Engineering a Next-Generation Glycoconjugate-Like Streptococcus pneumoniae Vaccine.

We detail the development of a next-generation Streptococcus pneumoniae liposomal encapsulation of polysaccharides (LEPS) vaccine, with design characteristics geared toward best-in-class efficacy. The first generation LEPS vaccine, which contained 20 encapsulated pneumococcal capsular polysaccharides (CPSs) and two surface-displayed virulence-associated proteins (GlpO and PncO), enabling prophylactic potency against 70+ serotypes of Streptococcus pneumoniae (the causative agent of pneumococcal disease), was rationally redesigned for advanced clinical readiness and best-in-class coverage. In doing so, the virulent-specific GlpO protein antigen was removed from the final formulation due to off-target immunogenicity toward bacterial species within the human microbiome, while directed protection was maintained by increasing the dose of PncO from 17 to 68 µg. LEPS formulation parameters also readily facilitated an increase in CPS valency (to a total of 24) and systematic variation in protein-liposome attachment mechanisms in anticipation of clinical translation. An additional safety assessment study demonstrated that LEPS does not exhibit appreciable toxicological effects even when administered at ten times the effective dose. In summary, this new design offers the broadest, safest, and most-complete protection while maintaining desirable glycoconjugate-like features, positioning the LEPS vaccine platform for clinical success and a global health impact.

Improving global vaccine accessibility.

Although the beneficial impact of vaccination is well established, developing countries often rely upon humanitarian support to obtain access to routine immunization schedules. Furthermore, philanthropic funders require that vaccines be supplied at prices that are drastically lower than that charged in developed nations. This pricing requirement arises due to both the massive volume of vaccines needed and the logistics necessary to ensure their integrity (i.e. the cold chain). Cost-prohibitive vaccine formulation strategies, especially for newer vaccines, along with the lack of infrastructure in developing nations can further complicate this process. Extensive research is being conducted to develop novel technological platforms that overcome each of these accessibility impediments. This review provides an overview of the humanitarian organizations and technological developments that are dedicated to improving global healthcare by increasing vaccine accessibility.

Overcoming Gene-Delivery Hurdles: Physiological Considerations for Nonviral Vectors.

With the use of contemporary tools and techniques, it has become possible to more precisely tune the biochemical mechanisms associated with using nonviral vectors for gene delivery. Consequently, nonviral vectors can incorporate numerous vector compositions and types of genetic cargo to develop diverse genetic therapies. Despite these advantages, gene-delivery strategies using nonviral vectors have poorly translated into clinical success due to preclinical experimental design considerations that inadequately predict therapeutic efficacy. Furthermore, the manufacturing and distribution processes are critical considerations for clinical application that should be considered when developing therapeutic platforms. In this review, we evaluate potential avenues towards improving the transition of gene-delivery technologies from in vitro assessment to human clinical therapy.

Scaffold diversification enhances effectiveness of a superlibrary of hyperthermophilic proteins.

The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in biotechnology and medicine. Typically, binders are isolated from a combinatorial library generated by mutating a single scaffold protein. In contrast, here we generated a "superlibrary" or "library-of-libraries" of 4 × 10(8) protein variants by mutagenesis of seven different hyperthermophilic proteins; six of the seven proteins have not been used as scaffolds prior to this study. Binding proteins for five different model targets were successfully isolated from this library. Binders obtained were derived from five out of the seven scaffolds. Strikingly, binders from this modestly sized superlibrary have affinities comparable or higher than those obtained from a library with 1000-fold higher sequence diversity but derived from a single stable scaffold. Thus scaffold diversification, i.e., randomization of multiple different scaffolds, is a powerful alternate strategy for combinatorial library construction.

Design of pH sensitive binding proteins from the hyperthermophilic Sso7d scaffold.

We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies - histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG(1-4)), and recognize hIgG(1-4) as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.

Open versus endovascular repair of popliteal artery aneurysms.

