Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques.

The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.

Vaccine induction of heterologous HIV-1-neutralizing antibody B cell lineages in humans.

A critical roadblock to HIV vaccine development is the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans. In people living with HIV-1, bnAbs take years to develop. The HVTN 133 clinical trial studied a peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs (NCT03934541). Here, we report MPER peptide-liposome induction of polyclonal HIV-1 B cell lineages of mature bnAbs and their precursors, the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains with lineage initiation after the second immunization. Neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids. This study demonstrates proof of concept for rapid vaccine induction of human B cell lineages with heterologous neutralizing activity and selection of antibody improbable mutations and outlines a path for successful HIV-1 vaccine development.

A new class of antibodies that overcomes a steric barrier to cross-group neutralization of influenza viruses.

Antibody titers that inhibit the influenza virus hemagglutinin (HA) from engaging its receptor are the accepted correlate of protection from infection. Many potent antibodies with broad, intra-subtype specificity bind HA at the receptor binding site (RBS). One barrier to broad H1-H3 cross-subtype neutralization is an insertion (133a) between positions 133 and 134 on the rim of the H1 HA RBS. We describe here a class of antibodies that overcomes this barrier. These genetically unrestricted antibodies are abundant in the human B cell memory compartment. Analysis of the affinities of selected members of this class for historical H1 and H3 isolates suggest that they were elicited by H3 exposure and broadened or diverted by later exposure(s) to H1 HA. RBS mutations in egg-adapted vaccine strains cause the new H1 specificity of these antibodies to depend on the egg adaptation. The results suggest that suitable immunogens might elicit 133a-independent, H1-H3 cross neutralization by RBS-directed antibodies.

Dorsal lip maturation and initial archenteron extension depend on Wnt11 family ligands.

Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes during the tailbud period. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues by imaging intact fixed embryos stained for anillin and microtubules. We found that the initial extension of the archenteron is correlated with blastopore lip maturation, and archenteron extension is dramatically disrupted by decreased Wnt11 family signaling. We were aided in our interpretation of the immunofluorescence by the novel, membrane proximal location of the cleavage furrow protein anillin in the epithelium of the blastopore lip and early archenteron.

Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg.

Fertilization releases the meiotic arrest and initiates the events that prepare the egg for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phosphoregulation is still unknown. We examined absolute protein and phosphosite dynamics of the fertilization response by mass spectrometry-based proteomics in electroactivated eggs. To do this, we developed an approach for calculating the stoichiometry of phosphosites from multiplexed proteomics that is compatible with dynamic, stable, and multisite phosphorylation. Overall, the data suggest that degradation is limited to a few low-abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium-activated kinases. We identify putative degradation targets and components of the slow block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.

Mapping the environmental fitness landscape of a synthetic gene circuit.

Gene expression actualizes the organismal phenotypes encoded within the genome in an environment-dependent manner. Among all encoded phenotypes, cell population growth rate (fitness) is perhaps the most important, since it determines how well-adapted a genotype is in various environments. Traditional biological measurement techniques have revealed the connection between the environment and fitness based on the gene expression mean. Yet, recently it became clear that cells with identical genomes exposed to the same environment can differ dramatically from the population average in their gene expression and division rate (individual fitness). For cell populations with bimodal gene expression, this difference is particularly pronounced, and may involve stochastic transitions between two cellular states that form distinct sub-populations. Currently it remains unclear how a cell population's growth rate and its subpopulation fractions emerge from the molecular-level kinetics of gene networks and the division rates of single cells. To address this question we developed and quantitatively characterized an inducible, bistable synthetic gene circuit controlling the expression of a bifunctional antibiotic resistance gene in Saccharomyces cerevisiae. Following fitness and fluorescence measurements in two distinct environments (inducer alone and antibiotic alone), we applied a computational approach to predict cell population fitness and subpopulation fractions in the combination of these environments based on stochastic cellular movement in gene expression space and fitness space. We found that knowing the fitness and nongenetic (cellular) memory associated with specific gene expression states were necessary for predicting the overall fitness of cell populations in combined environments. We validated these predictions experimentally and identified environmental conditions that defined a "sweet spot" of drug resistance. These findings may provide a roadmap for connecting the molecular-level kinetics of gene networks to cell population fitness in well-defined environments, and may have important implications for phenotypic variability of drug resistance in natural settings.

Computing the inducibility of B cell lineages under a context-dependent model of affinity maturation: Applications to sequential vaccine design.

