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In vat polymerization (VP) 3D printing, there is an urgent need to expand characterization efforts for resins derived from natural resources to counter the increasing consumption of fossil fuels required to synthesize conventional monomers. Here, we apply multiscale mechanical characterization techniques to interrogate a 3D printed biobased copolymer along a controlled range of monomer ratios. We varied the concentration of two dissimilar monomers to derive structural information about the polymer networks. Current research primarily considers the macroscale, but recent understanding of the process-induced anisotropy in 3D printed layers suggests a multiscale approach is critical. By combining typical macroscopic techniques with micro- and nanoscale analogues, clear correlations in the processing-structure-property relationships appeared. We observed that measured moduli were always greater via surface-localized methods, but property differences between formulations were easier to identify. As researchers continue to develop novel sustainable biopolymers that match or exceed the performance of commercial resins, it is vital to understand the multiscale relationships between the VP process, the structure of the formed polymer networks, and the resultant properties.

Abstract Lipid nanoparticles have demonstrated utility in hepatic delivery of a range of therapeutic modalities and typically deliver their cargo via low-density lipoprotein receptor-mediated endocytosis. For patients lacking sufficient low-density lipoprotein receptor activity, such as those with homozygous familial hypercholesterolemia, an alternate strategy is needed. Here we show the use of structure-guided rational design in a series of mouse and non-human primate studies to optimize a GalNAc-Lipid nanoparticle that allows for low-density lipoprotein receptor independent delivery. In low-density lipoprotein receptor-deficient non-human primates administered a CRISPR base editing therapy targeting the ANGPTL3 gene, the introduction of an optimized GalNAc-based asialoglycoprotein receptor ligand to the nanoparticle surface increased liver editing from 5% to 61% with minimal editing in nontargeted tissues. Similar editing was noted in wild-type monkeys, with durable blood ANGPTL3 protein reduction up to 89% six months post dosing. These results suggest that GalNAc-Lipid nanoparticles may effectively deliver to both patients with intact low-density lipoprotein receptor activity as well as those afflicted by homozygous familial hypercholesterolemia.

The degree of monomer conversion and polymerization shrinkage are two of the main reasons for potential adhesion failure between the tooth structure and the restoration substrate. To evaluate the degree of conversion and polymerization shrinkage of a newly developed self-adhesive resin cement, the degree of conversion (DC) was measured using FTIR under different activation modes, temperatures, and times. Volumetric shrinkage was tested using the AcuVol video imaging method. The experimental cement showed a higher DC than other cements under self-curing. The DC of the experimental cement was higher than that of other cements, except SpeedCem Plus under light curing. The experimental cement had a higher DC than other cements, except SpeedCem Plus in some conditions under dual curing. All self-adhesive cements had a higher DC at 37 °C than at 23 °C under self-curing, and there was no statistical difference between 23 °C and 37 °C under light curing. All self-adhesive cements showed a significantly higher DC at 10 min than at 5 min under self-curing. There was no statistical difference between 5 min and 10 min for most cements under dual curing. All self-adhesive cements statistically had the same volumetric shrinkage under light curing and self-curing. The newly developed self-adhesive resin cement exhibited a higher degree of conversion and similar volumetric shrinkage compared to these commercial self-adhesive resin cements.

Abstract Background Standardized assessments for dystrophic epidermolysis bullosa (DEB) are needed. This prospective, multicenter, 4-week, observational study was designed to evaluate DEB assessments for suitability as clinical trial endpoints. Methods Patients with confirmed DEB diagnosis and ≥ 5 measurable wounds were included. The primary outcome was change from baseline in wound surface area (WSA) of 5 selected wounds by 3-dimensional imaging. Secondary endpoints were change from baseline in clinician global assessment (CGA) of WSA, wound characteristics, disease-related questionnaires and instruments (disease severity, quality of life [QoL], pain and disability, and itch), and tolerability of procedures. Results Of 30 enrolled patients, 29 completed the study (of whom, 28 had recessive DEB). Median age was 17.8 years (range, 3.8–58.7). All patients developed new or recurrent wounds during the 4-week study. Of the wounds selected at baseline, 45/150 (30.0%) healed by week 2; an additional 38 healed by week 4, while 8 of those healed at week 2 had recurred by week 4 for a total of 75/150 (50.0%) healed wounds at week 4. Mean values for WSA, CGA, and disease-related questionnaire and instrument scores remained steady during this 4-week observational study. Of the 10 disease-related questionnaires and instruments assessed, the scores for the Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) and the Instrument for Scoring Clinical Outcomes for Research of Epidermolysis Bullosa (iscorEB) did not substantially overlap between moderate and severe disease. Between mild and moderate disease, only the EBDASI scores did not substantially overlap. Conclusions These results stress the dynamic nature of wounds, even during a 4-week period of observation, and suggest that a combination of clinician-assessed outcomes and patient-/caregiver-reported outcomes is needed to provide a comprehensive assessment of DEB severity and impact. In addition, these results support the use of EBDASI and iscorEB to monitor disease severity as both produced scores that did not substantially overlap between disease severity strata. Clinical trial registration ClinicalTrials.gov, NCT02178969 . Registered 4 June 2014, https://clinicaltrials.gov/ct2/show/NCT02178969 .

