A comprehensive understanding of the development and evolution of human B cell responses induced by pathogen exposure will facilitate the design of next-generation vaccines. Here, we utilized a high-throughput single B cell cloning technology to longitudinally track the human B cell response to the yellow fever virus 17D (YFV-17D) vaccine. The early memory B cell (MBC) response was mediated by both classical immunoglobulin M (IgM) (IgM+CD27+) and switched immunoglobulin (swIg+) MBC populations; however, classical IgM MBCs waned rapidly, whereas swIg+and atypical IgM+and IgD+MBCs were stable over time. Affinity maturation continued for 6 to 9 mo following vaccination, providing evidence for the persistence of germinal center activity long after the period of active viral replication in peripheral blood. Finally, a substantial fraction of the neutralizing antibody response was mediated by public clones that recognize a fusion loop-proximal antigenic site within domain II of the viral envelope glycoprotein. Overall, our findings provide a framework for understanding the dynamics and complexity of human B cell responses elicited by infection and vaccination.

Background: Adenoviruses (AdV) are broadly employed as gene delivery vectors. Results: Copy numbers of all AdV proteins were measured, and the release of proteins upon heat stress investigated. Conclusion:The viral protease plays a distinct role in the segmented release of AdV proteins. Significance: Our characterization by mass spectrometry provides new insight in HAdV disassembly during entry into host cells.

The identification of the adenovirus (AdV) protein that mediates endosome penetration during infection has remained elusive. Several lines of evidence from previous studies suggest that the membrane lytic factor of AdV is the internal capsid protein VI. While these earlier results imply a role for protein VI in endosome disruption, direct evidence during cell entry has not been demonstrated. To acquire more definitive proof, we engineered random mutations in a critical N-terminal amphipathic ␣-helix of VI in an attempt to generate AdV mutants that lack efficient membrane penetration and infection. Random mutagenesis within the context of the AdV genome was achieved via the development of a novel technique that incorporates both error-prone PCR and recombineering. Using this system, we identified a single mutation, L40Q, that significantly reduced infectivity and selectively impaired endosome penetration. Furthermore, we obtained biophysical data showing that the lack of efficient endosomalysis is associated with reduced insertion of the L40Q mutation in protein VI (VI-L40Q) into membranes. Our studies indicate that protein VI is the critical membrane lytic factor of AdV during cellular entry and reveal the biochemical basis for its membrane interactions.

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SUMMARY Passive administration of monoclonal antibodies (mAbs) is a promising therapeutic approach for Ebola virus disease (EVD). However, all mAbs and mAb cocktails that have entered clinical development are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against outbreak-causing Bundibugyo virus (BDBV) and Sudan virus (SUDV). Here, we advance MBP134, a cocktail of two broadly neutralizing human mAbs, ADI-15878 from an EVD survivor and ADI-23774 from the same survivor but specificity-matured for SUDV GP binding affinity, as a candidate pan-ebolavirus therapeutic. MBP134 potently neutralized all ebolaviruses and demonstrated greater protective efficacy than ADI-15878 alone in EBOV-challenged guinea pigs. A second-generation cocktail, MBP134AF, engineered to effectively harness natural killer (NK) cells afforded additional improvement relative to its precursor in protective efficacy against EBOV and SUDV in guinea pigs. MBP134AF is an optimized mAb cocktail suitable for evaluation as a pan-ebolavirus therapeutic in nonhuman primates.