A key challenge in B cell lineage-based vaccine design is understanding the inducibility of target neutralizing antibodies. We approach this problem through the use of detailed stochastic modeling of the somatic hypermutation process that occurs during affinity maturation. Under such a model, sequence mutation rates are context-dependent, rendering standard probability calculations for sequence evolution intractable. We develop an algorithmic approach to rapid, accurate approximation of key marginal sequence likelihoods required to inform modern sequential vaccine design strategies. These calculated probabilities are used to define an inducibility index for selecting among potential targets for immunogen design. We apply this approach to the problem of choosing targets for the design of boosting immunogens aimed at elicitation of the HIV broadly-neutralizing antibody DH270min11.

Engineered immunogens to elicit antibodies against conserved coronavirus epitopes.

Immune responses to SARS-CoV-2 primarily target the receptor binding domain of the spike protein, which continually mutates to escape acquired immunity. Other regions in the spike S2 subunit, such as the stem helix and the segment encompassing residues 815-823 adjacent to the fusion peptide, are highly conserved across sarbecoviruses and are recognized by broadly reactive antibodies, providing hope that vaccines targeting these epitopes could offer protection against both current and emergent viruses. Here we employ computational modeling to design scaffolded immunogens that display the spike 815-823 peptide and the stem helix epitopes without the distracting and immunodominant receptor binding domain. These engineered proteins bind with high affinity and specificity to the mature and germline versions of previously identified broadly protective human antibodies. Epitope scaffolds interact with both sera and isolated monoclonal antibodies with broadly reactivity from individuals with pre-existing SARS-CoV-2 immunity. When used as immunogens, epitope scaffolds elicit sera with broad betacoronavirus reactivity and protect as "boosts" against live virus challenge in mice, illustrating their potential as components of a future pancoronavirus vaccine.

Evolution of a single gene highlights the complexity underlying molecular descriptions of fitness.

Evolution by natural selection is the driving force behind the endless variation we see in nature, yet our understanding of how changes at the molecular level give rise to different phenotypes and altered fitness at the population level remains inadequate. The reproductive fitness of an organism is the most basic metric that describes the chance that an organism will succeed or fail in its environment and it depends upon a complex network of inter- and intramolecular interactions. A deeper understanding of the quantitative relationships relating molecular evolution to adaptation, and consequently fitness, can guide our understanding of important issues in biomedicine such as drug resistance and the engineering of new organisms with applications to biotechnology. We have developed the "weak link" approach to determine how changes in molecular structure and function can relate to fitness and evolutionary outcomes. By replacing adenylate kinase (AK), an essential gene, in a thermophile with a homologous AK from a mesophile we have created a maladapted weak link that produces a temperature-sensitive phenotype. The recombinant strain adapts to nonpermissive temperatures through point mutations to the weak link that increase both stability and activity of the enzyme AK at higher temperatures. Here, we propose a fitness function relating enzyme activity to growth rate and use it to create a dynamic model of a population of bacterial cells. Using metabolic control analysis we show that the growth rate exhibits thresholdlike behavior, saturating at high enzyme activity as other reactions in the energy metabolism pathway become rate limiting. The dynamic model accurately recapitulates observed evolutionary outcomes. These findings suggest that in vitro enzyme kinetic data, in combination with metabolic network analysis, can be used to create fitness functions and dynamic models of evolution within simple metabolic systems.

Optimal distribution of medical backpacks and health surveillance assistants in Malawi.

Despite recent progress, Malawi continues to perform poorly on key health indicators such as child mortality and life expectancy. These problems are exacerbated by a severe lack of access to health care. Health Surveillance Assistants (HSAs) help bridge this gap by providing community-level access to basic health care services. However, the success of these HSAs is limited by a lack of supplies and long distances between HSAs and patients. To address this issue, we used large-scale weighted p-median and capacitated facility location problems to create a scalable, three-tiered plan for optimal allocation of HSAs, HSA designated medical backpacks, and backpack resupply centers. Our analysis uses real data on the location and characteristics of hospitals, health centers, and the general population. In addition to offering specific recommendations for HSA, backpack, and resupply center locations, it provides general insights into the scope of the proposed HSA backpack program scale-up. In particular, it demonstrates the importance of local health centers to the resupply network. The proposed assignments are robust to changes in the underlying population structure, and could significantly improve access to medical supplies for both HSAs and patients.