A map of human protein interactions derived from co-expression of human mRNAs and their orthologs

The human protein interaction network will offer global insights into the molecular organization of cells and provide a framework for modeling human disease, but the network's large scale demands new approaches. We report a set of 7000 physical associations among human proteins inferred from indirect evidence: the comparison of human mRNA co-expression patterns with those of orthologous genes in five other eukaryotes, which we demonstrate identifies proteins in the same physical complexes. To evaluate the accuracy of the predicted physical associations, we apply quantitative mass spectrometry shotgun proteomics to measure elution profiles of 3013 human proteins during native biochemical fractionation, demonstrating systematically that putative interaction partners tend to co-sediment. We further validate uncharacterized proteins implicated by the associations in ribosome biogenesis, including WBSCR20C, associated with Williams–Beuren syndrome. This meta-analysis therefore exploits non-protein-based data, but successfully predicts associations, including 5589 novel human physical protein associations, with measured accuracies of 54±10%, comparable to direct large-scale interaction assays. The new associations' derivation from conserved in vivo phenomena argues strongly for their biological relevance

Longitudinal landscapes of serum antibody repertoires after influenza infection and vaccination

Vaccination is the most effective means of infectious disease prevention. Despite its success, however, we still lack a clear understanding of vaccine responses in humans. For example, influenza vaccines still leave a large fraction of population vulnerable. Over the past decade, single B-cell analysis and next-generation sequencing (NGS) technologies have become invaluable tools for studying the antibody repertoire to influenza. Such studies have led to discoveries of broadly-neutralizing antibodies (bNAbs), which can neutralize across multiple strains of influenza virus, promoting the notion of designing a universal vaccine that will elicit such antibodies. One of such isolated bNAbs, called FI6, showed remarkable ability to neutralize all of the influenza A virus strains through targeting the conserved epitope in the stem of hemagglutinin (HA). However, it remains unclear whether such bNAbs actually play a role in conferring protection against influenza since antibody proteins (not B-cells) need to circulate at physiologically relevant concentrations in serum to have implications in protection. Using high-resolution proteomics coupled with NGS, we quantitatively determined the serological antibody repertoire to CA09 HA (H1) at the individual clonotype-level in a donor (whom FI6 was isolated from) following influenza infection (in 2010 with pandemic CA09) and vaccination across five years (2010-2014 with seasonal flu vaccine). We analyzed the temporal changes of head-targeting and stem-binding antibodies, illustrating the gradual increase of stem-targeting antibodies following repeated exposures to CA09 HA. Following vaccination in 2014, \u3e60% of the repertoire consisted of one single clonotype of stem-binding antibody that was present at very low abundance in 2010. Our data demonstrate that the repetitive exposure to influenza skews the serological repertoire toward antibodies that target conserved epitopes, and these antibodies continue to be boosted every time the same epitopes are encountered. Once elicited, stem-binding antibodies displayed a tendency to persist in serum across multiple years while head-specific antibodies decayed quicker. The differential longevity of stem-binding and head-specific antibodies presented here has direct implications for the design of the future universal vaccine

Persistent antibody clonotypes dominate the serum response to influenza following repeated vaccination over multiple years

We used Ig-Seq, a liquid chromatography tandem mass spectrometry (LC-MS/MS)–based serum antibody proteomics methodology, to determine the clonal composition and dynamics of the H1N1 California/7/2009 (CA09) hemagglutinin (HA)-reactive antibody repertoire over 5 years in a well-characterized donor from whom a large number of homosubtypic and heterosubtypic neutralizing monoclonal antibodies had been previously isolated by B cell analysis. The donor was infected with the CA09 strain in 2009 and immunized annually for the next five years with seasonal influenza vaccine which contained the CA09 strain. We find that the serological repertoire in this donor was highly static, with a modest number (24) of persistent antibody clonotypes, detected in serum for at least 4 out of 5 years, accounting on average for 72.6 ± 10.0% of the repertoire to the CA09 HA. These persistent antibodies: (i) displayed a higher degree of somatic hypermutation relative to antibodies that could be detected in the serum transiently (i.e. lasted less than 1 year in serum); (ii) comprised a significant fraction that also bound to HA from a phylogenetically distant H5N1 A/Vietnam/1203/2004 (VT04) strain, a hallmark of stem-binding antibodies due to the lack of homology between CA09 and VT04 in the head region of HA and (iii) perhaps most strikingly, but consistent with the wealth of heterosubtypic neutralizing antibodies that had previously been identified from this donor, some of the most abundant persistent antibody clonotypes, including the dominant clone that accounted on average for 18.6 ± 12.3% of the serum titer across 5 years, neutralized both the CA09 and VT04 influenza strains. Our analysis highlights the magnitude of ‘serological imprinting’ in the donor’s serum response to CA09, indicates that seasonal vaccination can further reinforce a stable serological memory and finally suggests that once elicited, antibodies cross-reactive between CA09 and VT04 with heterosubtypic neutralization activity, thus likely to bind to HA-stem, can persist for many years, which is a fundamental goal of universal influenza vaccines

