In cancer patients, visual identification of sentinel lymph nodes (LNs) is achieved by the injection of dyes that bind avidly to endogenous albumin, targeting these compounds to LNs where they are efficiently filtered by resident phagocytes1,2. Here we translate this “albumin hitchhiking” approach to molecular vaccines, via the synthesis of amphiphiles (amph-vaccines) comprised of an antigen or adjuvant cargo linked to a lipophilic albumin-binding tail by a solubility-promoting polar polymer chain. Structurally-optimized CpG-DNA/peptide amph-vaccines exhibited dramatic increases in LN accumulation and decreased systemic dissemination relative to their parent compounds, leading to 30-fold increases in T-cell priming and enhanced anti-tumor efficacy while greatly reducing systemic toxicity. Amph-vaccines provide a simple, broadly-applicable strategy to simultaneously increase the potency and safety of subunit vaccines.

Summary Some cancers originate from a single mutation event in a single cell. Blood cancers known as myeloproliferative neoplasms (MPNs) are thought to originate when a driver mutation is acquired by a hematopoietic stem cell (HSC). However, when the mutation first occurs in individuals and how it affects the behavior of HSCs in their native context is not known. Here we quantified the effect of the JAK2 -V617F mutation on the self-renewal and differentiation dynamics of HSCs in treatment-naive individuals with MPNs and reconstructed lineage histories of individual HSCs using somatic mutation patterns. We found that JAK2- V617F mutations occurred in a single HSC several decades before MPN diagnosis—at age 9 ± 2 years in a 34-year-old individual and at age 19 ± 3 years in a 63-year-old individual—and found that mutant HSCs have a selective advantage in both individuals. These results highlight the potential of harnessing somatic mutations to reconstruct cancer lineages.

B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is the molecular target for many vaccines and antibody-based prophylactics aimed at bringing COVID-19 under control. Such a narrow molecular focus raises the specter of viral immune evasion as a potential failure mode for these biomedical interventions. With the emergence of new strains of SARS-CoV-2 with altered transmissibility and immune evasion potential, a critical question is this: how easily can the virus escape neutralizing antibodies (nAbs) targeting the spike RBD? To answer this question, we combined an analysis of the RBD structure-function with an evolutionary modeling framework. Our structure-function analysis revealed that epitopes for RBD-targeting nAbs overlap one another substantially and can be evaded by escape mutants with ACE2 affinities comparable to the wild type, that are observed in sequence surveillance data and infect cells in vitro. This suggests that the fitness cost of nAb-evading mutations is low. We then used evolutionary modeling to predict the frequency of immune escape before and after the widespread presence of nAbs due to vaccines, passive immunization or natural immunity. Our modeling suggests that SARS-CoV-2 mutants with one or two mildly deleterious mutations are expected to exist in high numbers due to neutral genetic variation, and consequently resistance to vaccines or other prophylactics that rely on one or two antibodies for protection can develop quickly -and repeatedly- under positive selection. Predicted resistance timelines are comparable to those of the decay kinetics of nAbs raised against vaccinal or natural antigens, raising a second potential mechanism for loss of immunity in the population. Strategies for viral elimination should therefore be diversified across molecular targets and therapeutic modalities.

The rapid emergence and expansion of novel SARS-CoV-2 variants threatens our ability to achieve herd immunity for COVID-19. These novel SARS-CoV-2 variants often harbor multiple point mutations, conferring one or more evolutionarily advantageous traits, such as increased transmissibility, immune evasion and longer infection duration. In a number of cases, variant emergence has been linked to long-term infections in individuals who were either immunocompromised or treated with convalescent plasma. In this paper, we used a stochastic evolutionary modeling framework to explore the emergence of fitter variants of SARS-CoV-2 during long-term infections. We found that increased viral load and infection duration favor emergence of such variants. While the overall probability of emergence and subsequent transmission from any given infection is low, on a population level these events occur fairly frequently. Targeting these low-probability stochastic events that lead to the establishment of novel advantageous viral variants might allow us to slow the rate at which they emerge in the patient population, and prevent them from spreading deterministically due to natural selection. Our work thus suggests practical ways to achieve control of long-term SARS-CoV-2 infections, which will be critical for slowing the rate of viral evolution.

As many prophylactics targeting SARS-CoV-2 are aimed at the spike protein receptor-binding domain (RBD), we examined the risk of immune evasion from previously published RBD-targeting neutralizing antibodies (nAbs). Epitopes for RBD-targeting nAbs overlap one another substantially and can give rise to escape mutants with ACE2 affinities comparable to wild type that still infect cells in vitro. Based on this demonstrated mutational tolerance of the RBD, we used evolutionary modeling to predict the frequency of immune escape before and after the widespread presence of nAbs raised by vaccines, administered as prophylactics, or produced through natural immunity. Our modeling suggests that SARS-CoV-2 mutants with one or two mildly deleterious mutations are expected to exist in high numbers due to neutral genetic variation, and likewise resistance to single or double antibody combinations will develop quickly under positive selection.One Sentence SummarySARS-CoV-2 will evolve quickly to evade widely deployed spike RBD-targeting monoclonal antibodies, requiring combinations with at least three antibodies to suppress viral immune evasion.

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Some cancers originate from a single mutation event in a single cell. For example, blood cancers known as myeloproliferative neoplasms (MPN) are thought to originate through the acquisition of a driver mutation (most commonly JAK2-V617F) in a hematopoietic stem cell (HSC). However, when the mutation first occurs in individual patients and how it impacts the behavior of HSCs in their native context is not known. Here we quantified the impact of the JAK2-V617F mutation on the proliferation dynamics of HSCs and the differentiation trajectories of their progenies in individual MPN patients. We reconstructed the lineage history of individual HSCs obtained from MPN patients using the patterns of spontaneous somatic mutations accrued in their genomes over time. Strikingly, we found that the JAK2-V617F mutation occurred in a single HSC several decades before MPN diagnosis - at age 9±2 years in a 34-year-old patient, and at age 19±3 years in a 63-year-old patient. For each patient, we inferred the number of mutated HSCs over time and computed their fitness. The population of JAK2-mutated HSCs grew exponentially by 63±15% and 44±13% every year in the two patients, respectively. To contrast the differentiation trajectories of the JAK2-mutated HSCs with those of healthy HSCs, we simultaneously measured the full transcriptome and somatic mutations in single hematopoietic stem and progenitor cells (HSPCs). We found that the fraction of JAK2-mutant HSPCs varied significantly across different myeloid cell types within the same patient. The erythroid progenitor cells were often entirely JAK2-mutant, even when the peripheral blood JAK2-V617F allele burden was low. The novel biological insights uncovered by this work have implications for the prevention and treatment of MPN, as well as the accurate assessment of disease burden in patients. The technology platforms and computational frameworks developed here are broadly applicable to other types of hematological malignancies and cancers.