Introduction: Triage is a sorting process to quickly assess patients upon their arrival in the emergency department which helps to stream them to an appropriate location and adequate treatment. Triage assessment helps in recognising the commonly presenting childhood emergencies to optimise quality of care delivered in the Paediatric Emergency Department. Priority attention can be given to the critically ill or injured patients as how long the patient can safely wait, is predicted by triage. Aim: To provide data on disease spectrum and triage assessment of children presenting to an exclusive Paediatric Emergency Department. Materials and Methods: This was a cross-sectional study which was conducted from October 2020 to September 2021 over a period of one year in the Paediatric Emergency Department at Niloufer Hospital, a tertiary care children hospital in Hyderabad, Telangana, India. All children in the age group of one month to twelve years triaged by five-level Canadian triage and acute scale were included in the study. Data was collected from the register maintained at the Emergency Department. Descriptive statistics was used to analyse the data. Micosoft excel sheets were used for recording data. Results: During the study period, 7986 children were admitted with 5718 (71.6%) males and 2268 (28.4%) females. A total of 4352 (54.5%) patients were less than one year age. Neurological emergencies, acute febrile illnesses, respiratory illnesses were most common reasons for emergency visits. The number of chidren presenting with triage level 1,2,3 were n=4369 (54.71%). Conclusion: Majority of the patients attending the Emergency Department were less than one year old and acute febrile illnesses and seizures were the most common causes for admissions. Triaging and priorisation of pediatric emergencies is strogly recommended for early recognition of life threatening illnesses and to improve outcomes. Specially trained nurses, healthcare professionals trained in Basic Life Support (BLS) and Paediatric Advanced Life Support (PALS) will go a long way in reducing morbidity and mortality.

Objective. To evaluate a newly developed self-adhesive resin cement on physical, mechanical, and adhesive properties and compare it with other commercial self-adhesive resin cements. Materials and Methods. Experimental self-adhesive resin cement (SARC) was formulated by our proprietary adhesive resin and filler technology. Maxcem Elite, RelyX Unicem 2, SpeedCem Plus, SmartCEM 2, and Calibra Universal 2 were selected for comparison. Working and setting times, film thickness, water sorption and solubility, flexural strength, and modulus were measured in accordance with ISO-4049. Consistency was tested according to modified ISO 4823. Shear bond strengths were conducted according to ISO 29022. The data were analyzed by one-way ANOVA and post hoc Tukey’s tests (p ≤ 0.05). Results. All cements showed about 2–4 min working time and about 3–6 min setting time except that RelyX Unicem 2 has a longer working time (9’58”) and setting time (10’18”). All cements meet ISO standards for film thickness (≤50 µm), water sorption (≤40 µg/mm3) except Maxcem Elite (46.19 µg/mm3), and water solubility (≤7.5 µg/mm3) except SmartCEM 2 (11.35 µg/mm3) and Calibra Universal (9.87 µg/mm3). Experimental SARC showed significantly higher flexural strength and modulus than other cements (p 0.05). For light-curing, Experimental SARC showed significantly higher bond strength than other cements (p 0.05). For dual-curing, the bond strength of Experimental SARC is significantly higher than that of other cements (p

Reservoir siting, permitting, construction and operation can, at times, face insurmountable challenges. Mosul Dam, a multipurpose dam located in Northern Iraq, is constructed on a dissolvable anhydrite, gypsum and limestone formation. To mitigate long-term impacts to the foundation, the dam designers opted for a structural solution. They incorporated into the design a grouting gallery for long-term maintenance grouting of the foundation cutoff grout curtain. In 2016, concerns with the risk posed by the deteriorating foundation resulted in the execution of the Mosul Dam emergency grouting and maintenance project. This paper illustrates how the international community proactively came together to prevent an incident of potential tragic proportions well before it could manifest itself. The emergency grouting work is an excellent example of Governments investing in preventing a disaster rather than investing in fixing a disaster. The paper presents the general approach adopted for the execution of the emergency project and discusses how the scope of work (SOW) evolved to address the myriad of requirements, including the training and transfer of information and equipment to the Iraqi Ministry of Water Resources that could not have been foreseen during the scoping of the work. Additionally, this paper illustrates the success that can be achieved when team members share a strong commitment to cooperation and a determination to succeed.

Abstract: Adjuvants have a critical role for improving vaccine efficacy against many pathogens, including HIV. Here, using transcriptional RNA profiling and systems serology, we assessed how distinct innate pathways altered HIV-specific antibody responses in nonhuman primates (NHPs) using 8 clinically based adjuvants. NHPs were immunized with a glycoprotein 140 HIV envelope protein (Env) and insoluble aluminum salts (alum), MF59, or adjuvant nanoemulsion (ANE) coformulated with or without Toll-like receptor 4 (TLR4) and 7 agonists. These were compared with Env administered with polyinosinic-polycytidylic acid:poly-L-lysine, carboxymethylcellulose (pIC:LC) or immune-stimulating complexes. Addition of the TLR4 agonist to alum enhanced upregulation of a set of inflammatory genes, whereas the TLR7 agonist suppressed expression of alum-responsive inflammatory genes and enhanced upregulation of antiviral and interferon (IFN) genes. Moreover, coformulation of the TLR4 or 7 agonists with alum boosted Env-binding titers approximately threefold to 10-fold compared with alum alone, but remarkably did not alter gene expression or enhance antibody titers when formulated with ANE. The hierarchy of adjuvant potency was established after the second of 4 immunizations. In terms of antibody durability, antibody titers decreased ∼10-fold after the final immunization and then remained stable after 65 weeks for all adjuvants. Last, Env-specific Fc-domain glycan structures and a series of antibody effector functions were assessed by systems serology. Antiviral/IFN gene signatures correlated with Fc-receptor binding across all adjuvant groups. This study defines the potency and durability of 8 different clinically based adjuvants in NHPs and shows how specific innate pathways can alter qualitative aspects of Env antibody function.

Background: Glucose metabolism links closely to cholesterol metabolism. Posttransplant diabetes mellitus (PTDM) adversely affects posttransplant outcomes, but its risk factors in relation to cholesterol metabolism have not been fully delineated. The apolipoprotein B/A1 (Apo B/A1) ratio, which is associated with insulin resistance, has not been evaluated in kidney transplant recipients as a risk factor for PTDM. Objective: The objective of this study was to determine whether serum apolipoprotein profiles predict late PTDM, defined as a new onset diabetes occurring greater than 3 months posttransplant. Design: Retrospective chart review of a prevalent population of kidney transplant recipients. Setting: Large transplant center in Ontario, Canada. Patients: We identified 1104 previously nondiabetic adults who received a kidney transplant between January 1, 1998, and December 1, 2015, and were followed at 1 transplant center. Measurements: Recipients provided testing for serum apolipoprotein B (Apo B) and apolipoprotein A1 (Apo A1) concentrations from 2010, either at 3 months posttransplant for new transplant recipients or the next clinic visit for prevalent recipients. Late PTDM defined using Canadian Diabetes Association criteria as occurring ≥3 months posttransplant was recorded until May 1, 2016. Methods: All analyses were conducted with R, version 3.4.0 (The R Foundation for Statistical Computing). Comparisons were made using Student t test, Fisher exact test or chi-square test, Kaplan-Meier methodology with the logrank test, or Cox proportional hazards analysis as appropriate. Covariates for the multivariate Cox proportional hazards models of PTDM as the outcome variable were selected based on significance of the univariate associations and biological plausibility. Results: There were 53 incident late PTDM cases, or 1.71 cases per 100 patient-years. Incident late PTDM differed between the highest and lowest quartiles for Apo B/A1 ratio, 2.47 per 100 patient-years vs 0.88 per 100 patient-years ( P = .005 for difference). In multiple Cox regression analysis, first measured serum Apo B/A1 concentration better predicted subsequent PTDM than low-density lipoprotein cholesterol (LDL-C; hazard ratio [HR] = 7.80 per unit increase, P = .039 vs HR = 1.05 per unit increase, P = .774). Non-high-density lipoprotein cholesterol (HDL-C) concentrations also did not predict PTDM ( P = .136). By contrast to Apo B, Apo A1 was protective against PTDM in statin users (HR = 0.17 per unit increase, P = .016). Limitations: Posttransplant diabetes mellitus cases occurring before apolipoprotein testing was implemented were not included in the analysis. Conclusions: Apolipoproteins B and A1 better predict late PTDM than conventional markers of cholesterol metabolism.

