Emma M. Beadle

We present a method for sequencing polyclonal IgG enriched from human plasma, employing a combination of de novo sequencing, proteomics, bioinformatics, protein separation, sequencing, and peptide separations. Our study analyzes a single patient’s IgG antibody response triggered by the Moderna Spikevax mRNA COVID-19 vaccine. From the sequencing data of the natural polyclonal response to vaccination, we generated 12 recombinant antibodies. Six derived recombinant antibodies, including four generated with de novo sequencing, exhibited similar or higher binding affinities than the original natural polyclonal antibody. Our neutralization tests revealed that the six antibodies possess neutralizing capabilities against the target antigen. This research provides insights into sequencing polyclonal IgG antibodies while highlighting the effectiveness and potential of our approach in generating recombinant antibodies with robust binding affinity and neutralization capabilities. Our proposed approach is an advancement in characterizing the IgG response by directly investigating the circulating pool of IgG without relying exclusively on the B-cell repertoire or population. This is crucial as the B-cell analysis may not accurately represent the circulating antibodies. Interestingly, a large proportion (80 to 90%) of the human antibody sequences generated against SARS-CoV-2 in the literature have been derived solely from B-cell analysis. Therefore, the ability to offer a different perspective is crucial in gaining a comprehensive understanding of the IgG response. Significance Statement: We investigate human IgG targeting the receptor binding domain using de novo proteomics. The peripheral B-cell repertoire may not adequately cover all the circulating IgG for human IgG sequencing. Our approach overcomes this limitation by using a de novo protein sequencing on top of standard proteomics. We obtained distinct de novo sequences, showcasing our method’s potential. The recombinant proteins we generate possess traits comparable to or surpassing the naturally occurring polyclonal antibodies (pAbs). This study highlights similarities and differences between IgG populations in blood and circulating B-cells, which is crucial for future biologics development.

Cells rapidly adapt to survive harsh environmental conditions through the potent upregulation of molecular chaperones or heat shock proteins (HSPs). The inducible members of the HSP70 family are the fastest and most transcriptionally induced chaperone upon stress. The HSP70 mRNA life cycle regulation in the cytoplasm is unique because it is translated during stress when general translation is repressed and rapidly degraded once conditions are optimal for growth. Contrary to the role of the HSP70 mRNA 5’ untranslated region in maximizing the synthesis of HSP70, we discovered that the coding sequence (CDS) represses its translation through the ribosome quality control (RQC) mechanism. The CDS of the most inducible HSP70 in Saccharomyces cerevisiae, SSA4 , is uniquely biased with low-frequency codons that promote ribosome stalling during heat stress. The stalled ribosomes are recognized by RQC components Asc1p and Hel2p and two ribosome proteins, Rps28A and Rps19B, that we identified as new RQC components. Surprisingly, RQC does not signal the degradation of the SSA4 mRNA by no-go-decay (NGD). Instead, Asc1p destabilizes the SSA4 mRNA during recovery from heat stress by a mechanism independent of its ribosome binding and SSA4 CDS codon optimality. Therefore, Asc1p operates two synergistic mechanisms that converge to regulate the life cycle of HSP70 mRNA during stress and recovery. Our research identifies Asc1p as a critical regulator of the stress response and RQC as the system tuning HSP70 synthesis.