1 is a formidable hurdle for the development of an HIV-1 vaccine. Elicitation of broadly neutralizing antibodies (bNAbs) would offer a solution, but so far immunization strategies have failed to efficiently elicit bNAbs. To overcome these obstacles, it is important to understand the immune responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens. To gain more insight, we characterized monoclonal antibodies (MAbs) isolated from rabbits immunized with Env SOSIP trimers based on the clade B isolate AMC008. Four rabbits that were immunized three times with AMC008 trimer developed robust autologous and sporadic low-titer heterologous neutralizing responses. Seventeen AMC008 trimer-reactive MAbs were isolated using antigen-specific single B-cell sorting. Four of these MAbs neutralized the autologous AMC008 virus and several other clade B viruses. When visualized by electron microscopy, the complex of the neutralizing MAbs with the AMC008 trimer showed binding to the gp41 subunit with unusual approach angles, and we observed that their neutralization ability depended on their capacity to induce Env trimer dissociation. Thus, AMC008 SOSIP trimer immunization induced clade B-neutralizing MAbs with unusual approach angles with neutralizing effects that involve trimer destabilization. Optimizing these responses might provide an avenue to the induction of trimer-dissociating bNAbs. IMPORTANCE Roughly 32 million people have died as a consequence of HIV-1 infection since the start of the epidemic, and 1.7 million people still get infected with HIV-1 annually. Therefore, a vaccine to prevent HIV-1 infection is urgently needed. Current HIV-1 immunogens are not able to elicit the broad immune responses needed to provide protection against the large variation of HIV-1 strains circulating globally. A better understanding of the humoral immune responses elicited by immunization with state-of-the-art HIV-1 immunogens should facilitate the design of improved HIV-1 vaccine candidates. We identified antibodies with the ability to neutralize multiple HIV-1 viruses by destabilization of the envelope glycoprotein. Their weak but consistent cross-neutralization ability indicates the potential of this epitope to elicit broad responses. The trimer-destabilizing effect of the neutralizing MAbs, combined with detailed characterization of the neutralization epitope, can be used to shape the next generation of HIV-1 immunogens to elicit improved humoral responses after vaccination.

Neutralising antibodies in Spike mediated SARS-CoV-2 adaptation.

SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2, and amino acid variation in Spike is increasingly appreciated. Given both vaccines and therapeutics are designed around Wuhan-1 Spike, this raises the theoretical possibility of virus escape, particularly in immunocompromised individuals where prolonged viral replication occurs. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences by both short and long read technologies over 23 time points spanning 101 days. Although little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days, N501Y in Spike was transiently detected at day 55 and V157L in RdRp emerged. However, following convalescent plasma we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro, the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility, but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type and appeared to compensate for the reduced infectivity of D796H. Consistent with the observed mutations being outside the RBD, monoclonal antibodies targeting the RBD were not impacted by either or both mutations, but a non RBD binding monoclonal antibody was less potent against ΔH69/ΔV70 and the double mutant. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with reduced susceptibility to neutralising antibodies.

Differentiated nursing practice: assessing the state-of-the-science.

The authors present their findings following an exhaustive literature review of research on differentiated nursing practice (DNP) that used a number of tools to measure various aspects of DNP that are applicable across the health care delivery continuum. Issues related to DNP include: optimal nursing care, matching patient needs with nurse competencies, effective use of nursing resources, equitable compensation, career satisfaction, loyalty to employers, and enhanced prestige of the nursing profession. One 1992 Massachusetts study of a three-role oncology unit project (including patient care manager, clinical nurse, and patient care technician), showed positive change in five criteria including: standards of nursing care, actual care hours, average labor costs, job satisfaction and patient satisfaction. A 1990 Arizona study that included unit assistants concluded that DNP supported a decline in the use of supplemental staff and staff overtime which led to cost savings, and increases in the actual hours of care and nurse satisfaction.

Treatment of cerebral ischemia with Dextran-40 or Fluosol DA 20%.