Molecular understanding of the serum antibody repertoires after seasonal influenza vaccination among different age cohorts

Numerous influenza vaccination studies based on bulk serology have indicated that the antibody responses to the vaccine markedly decrease in the elderly. However, whether such decline results from the changes in the overall quantity or the quality of the circulating antibodies in serum remains unknown. Utilizing novel antibody repertoire profiling technologies, combining tandem mass spectrometry (LC-MS/MS) and high-throughput sequencing, we investigated the influenza-specific serological repertoires of 10 donors ranging from 26 to 70 years old vaccinated with Fluzone® 2013-2014 and/or 2014-2015. In particular, we determined the serum antibodies that are specific to the H1 or H3 component of the vaccine or cross-reactive between the two (H1+H3) and examined their relative quantitative distributions. Our data indicate that the proportion of H1+H3 antibodies significantly increases in the elderly and that the somatic hypermutation rates of the influenza-specific antibodies are higher in the elderly. These results suggest that the repeated exposure to the different virus subtypes could have led to the prolonged selection of H1+H3 antibodies targeting highly conserved epitopes. To evaluate the potency of the antibodies circulating in different age groups, we recombinantly expressed a number of representative monoclonal antibodies isolated from the donors in different age groups for further characterizations. Overall, our analysis suggests that the influenza-specific repertoire in the elderly may converge toward shared epitopes but the quality of the antibodies can be superior in terms of cross-reactivity. However, because the antibody repertoire “shrinks” as we age while targeting more conserved epitopes across different influenza subtypes, it is possible that the elderly is particularly susceptible to significantly altered strains. Collectively, profiling vaccine induced serological repertoires among different age cohorts can provide unprecedented insights regarding humoral immunity associated with age and a potential explanation for the vulnerability of the elderly

Proteomic and protein interaction network analysis of human T lymphocytes during cell-cycle entry

Proteomic analysis of T cells emerging from quiescence identifies dynamic network-level changes in key cellular processes. Disruption of two such processes, ribosome biogenesis and RNA splicing, reveals that the programs controlling cell growth and cell-cycle entry are separable

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following SARS-CoV-2 infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor-binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an N-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multi-donor class of “public” antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that “public” NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape

The Genomics of Disulfide Bonding and Protein Stabilization in Thermophiles

Thermophilic organisms flourish in varied high-temperature environmental niches that are deadly to other organisms. Recently, genomic evidence has implicated a critical role for disulfide bonds in the structural stabilization of intracellular proteins from certain of these organisms, contrary to the conventional view that structural disulfide bonds are exclusively extracellular. Here both computational and structural data are presented to explore the occurrence of disulfide bonds as a protein-stabilization method across many thermophilic prokaryotes. Based on computational studies, disulfide-bond richness is found to be widespread, with thermophiles containing the highest levels. Interestingly, only a distinct subset of thermophiles exhibit this property. A computational search for proteins matching this target phylogenetic profile singles out a specific protein, known as protein disulfide oxidoreductase, as a potential key player in thermophilic intracellular disulfide-bond formation. Finally, biochemical support in the form of a new crystal structure of a thermophilic protein with three disulfide bonds is presented together with a survey of known structures from the literature. Together, the results provide insight into biochemical specialization and the diversity of methods employed by organisms to stabilize their proteins in exotic environments. The findings also motivate continued efforts to sequence genomes from divergent organisms

Sera Antibody Repertoire Analyses Reveal Mechanisms of Broad and Pandemic Strain Neutralizing Responses after Human Norovirus Vaccination

Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire—pre- and post-vaccination—and select several anti-body clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen