An HIV-1 broadly neutralizing antibody overcomes structural and dynamic variation through highly focused epitope targeting.

The existence of broadly cross-reactive antibodies that can neutralize diverse HIV-1 isolates (bnAbs) has been appreciated for more than a decade. Many high-resolution structures of bnAbs, typically with one or two well-characterized HIV-1 Env glycoprotein trimers, have been reported. However, an understanding of how such antibodies grapple with variability in their antigenic targets across diverse viral isolates has remained elusive. To achieve such an understanding requires first characterizing the extent of structural and antigenic variation embodied in Env, and then identifying how a bnAb overcomes that variation at a structural level. Here, using hydrogen/deuterium-exchange mass spectrometry (HDX-MS) and quantitative measurements of antibody binding kinetics, we show that variation in structural ordering in the V1/V2 apex of Env across a globally representative panel of HIV-1 isolates has a marked effect on antibody association rates and affinities. We also report cryo-EM reconstructions of the apex-targeting PGT145 bnAb bound to two divergent Env that exhibit different degrees of structural dynamics throughout the trimer structures. Parallel HDX-MS experiments demonstrate that PGT145 bnAb has an exquisitely focused footprint at the trimer apex where binding did not yield allosteric changes throughout the rest of the structure. These results demonstrate that structural dynamics are a cryptic determinant of antigenicity, and mature antibodies that have achieved breadth and potency in some cases are able to achieve their broad cross-reactivity by "threading the needle" and binding in a highly focused fashion, thus evading and overcoming the variable properties found in Env from divergent isolates.

Quantitative chemical imaging of breast calcifications in association with neoplastic processes.

Calcifications play an essential role in early breast cancer detection and diagnosis. However, information regarding the chemical composition of calcifications identified on mammography and histology is limited. Detailed spectroscopy reveals an association between the chemical composition of calcifications and breast cancer, warranting the development of novel analytical tools to better define calcification types. Previous investigations average calcification composition across broad tissue sections with no spatially resolved information or provide qualitative visualization, which prevents a robust linking of specific spatially resolved changes in calcification chemistry with the pathologic process. Method: To visualize breast calcification chemical composition at high spatial resolution, we apply hyperspectral stimulated Raman scattering (SRS) microscopy to study breast calcifications associated with a spectrum of breast changes ranging from benign to neoplastic processes, including atypical ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma. The carbonate content of individual breast calcifications is quantified using a simple ratiometric analysis. Results: Our findings reveal that intra-sample calcification carbonate content is closely associated with local pathological processes. Single calcification analysis supports previous studies demonstrating decreasing average carbonate level with increasing malignant potential. Sensitivity and specificity reach >85% when carbonate content level is used as the single differentiator in separating benign from neoplastic processes. However, the average carbonate content is limiting when trying to separate specific diagnostic categories, such as fibroadenoma and invasive ductal carcinoma. Second harmonic generation (SHG) data can provide critical information to bridge this gap. Conclusion: SRS, combined with SHG, can be a valuable tool in better understanding calcifications in carcinogenesis, diagnosis, and possible prognosis. This study not only reveals previously unknown large variations of breast microcalcifications in association with local malignancy but also corroborates the clinical value of linking microcalcification chemistry to breast malignancy. More importantly, it represents an important step in the development of a label-free imaging strategy for breast cancer diagnosis with tremendous potential to address major challenges in diagnostic discordance in pathology.

Intraoperative assessment of skull base tumors using stimulated Raman scattering microscopy.

Intraoperative consultations, used to guide tumor resection, can present histopathological findings that are challenging to interpret due to artefacts from tissue cryosectioning and conventional staining. Stimulated Raman histology (SRH), a label-free imaging technique for unprocessed biospecimens, has demonstrated promise in a limited subset of tumors. Here, we target unexplored skull base tumors using a fast simultaneous two-channel stimulated Raman scattering (SRS) imaging technique and a new pseudo-hematoxylin and eosin (H&E) recoloring methodology. To quantitatively evaluate the efficacy of our approach, we use modularized assessment of diagnostic accuracy beyond cancer/non-cancer determination and neuropathologist confidence for SRH images contrasted to H&E-stained frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections. Our results reveal that SRH is effective for establishing a diagnosis using fresh tissue in most cases with 87% accuracy relative to H&E-stained FFPE sections. Further analysis of discrepant case interpretation suggests that pseudo-H&E recoloring underutilizes the rich chemical information offered by SRS imaging, and an improved diagnosis can be achieved if full SRS information is used. In summary, our findings show that pseudo-H&E recolored SRS images in combination with lipid and protein chemical information can maximize the use of SRS during intraoperative pathologic consultation with implications for tissue preservation and augmented diagnostic utility.