Fine-mapping the immunodominant antibody epitopes on consensus sequence-based HIV-1 envelope trimer vaccine candidates.

The HIV-1 envelope glycoprotein (Env) trimer is the key target for vaccines aimed at inducing neutralizing antibodies (NAbs) against HIV-1. The clinical candidate immunogen ConM SOSIP.v7 is a stabilized native-like HIV-1 Env trimer based on an artificial consensus sequence of all HIV-1 isolates in group M. In preclinical studies ConM SOSIP.v7 trimers induced strong autologous NAb responses in non-human primates (NHPs). To fine-map these responses, we isolated monoclonal antibodies (mAbs) from six cynomolgus macaques that were immunized three times with ConM SOSIP.v7 protein and boosted twice with the closely related ConSOSL.UFO.664 immunogen. A total of 40 ConM and/or ConS-specific mAbs were isolated, of which 18 were retrieved after the three ConM SOSIP.v7 immunizations and 22 after the two immunizations with ConSOSL.UFO.664. 22 mAbs (55%) neutralized the ConM and/or ConS virus. Cross-neutralization of ConS virus by approximately one-third of the mAbs was seen prior to ConSOSL.UFO.664 immunization, albeit with modest potency. Neutralizing antibodies predominantly targeted the V1 and V2 regions of the immunogens, with an apparent extension towards the V3 region. Thus, the V1V2V3 region is immunodominant in the potent NAb response elicited by two consensus sequence native-like HIV-1 Env immunogens. Immunization with these soluble consensus Env proteins also elicited non-neutralizing mAbs targeting the trimer base. These results inform the use and improvement of consensus-based trimer immunogens in combinatorial vaccine strategies.

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.

COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models.

One year into the Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), effective treatments are still needed 1-3 . Monoclonal antibodies, given alone or as part of a therapeutic cocktail, have shown promising results in patients, raising the hope that they could play an important role in preventing clinical deterioration in severely ill or in exposed, high risk individuals 4-6 . Here, we evaluated the prophylactic and therapeutic effect of COVA1-18 in vivo , a neutralizing antibody isolated from a convalescent patient 7 and highly potent against the B.1.1.7. isolate 8,9 . In both prophylactic and therapeutic settings, SARS-CoV-2 remained undetectable in the lungs of COVA1-18 treated hACE2 mice. Therapeutic treatment also caused a dramatic reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg - 1 one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 had a very strong antiviral activity in the upper respiratory compartments with an estimated reduction in viral infectivity of more than 95%, and prevented lymphopenia and extensive lung lesions. Modelling and experimental findings demonstrate that COVA1-18 has a strong antiviral activity in three different preclinical models and could be a valuable candidate for further clinical evaluation.

Oceanic protists with different forms of acquired phototrophy display contrasting biogeographies and abundance.

This first comprehensive analysis of the global biogeography of marine protistan plankton with acquired phototrophy shows these mixotrophic organisms to be ubiquitous and abundant; however, their biogeography differs markedly between different functional groups. These mixotrophs, lacking a constitutive capacity for photosynthesis (i.e. non-constitutive mixotrophs, NCMs), acquire their phototrophic potential through either integration of prey-plastids or through endosymbiotic associations with photosynthetic microbes. Analysis of field data reveals that 40-60% of plankton traditionally labelled as (non-phototrophic) microzooplankton are actually NCMs, employing acquired phototrophy in addition to phagotrophy. Specialist NCMs acquire chloroplasts or endosymbionts from specific prey, while generalist NCMs obtain chloroplasts from a variety of prey. These contrasting functional types of NCMs exhibit distinct seasonal and spatial global distribution patterns. Mixotrophs reliant on 'stolen' chloroplasts, controlled by prey diversity and abundance, dominate in high-biomass areas. Mixotrophs harbouring intact symbionts are present in all waters and dominate particularly in oligotrophic open ocean systems. The contrasting temporal and spatial patterns of distribution of different mixotroph functional types across the oceanic provinces, as revealed in this study, challenges traditional interpretations of marine food web structures. Mixotrophs with acquired phototrophy (NCMs) warrant greater recognition in marine research.

[Calcaneus fractures]. / Kalkaneusfrakturen.

Fractures of the calcaneus generally occur in the setting of high-energy trauma, resulting in complex, three-dimensionally oriented fracture patterns. Surgical treatment is typically indicated for displaced intra-articular fractures, permitting restoration of calcaneal height, width and overall morphology, in addition to the posterior facet articular surface where possible, and enabling late in situ arthrodesis as a means of salvage in the event of post-traumatic arthritis. The present article briefly discusses our preferred methods for the management of calcaneal fractures. An English full text version of this article is available at SpringerLink as supplemental.

Inhibiting HIV-1 entry with fusion inhibitors.

In recent years, tremendous progress has been made in understanding the HIV-1 entry process in which the viral and cellular membranes are fused, resulting in the subsequent delivery of the viral genome into the host cell. The mechanistic insight gained from these studies has led to the formulation of exciting new approaches for therapeutic intervention. One of the first and clinically most advanced drugs to emerge from this effort is the fusion inhibitor T20. T20 acts by freezing a transient structural intermediate of the HIV-1 fusion process, thus blocking an essential step in viral entry. With phase III clinical trials already well underway, the success of T20 indicates that targeting of the viral entry process will soon be an important component of antiretroviral therapy. This review addresses this rapidly developing area of HIV research, with a focus on the mechanistic role of fusion inhibitors targeted to the HIV-1 gp41 transmembrane glycoprotein. We will review the results of recent clinical trials with T20 and discuss possible mechanisms of viral escape through the evolution of drug-resistant HIV-1 variants. We will also discuss ongoing research on fusion inhibitor susceptibility testing and the development of new improved fusion inhibitors.

Evolution of AZT resistance in HIV-1: the 41-70 intermediate that is not observed in vivo has a replication defect.

The human immunodeficiency virus type 1 (HIV-1) is notorious for its ability to evolve drug-resistance in patients treated with potent antivirals. Resistance to inhibitors of the viral reverse transcriptase (RT) enzyme is frequently mediated by a single amino acid substitution within RT. Resistance against the nucleoside analogue AZT is remarkable in that multiple amino acid changes accumulate over time to yield virus variants with high-level drug resistance. We now report that in addition to drug-resistance properties, the relative replication capacity of the virus variants affects the evolution of AZT resistance. Some of the typical AZT-resistance mutations have a negative impact on virus replication, and the 41-70 double mutant was found to represent a particularly poor virus. Furthermore, introduction of additional AZT-resistance mutations (41-70-215) leads to nearly complete restoration of virus replication. These results may explain the absence of the 41-70 double mutant in clinical samples and indicate that the evolution of AZT resistance is also influenced by virus replication parameters. Prolonged passage of the replication-impaired 41-70 virus in the absence of AZT yielded several fast-replicating variants. These revertants have compensatory changes in the RT polymerase, some of which have been observed previously in AZT-treated patients. Because we could select for these changes without drug pressure, these changes are likely to improve the RT enzyme function and the HIV-1 replication capacity.

A prospective evaluation of the clinical utility of the lower-extremity injury-severity scores.

BACKGROUND: High-energy trauma to the lower extremity presents challenges with regard to reconstruction and rehabilitation. Failed efforts at limb salvage are associated with increased patient mortality and high hospital costs. Lower-extremity injury-severity scoring systems were developed to assist the surgical team with the initial decision to amputate or salvage a limb. The purpose of the present study was to prospectively evaluate the clinical utility of five lower-extremity injury-severity scoring systems. METHODS: Five hundred and fifty-six high-energy lower-extremity injuries were prospectively evaluated with use of five injury-severity scoring systems for lower-extremity trauma designed to assist in the decision-making process for the care of patients with such injuries. Four hundred and seven limbs remained in the salvage pathway six months after the injury. The sensitivity, specificity, and area under the receiver operating characteristic curve were calculated for the Mangled Extremity Severity Score (MESS); the Limb Salvage Index (LSI); the Predictive Salvage Index (PSI); the Nerve Injury, Ischemia, Soft-Tissue Injury, Skeletal Injury, Shock, and Age of Patient Score (NISSSA); and the Hannover Fracture Scale-97 (HFS-97) for ischemic and nonischemic limbs. The scores were analyzed in two ways: including and excluding limbs that required immediate amputation. RESULTS: The analysis did not validate the clinical utility of any of the lower-extremity injury-severity scores. The high specificity of the scores in all of the patient subgroups did confirm that low scores could be used to predict limb-salvage potential. The converse, however, was not true. The low sensitivity of the indices failed to support the validity of the scores as predictors of amputation. CONCLUSIONS: Lower-extremity injury-severity scores at or above the amputation threshold should be cautiously used by a surgeon who must decide the fate of a lower extremity with a high-energy injury.

Nutrient Acquisition and Population Growth of a Mixotrophic Alga in Axenic and Bacterized Cultures.

Axenic growth of a mixotrophic alga, Ochromonas sp., was compared in several inorganic and organic media, and in the presence of live bacteria under nutrient-replete and low-nutrient conditions. Axenic growth in the light was negligible in inorganic media with or without the addition of glucose. Addition of vitamins increased growth rate, but average cell size declined, resulting in no net increase in biomass. Supplementing axenic cultures with a more complex organic substrate resulted in moderate growth and higher maximal abundance (and biomass) than in the inorganic media with added vitamins. The absence of light did not greatly affect population growth rate in the presence of complex dissolved organic compounds, although cell size was significantly greater in the light than in the dark. The highest growth rates for the alga (up to 2.6 d-1) were measured in treatments containing live bacteria. Increases in cell number of Ochromonas sp. in the presence of bacterial prey were similar in the light and dark, although chloroplast and cell sizes differed. Bacterial abundance was reduced and dissolved phosphorus and ammonia were rapidly released in bacterized cultures in the light and dark, indicating high rates of bacterial ingestion and suggesting an inability of the alga to store or utilize N and P in excess of the quantities required for heterotrophic growth. Low-nutrient conditions in the presence of bacteria were promoted by adding glucose to stimulate bacterial growth and the uptake of N and P released by algal phagotrophy. Subsequent decreases in dissolved N and P following the addition of glucose corresponded to a second period of rapid growth of the alga in both light and dark. This result, combined with evidence for slow axenic growth of this strain, indicated that nutrient acquisition for this species in the presence of bacteria was accomplished primarily via ingestion of bacteria.

Characterization of patients with high-energy lower extremity trauma.

PURPOSE: (a) to report the demographic, socioeconomic, behavioral, social, and vocational characteristics of patients enrolled in a study to examine outcomes after high-energy lower extremity trauma (HELET) and to compare them with the general population; (b) to determine whether characteristics of patients undergoing limb salvage versus amputation after HELET are significantly different from each other. DESIGN AND STUDY POPULATION: A prospective study of 601 patients admitted with high-energy lower extremity trauma to eight Level I trauma centers. PROCEDURES: Patients were evaluated during the initial hospitalization. They are being followed up for 24 months postinjury. Study patients are compared with the general population by using census information, population survey data, and published norms. Characteristics of patients undergoing limb salvage versus amputation are also compared. RESULTS: Most patients were male (77 percent), white (72 percent), and between the ages of twenty and forty-five years (71 percent). Seventy percent graduated from high school (compared with 86 percent nationally) (p < 0.05). One fourth lived in households with incomes below the federal poverty line, compared with 16 percent nationally (p < 0.05). The percentage with no health insurance (38 percent) was also higher than in the general population (20 percent) (p < .05). The percentage of heavy drinkers was over two times higher than reported nationally (p < 0.01). Study patients were slightly more neurotic and extroverted and less open to new experiences. When patient characteristics were compared for those undergoing amputation versus limb salvage, no significant differences were found among any of the variables (p > 0.05). CONCLUSION: In conclusion, LEAP patients differ in important ways from the general population. However, the decision to amputate verus reconstruct does not appear to be significantly influenced by patient characteristics.

Evaluation and treatment of high subtrochanteric femur fractures.

This retrospective study reviews the biomechanical factors and surgical approaches for the treatment of high subtrochanteric femur fractures to determine whether management should be directed toward using an intramedullary or extramedullary device. Results have demonstrated that both plates and intramedullary implants work equally well, with intramedullary implants resulting in significant decreases in surgical times and blood losses. Because intramedullary devices can now be placed through percutaneous trochanteric insertions, they have become more attractive for the management of these injuries. However, intramedullary devices may be difficult to use in fractures presenting with a trochanteric extension, and adjunctive reduction techniques are still required to obtain fracture reductions and prevent varus malunions. When deciding on which technique to use, surgeons should evaluate the fracture pattern and determine whether the implants and techniques that are familiar to them will allow good functional outcomes, high rates of unions, and low rates of complications.

Bohler incision: an extensile anterolateral approach to the foot and ankle.

Incisions in the foot and ankle should allow efficient surgical approaches to the anatomic structures being addressed while preventing chronic neuritic symptoms, extensive scar contractures, soft tissue ischemia or necrosis, and chronic edema. The purpose of this report is to describe the Böhler incision, an extensile approach that provides the surgeon with easy access to the anterior surface of the distal tibia, the anterior talar dome, talar neck, talonavicular, subtalar, and calcaneocuboid joints by allowing direct visualization of these areas. The incision can be extended in both directions, if needed, or it can be used at either end, produces few complications, and closes with a cosmetically acceptable scar that does not produce pressure with shoe wear.

Percutaneous plating in the lower extremity.

Since the late 1950s, open reduction and internal fixation has been advocated to restore bone anatomy and enable early mobilization. This approach often necessitated extensive dissection and tissue devitalization, creating an environment less favorable for fracture union and more prone to bone infection. As a result, other methods, such as intramedullary nailing, have become the standard treatment for most diaphyseal fractures of the femur and tibia. However, internal fixation with plates and screws remains the treatment of choice for most periarticular fractures and other complex fractures inadequately stabilized by intramedullary nailing. Recently, more "biologic" methods of reduction involving the use of indirect techniques and new plate designs have been developed in an attempt to preserve the blood supply to the injured bone, improve the rate of fracture healing, decrease the need for bone grafting, and lower the incidence of infection and other complications. Percutaneous plating appears to be the next step in the evolution of biologic plating. With these techniques, the fracture is reduced indirectly, and plates are placed into submuscular or subcutaneous tunnels through limited skin incisions. This may result in less surgical trauma to tissues and further improvements in clinical results compared with current methods of plate insertion.

Subtalar arthrodesis for complications of intra-articular calcaneal fractures.

Eighty six subtalar arthrodeses performed between 1985 and 1996 for complications associated with intra-articular calcaneal fractures were retrospectively evaluated. Patients were divided into three Groups: (I) 59 patients with calcaneal malunions (II) 13 patients with failed open reduction and internal fixation, and (III) eight patients undergoing open reductions and primary fusion for highly comminuted fractures. In each scenario, internal fixation was achieved with cancellous lag screws. Bone graft material consisted of either autogenous iliac crest graft, local graft obtained from the lateral wall exostectomy of the malunion, or freeze-dried cancellous allograft. Fusions in Groups II and III were performed in situ. Fusions in Group I were performed either in situ or utilizing a variety of reconstructive procedures depending upon the type of malunion encountered. Eighty three of the 86 fusion attempts were successful following the initial operations for a union rate of 96%. Fusion rates were similar regardless of the graft material used. Complications included four varus malunions, four cases of osteomyelitis, and two cases of reflex sympathetic dystrophy. A statistically significant shorter hospital stay was found for patients not undergoing iliac crest bone graft procedures. Eighty patients with at least two year follow up achieved a mean American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot score of 75.0. Scores were similar for all three groups and for the various types of reconstructive procedures used. No correlation was found between postoperative talar declination angle and the AOFAS ankle-hindfoot score. Worker's compensation patients tended to have a poorer clinical outcome.

The effects, risks, and guidelines for radiation use in orthopaedic surgery.

Radiation is used during orthopaedic surgery in more than 15 million studies performed yearly. The biologic effects of radiation have been shown to inhibit mitosis by producing irrepairable deoxyribonucleic acid double strand breaks or create structural changes by damaging the nucleus, thereby producing potential genetic transmissions. Although human cells are thought to be resistant to malignant change and no studies have shown toxic effects resulting from long-term exposure to low-dose radiation, risks still are assumed. To decrease all risks, radiographic units should undergo periodic calibration, surgeons should wear protective devices, increase their working distance from the x-ray beam, and limit their duration of radiation exposure by making certain that they follow the guidelines set forth by the National Council for Radiation Protection and Measurement.

Variable-loop-deleted variants of the human immunodeficiency virus type 1 envelope glycoprotein can be stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits.

We have described an oligomeric gp140 envelope glycoprotein from human immunodeficiency virus type 1 that is stabilized by an intermolecular disulfide bond between gp120 and the gp41 ectodomain, termed SOS gp140 (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol. 74:627-643, 2000). In this protein, the protease cleavage site between gp120 and gp41 is fully utilized. Here we report the characterization of gp140 variants that have deletions in the first, second, and/or third variable loop (V1, V2, and V3 loops). The SOS disulfide bond formed efficiently in gp140s containing a single loop deletion or a combination deletion of the V1 and V2 loops. However, deletion of all three variable loops prevented formation of the SOS disulfide bond. Some variable-loop-deleted gp140s were not fully processed to their gp120 and gp41 constituents even when the furin protease was cotransfected. The exposure of the gp120-gp41 cleavage site is probably affected in these proteins, even though the disabling change is in a region of gp120 distal from the cleavage site. Antigenic characterization of the variable-loop-deleted SOS gp140 proteins revealed that deletion of the variable loops uncovers cryptic, conserved neutralization epitopes near the coreceptor-binding site on gp120. These modified, disulfide-stabilized glycoproteins might be useful as immunogens.

A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure.

The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virion-associated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.