A cat stroke model was used to evaluate the efficacy of Dextran-40 (DEX) or Fluosol-DA 20% (FDA) in the treatment of focal cerebral ischemia. The animals were assigned randomly to one of three treatment groups: control, isovolemic hemodilution with DEX or isovolemic hemodilution with FDA. The oxidation state of cytochrome aa3 was measured in-vivo using near infrared reflectance spectrophotometry. The cerebral edema was measured by magnetic resonance imaging (MRI). The MRI edema indices for the three groups revealed that the FDA group had less edema (p less than 0.05), approaching that of non-stroke controls. The relative oxidation state of aa3 for the DEX group declined both during and after hemodilution. At the ninth hour post stroke the FDA group was better (aa3 more oxidized. p less than 0.025). Changes in blood and plasma components were reflective of the extent of hemodilution. Whole blood viscosity analysis revealed a difference (p less than 0.05) at the lower shear rates comparing DEX to FDA with FDA being higher than DEX. Two animals in each of the groups were allowed to awaken at the end of the procedure for functional assessment. These observations suggest that hemodilution with FDA following stroke significantly reduces early post-ischemic cerebral edema, improves oxidation in the peri-infarct area and appears to minimize functional deficits.

Acute severe carbon monoxide exposure in the rat: effects of hyperglycemia and hypoglycemia on mortality, recovery, and neurologic deficit.

Human and animal studies suggest a poorer outcome in the presence of abnormal blood glucose concentration during cerebral hypoxia-ischemia. It is unknown whether this is also the case in acute severe carbon monoxide poisoning. Using Levine-prepared rats, three groups were established and exposed to CO to answer this question: (1) hyperglycemics resulting from the administration of a 50% glucose solution, (2) hypoglycemics resulting from the administration of normal saline, and (3) untreated controls. The rats inhaled 2400 ppm CO for 90 min in the absence of anesthesia. Blood glucose was raised to a mean value of 402 mg/dL just prior to CO exposure in group 1. This resulted in an increased mortality rate (i.e., 54%), and during 4 h of room air recovery an impaired ability to regain body temperature, an increased plasma lactate dehydrogenase activity, and an increased neurologic deficit as compared with group 3. Hypoglycemia, which developed during CO exposure in group 2 (mean minimum glucose after 90 min, 44 mg/dL), resulted in an increased mortality rate (i.e., 46%), and during 4 h of room air recovery an impaired ability to regain body temperature and an increased neurologic deficit as compared with group 3. Blood glucose concentration in the rats in groups 2 and 3 that died during or shortly after CO exposure was significantly depressed relative to the survivors of those groups. Plasma insulin activity was elevated during CO exposure in group 1 as compared with group 3, but fell during recovery; insulin remained low throughout CO exposure and recovery in group 2. The results demonstrate the deleterious effects of both a very high and a very low blood glucose concentration during acute CO exposure.

MRI quantitation of edema in focal cerebral ischemia in cats: correlation with cytochrome aa3 oxidation state.

1H MRI permits detection of edema in the brain. In a middle cerebral artery stroke model in the cat, we found a significant correlation between an edema index based on MRI and a sensitive metabolic index of ischemia, the in vivo oxidation status of mitochondrial cytochrome aa3 determined by near-infrared reflectance spectrophotometry (r = -0.70, alpha = 0.001). This result suggests that a simple, noninvasive study using MRI can provide an index of the extent of ischemic damage in an experimental acute stroke model.

Role of Fluosol-DA 20% in prehospital resuscitation.

Fluosol-DA 20% (FDA-20) resuscitation has been proposed for prehospital therapy of hemorrhagic shock (HS). Acute HS (mean arterial pressure 60 mm Hg X 90 min, then 40 mm Hg X 30 min) in 24 splenectomized dogs was treated with 50 ml/kg of lactated Ringer's solution (RL) plus a volume equal to 105% of shed blood volume of FDA-20 (group 1), FDA-20 carrier Annex solution (group 2), or RL (group 3). Cardiopulmonary, hemopoietic, hemodynamic, and organ function parameters were measured preshock, at the end of shock, and at 30 and 60 min, and 24 h after resuscitation. FDA-20 produced effective volume expansion, oxygen delivery, and oxygen consumption. The FDA-20 appeared to affect organ function and cells adversely as reflected by a fall in red cell mass and platelet levels and by a rise in liver enzymes, BUN, and serum creatinine. Both the FDA-20 and Annex solution led to a reduction in serum proteins including serum albumin, serum globulin, immunoglobulin-G, and fibrinogen. These reductions are probably due to an oncotically driven factor which appears to maintain an optimal lymph to plasma oncotic ratio. The hydroxyethyl starch in the FDA-20 and the Annex solution, thus, would drive the plasma proteins into the interstitial space. The prolonged prothrombin times and the activated partial thromboplastin times after FDA-20 may be due, in part, to a reduction in the coagulation proteins, although these were not measured. Pending further studies designed to assess the effects of FDA-20 on possible cellular and multiple organ toxicity, clinical studies are premature and unwarranted.

Regenerative responses to exchange perfusion.

Isovolemic exchange perfusion of conscious normal and splenectomized rats to a Hct of +/- 3% with the perfluorocarbon based oxygen transport fluid, Fluosol-DA 20%, is characterized by: 1) a greater than projected depression of fibrinogen and the plasma globulins and 2) a rapid regeneration of these and certain other plasma proteins. Similar responses were observed in a study of PFC resuscitation of hemorrhagic shock in splenectomized dogs in which there was a selective depression of the platelets, the plasma globulins, IgG and fibrinogen. In the rats, the red cells and platelets required 14-21 days to return to control levels while the leukocytes returned to normal in 1-2 days. The globulins and fibrinogen exhibited a transient rebound response at 3 and 12 hours post exchange respectively with total protein levels restored to control levels at 48 hours. In the shock study, the leukocytes, which remained at control levels throughout the shock period and for 1 hour post resuscitation were 2.5x control levels at 24 hours. The platelets which were depressed to 20% of control levels following resuscitation remained depressed through the 24 hour time course of the study. Implicit in these results is the possibility that; A) thrombosis and B) immunosuppression may be caused by some component(s) of the perfusion/resuscitation fluid.

Endothelial response to perfluorochemical perfusion.

The purpose of this study was to evaluate, in vivo, the effect of perfluorochemical (PFC) blood substitutes on arterial endothelium following total blood replacement. Conscious-female-Sprague-Dawley rats (150-200 gms) were isovolumically exchange perfused with 3 blood volumes of PFC emulsion in an oxygen chamber. The exchange was performed at 0.5 ml/min via an indwelling-intracardiac-double-lumen catheter. One hour after the exchange, the animal was sacrificed and the circulatory system flushed free of blood and PFC emulsion with Dulbecco's solution, followed by perfusion fixation with glutaraldehyde solution. The heart, lungs and thoracic aorta were excised. The pulmonary arterial endothelial cell response was determined by scanning electron microscopy (SEM) following acute exposure to PFC emulsions under conditions of varied pH, oncotic pressure and emulsion age. These endothelial surfaces exhibited increased microvilli, leukocyte adhesion, fibrin deposition and subendothelium exposure. The magnitude of these abnormal responses varied with the degree of alkalinity and reduced oncotic pressure of the perfusate.

A method for evaluation of blood substitutes in the conscious animal.

Physiological evaluation of new and potential blood replacement agents has not kept pace with the development of such agents. Current procedures involve partial or total blood replacement in the anesthetized animal. This introduces the variable of anesthesia and eliminates the ability to observe behavior changes during blood replacement. Clinically, many patients receive blood or will receive artificial agents while sedated or under anesthesia, whereas others will be conscious. It is essential that evaluative studies be performed on the awake animal using procedures that are nontraumatic and nonrestrictive. A technique for isovolumic exchange perfusion utilizing an indwelling, heparin-coated, double-lumen catheter in the right atrium of a conscious rat is described. This animal model system permits continuous pre- and postperfusion monitoring. Nearly total blood replacement with perfluorochemical blood substitutes causes no discerniable discomfort or adverse reactions in the animal. Such animals thrive and replace missing hematologic components in 1-3 wk. The technique described can, with minimal modification, be used for isovolumic exchange perfusion of larger animals.

Purification and characterization of human and bovine platelet factor 4.

Platelet antiheparin, platelet factor 4, was isolated from freeze-thaw lysates of fresh bovine and outdated human platelet concentrates by a single step affinity chromatographic procedure. The yields of PF4 were 93 microgram and 142 microgram/ml of human and bovine platelets respectively. Antiheparin activity of the products were 558 units/mg for the bovine isolate and 489 units/mg for the human material. The bovine product is a single chain polypeptide with an apparent molecular weight of 12,300. Amino acid composition indicates 107-109 residues compared to the smaller human product which has an apparent molecular weight of 8,000 for a 70 residue polypeptide. The intact polypeptide was resistant to enzymatic hydrolysis as opposed to the reduced-alkylated derivative which was susceptible to hydrolysis in the presence and absence of heparin.