Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning.

Understanding the specificities of human serum antibodies that broadly neutralize HIV can inform prevention and treatment strategies. Here, we describe a deep mutational scanning system that can measure the effects of combinations of mutations to HIV envelope (Env) on neutralization by antibodies and polyclonal serum. We first show that this system can accurately map how all functionally tolerated mutations to Env affect neutralization by monoclonal antibodies. We then comprehensively map Env mutations that affect neutralization by a set of human polyclonal sera that neutralize diverse strains of HIV and target the site engaging the host receptor CD4. The neutralizing activities of these sera target different epitopes, with most sera having specificities reminiscent of individual characterized monoclonal antibodies, but one serum targeting two epitopes within the CD4-binding site. Mapping the specificity of the neutralizing activity in polyclonal human serum will aid in assessing anti-HIV immune responses to inform prevention strategies.

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike.

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here, we describe a deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We apply this platform to produce libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ∼7,000 distinct amino acid mutations in the context of up to ∼135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor-binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ∼105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning.

Understanding the specificities of human serum antibodies that broadly neutralize HIV can inform prevention and treatment strategies. Here we describe a deep mutational scanning system that can measure the effects of combinations of mutations to HIV envelope (Env) on neutralization by antibodies and polyclonal serum. We first show that this system can accurately map how all functionally tolerated mutations to Env affect neutralization by monoclonal antibodies. We then comprehensively map Env mutations that affect neutralization by a set of human polyclonal sera known to target the CD4-binding site that neutralize diverse strains of HIV. The neutralizing activities of these sera target different epitopes, with most sera having specificities reminiscent of individual characterized monoclonal antibodies, but one sera targeting two epitopes within the CD4 binding site. Mapping the specificity of the neutralizing activity in polyclonal human serum will aid in assessing anti-HIV immune responses to inform prevention strategies.

Age-dependent heterogeneity in the antigenic effects of mutations to influenza hemagglutinin.

Human influenza virus evolves to escape neutralization by polyclonal antibodies. However, we have a limited understanding of how the antigenic effects of viral mutations vary across the human population, and how this heterogeneity affects virus evolution. Here we use deep mutational scanning to map how mutations to the hemagglutinin (HA) proteins of the A/Hong Kong/45/2019 (H3N2) and A/Perth/16/2009 (H3N2) strains affect neutralization by serum from individuals of a variety of ages. The effects of HA mutations on serum neutralization differ across age groups in ways that can be partially rationalized in terms of exposure histories. Mutations that fixed in influenza variants after 2020 cause the greatest escape from sera from younger individuals. Overall, these results demonstrate that influenza faces distinct antigenic selection regimes from different age groups, and suggest approaches to understand how this heterogeneous selection shapes viral evolution.

A biophysical model of viral escape from polyclonal antibodies.

A challenge in studying viral immune escape is determining how mutations combine to escape polyclonal antibodies, which can potentially target multiple distinct viral epitopes. Here we introduce a biophysical model of this process that partitions the total polyclonal antibody activity by epitope and then quantifies how each viral mutation affects the antibody activity against each epitope. We develop software that can use deep mutational scanning data to infer these properties for polyclonal antibody mixtures. We validate this software using a computationally simulated deep mutational scanning experiment and demonstrate that it enables the prediction of escape by arbitrary combinations of mutations. The software described in this paper is available at https://jbloomlab.github.io/polyclonal.

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike.

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here we describe a new deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We demonstrate this new platform by making libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ~7000 distinct amino-acid mutations in the context of up to ~135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ~10 5 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems.

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.

Delayed fractional dosing with RTS,S/AS01 improves humoral immunity to malaria via a balance of polyfunctional NANP6- and Pf16-specific antibodies.

BACKGROUND: Malaria remains a key cause of mortality in low-income countries. RTS,S/AS01 is currently the most advanced malaria vaccine, demonstrating ∼50% efficacy in controlled human malaria infection (CHMI) studies in malaria-naive adults and ∼30%-40% efficacy in field trials in African infants and children. However, a higher vaccine efficacy is desirable. METHODS: Modification of the vaccine regimen in a CHMI trial in malaria-naive individuals resulted in significant increase in protection. While three equal monthly RTS,S/AS01 doses (RRR) were used originally, the administration of a delayed third dose with 20% of the original antigen dose (RRr) resulted in ∼87% protection, linked to enhanced antibody affinity maturation. Here, we sought to identify a novel molecular basis for this higher protective efficacy using Systems Serology. FINDINGS: We demonstrate that the delayed fractional dose maintains monocyte phagocytosis and NK activation mediated by NANP6-specific antibodies, key correlates of protection for the RRR regimen. However, it is also marked by a higher breadth of C-term Fc effector functions, including enhanced phagocytosis. The RRr regimen breaches immunodominance of the humoral immune response, inducing a balanced response across the C-terminal (Pf16) and NANP region of CSP, both of which were linked to protection. CONCLUSIONS: Collectively, these data point to an unexpectedly concordant evolution in Fab avidity and expanded C-term Fc effector functions, providing novel insights into the basis for higher protection conferred by the delayed fractional dose in malaria-naive individuals. FUNDING: This research was supported by PATH's Malaria Vaccine Initiative and the MGH Research Scholars program.