Early studies have shown that moderate levels of calcium overload can cause lower oxidative phosphorylation rates. However, the mechanistic interpretations of these findings were inadequate. And while the effect of excessive calcium overload on mitochondrial function is well appreciated, there has been little to no reports on the consequences of low to moderate calcium overload. To resolve this inadequacy, mitochondrial function from guinea pig hearts was quantified using several well-established methods including high-resolution respirometry and spectrofluorimetry and analyzed using mathematical modeling. We measured key mitochondrial variables such as respiration, mitochondrial membrane potential, buffer calcium, and substrate effects for a range of mitochondrial calcium loads from near zero to levels approaching mitochondrial permeability transition. In addition, we developed a computer model closely mimicking the experimental conditions and used this model to design experiments capable of eliminating many hypotheses generated from the data analysis. We subsequently performed those experiments and determined why mitochondrial ADP-stimulated respiration is significantly lowered during calcium overload. We found that when calcium phosphate levels, not matrix free calcium, reached sufficient levels, complex I activity is inhibited, and the rate of ATP synthesis is reduced. Our findings suggest that calcium phosphate granules form physical barriers that isolate complex I from NADH, disrupt complex I activity, or destabilize cristae and inhibit NADH-dependent respiration.

The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2-20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant.

The prevalence of extracorporeal cardiopulmonary resuscitation is increasing worldwide as more health care centers develop the necessary infrastructure, protocols, and technical expertise required to provide mobile extracorporeal life support with short notice. Strict adherence to patient selection guidelines in the setting of out-of-hospital cardiac arrest, as well as in-hospital cardiac arrest, allows for improved survival with neurologically favorable outcomes in a larger patient population. This review discusses the preferred approaches, cannulation techniques, and available support devices ideal for the various clinical situations encountered during the treatment of cardiac arrest and refractory cardiogenic shock. [ABSTRACT FROM AUTHOR]

Rupture of the ventricular septum during acute myocardial infarction usually occurs within the first week. The event is usually followed by low cardiac output, heart failure, and multiorgan failure. Despite the many advances in the nonoperative treatment of heart failure and cardiogenic shock, including the intra-aortic balloon pump and a multitude of new inotropic agents and vasodilators, these do not supplant the need for operative intervention in these critically ill patients. This article describes the successful use of extracorporeal membrane oxygenation support as a bridge to recovery postoperatively in a patient with a large infarctproduced ventricular septal defect. [ABSTRACT FROM AUTHOR]

Objective: Intra-aortic balloon pumps are traditionally inserted through the femoral artery, limiting the patient''s mobility. We used alternate approaches of intra-aortic balloon pump insertion to provide temporary and minimally invasive support for patients with decompensating, end-stage heart failure. The present study describes the outcomes with closed-chest, transthoracic intra-aortic balloon pumps by way of the subclavian artery. Methods: During a 3-year period, 20 patients underwent subclavian artery–intra-aortic balloon pump in the setting of end-stage heart failure. The balloon was inserted through a polytetrafluoroethylene graft sutured to the right subclavian artery in 19 patients (95%) and to the left subclavian artery in 1 patient (5%). The goal of support was to bridge to transplantation in 17 patients (85%) and bridge to recovery in 3 patients (15%). The primary outcome measure was death during subclavian artery–intra-aortic balloon pump support. The secondary outcomes included survival to the intended endpoint of bridge to transplantation/bridge to recovery, complications during subclavian artery–intra-aortic balloon pump support (eg, stroke, limb ischemia, brachial plexus injury, dissection, bleeding requiring reoperation, and device-related infection), emergent surgery for worsening heart failure, and ambulation during intra-aortic balloon pump support. Results: The duration of balloon support ranged from 3 to 48 days (mean, 17.3 ± 13.1 days). No patients died during subclavian artery–intra-aortic balloon pump support. Of the 20 patients, 14 (70%) were successfully bridged to transplant or left ventricular-assist device. Two patients (10%) required emergent left ventricular-assist device for worsening heart failure. Conclusions: An intra-aortic balloon pump inserted through the subclavian artery is a simple, minimally invasive approach to mechanical support and is associated with limited morbidity and facilitates ambulation in patients with end-stage heart failure. [Copyright &y& Elsevier]