A 72-year-old male presents with a large asymptomatic aneurysm of his left popliteal artery. He has a history of noninsulin dependent diabetes, hypertension, and a prior history of a percutaneous intervention for a coronary artery stenosis. He is anatomically and physiologically a candidate for surgical or endovascular repair of his aneurysm. The following debate attempts to resolve whether open repair remains the gold standard for the treatment of popliteal artery aneurysms.

Health-related quality of life in survivors of open ruptured abdominal aortic aneurysm repair: a matched, controlled cohort study.

OBJECTIVE: The objective of this study was to document the health-related quality of life (HRQOL) for patients who survived operative repair of a ruptured abdominal aortic aneurysm (RAAA) and to compare this with a matched group of patients who survived elective operative repair of an abdominal aortic aneurysm (EAAA). METHODS: A matched, controlled cohort study of HRQOL was used to compare patients surviving RAAA with an EAAA control group. The study was conducted at two university-affiliated vascular tertiary care referral centers. Survivors of RAAA and EAAA during an 8.5-year period were identified and followed up. The RAAA and EAAA control patients were matched for age, serum creatinine concentration, gender, and duration of follow-up since surgery. HRQOL was measured with the Medical Outcomes Study Short Form-36 Health Survey (SF-36). Scores for the EAAA and RAAA cohorts were also compared with age-corrected SF-36 population scores. RESULTS: Of 267 patients operated for RAAA during the study period, 130 (49%) survived to hospital discharge. Death after discharge was documented in 35 patients, leaving a potential study population of 95 RAAA survivors. Thirteen were lost to follow-up, seven refused to participate, and four patients were not able to participate. The SF-36 was completed by 71 RAAA patients (75% of surviving RAAA patients). The 71 RAAA survivors and 189 EAAA control patients were similar for seven of eight domains of the SF-36: Physical Function, Role-Physical, Bodily Pain, General Health, Vitality, Mental Health, and Role-Emotional. There was also no difference in the Physical Health Summary and Mental Health Summary scores. The social function component of the SF-36 demonstrated a statistically significant decline in the EAAA group. Both the EAAA and RAAA SF-36 individual and summary scores compared favorably with population norms that were adjusted only for age. CONCLUSION: Long-term survivors of RAAA enjoy a HRQOL that does not differ significantly from EAAA survivors. Scores for both groups compare favorably with population scores adjusted only for age.

Heparin prophylaxis for deep venous thrombosis in a patient with multiple injuries: an evidence-based approach to a clinical problem.

OBJECTIVE: To demonstrate a clinical decision-making process by which to determine if heparin prophylaxis for deep venous thrombosis (DVT) is appropriate in a specific patient with multiple injuries. DATA SOURCES: A Medline search of the literature. Search terms included trauma, heparin, deep venous thrombosis, thrombophlebitis, phlebitis, and trauma. STUDY SELECTION: Eleven studies were selected from 789 publications using published criteria. Incidence, risk and potential for prophylaxis were established through a structured review process. DATA EXTRACTION: After the structured review, a small number of studies were available for the consideration of incidence (2), natural history (4) and prophylactic therapy (2). DATA SYNTHESIS: The incidence of DVT in a patient with such multiple injuries is significant (58%-63%). The resulting risk of pulmonary embolism was 4.3% with an associated 20% death rate. Prophylaxis with low molecular weight heparin is associated with a statistically and clinically significant risk reduction for DVT when compared with unfractionated heparin and untreated controls. CONCLUSIONS: Few of the multiple available studies concerning trauma, DVT and pulmonary embolism meet reasonable standards to establish clinical validity. Available guidelines for literature evaluation allow surgeons to select relevant articles for consideration. Patients with multiple trauma appear to be at significant risk for DVT. The death rate associated with subsequent pulmonary embolism is significant. There is reasonably good evidence to suggest that low molecular weight heparin will reduce this likelihood without a significant risk of treatment complications.