Venoarterial extracorporeal membrane oxygenation (ECMO) can provide temporary cardiopulmonary support for patients in hemodynamic extremis or refractory heart failure until more durable therapies--such as cardiac transplantation or a left ventricular assist device--can be safely implemented. Conventional ECMO cannulation strategies commonly employ the femoral artery and vein, constraining the patients to the supine position for the duration of ECMO support. We have recently adopted a modified cannulation approach to promote patient mobility, rehabilitation, and faster recovery and to mitigate complications associated with femoral arterial cannulation, such as limb ischemia and compartment syndrome. This technique involves cannulation of the subclavian artery and vein. The current case report details our recent experience with this approach in a critically ill patient awaiting cardiac transplantation. [ABSTRACT FROM AUTHOR]

Antibody-drug conjugates unite the specificity and long circulation time of an antibody with the toxicity of a chemical cytostatic or otherwise active drug using appropriate chemical linkers to reduce systemic toxicity and increase therapeutic index. This combination of a large biological molecule and a small molecule creates an increase in complexity. Multiple production processes are required to produce the native antibody, the drug and the linker, followed by conjugation of afore mentioned entities to form the final antibody-drug conjugate. The connected processes further increase the number of points of control, resulting in necessity of additional specifications and intensified analytical characterization. By combining scientific understanding of the production processes with risk-based approaches, quality can be demonstrated at those points where control is required and redundant comparability studies, specifications or product characterization are avoided. Over the product development lifecycle, this will allow process qualification to focus on those areas critical to quality and prevent redundant studies. The structure of the module 3 common technical document for an ADC needs to reflect each of the production processes and the combined overall approach to quality. Historically, regulatory authorities have provided varied expectations on its structure. This paper provides an overview of essential information to be included and shows that multiple approaches work as long as adequate cross-referencing is included., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

As one of the leading causes of central vision loss in elderly population, worldwide cases of age-related macular degeneration (AMD) have seen a dramatic increase over the past several years. Treatment regimens for AMD, especially with biological agents, are complicated due to anatomical and physiological barriers, as well as administration of high doses and frequent regimens. Some clinical examples include monthly intravitreal administration of anti-VEGF antibody ranibizumab (Lucentis®) from Genentech and aflibercept (Eylea®) from Regeneron Pharmaceuticals. Long-acting sustained intraocular drug delivery provides promising solutions, such as Vitrasert® from Bausch & Lomb, an intravitreal biodegradable polymeric implant made from poly(D,L-lactic co glycolic acid) (PLGA), and can be used as a guiding reference to formulate sustained delivery systems. In this review, we discuss the anatomy and physiology of the eye, barriers to delivery, pathology of AMD, opportunities for biological therapeutics, and future prospects of intraocular delivery strategies that are in development for treatment of AMD., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Adjuvants are necessary to enable vaccine development against a significant number of challenging pathogens for which effective vaccines are not available. We engineered a novel small-molecule immune potentiator, a benzonaphthyridine agonist targeting toll-like receptor 7 (TLR7), as a vaccine adjuvant. TLR7 agonist (TLR7a) was engineered to be adsorbed onto aluminum hydroxide (AlOH), and the resulting AlOH/TLR7a was evaluated as a vaccine adjuvant. AlOH/TLR7a exploits the flexibility of AlOH formulations, has an application in many vaccine candidates, and induced good efficacy and safety profiles against all tested antigens (bacterial- and viral-derived protein antigens, toxoids, glycoconjugates, and so forth) in many animal models, including nonhuman primates. In this article, we describe the outcome of the physicochemical characterization of AlOH/TLR7a. Reverse-phase ultra performance liquid chromatography, confocal microscopy, flow cytometry, zeta potential, and phosphophilicity assays were used as tools to demonstrate the association of TLR7a to AlOH and to characterize this novel formulation. Raman spectroscopy, nuclear magnetic resonance, and mass spectroscopy were also used to investigate the interaction between TLR7a and AlOH (data not shown). This pivotal work paved the way for AlOH/TLR7a to progress into the clinic for evaluation as an adjuvant platform for vaccines against challenging preventable diseases., (Copyright © 2018 American Pharmacists Association®. All rights reserved.)

The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2-20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant.