HIV-1 neutralizing antibodies in SHIV-infected macaques recapitulate structurally divergent modes of human V2 apex recognition with a single D gene.

Broadly neutralizing antibodies targeting the V2 apex of the HIV-1 envelope trimer are among the most common specificities elicited in HIV-1-infected humans and simian-human immunodeficiency virus (SHIV)-infected macaques. To gain insight into the prevalent induction of these antibodies, we isolated and characterized 11 V2 apex-directed neutralizing antibody lineages from SHIV-infected rhesus macaques. Remarkably, all SHIV-induced V2 apex lineages were derived from reading frame two of the rhesus DH3-15*01 gene. Cryo-EM structures of envelope trimers in complex with antibodies from nine rhesus lineages revealed modes of recognition that mimicked three canonical human V2 apex-recognition modes. Notably, amino acids encoded by DH3-15*01 played divergent structural roles, inserting into a hole at the trimer apex, H-bonding to an exposed strand, or forming part of a loop scaffold. Overall, we identify a DH3-15*01-signature for rhesus V2 apex broadly neutralizing antibodies and show that highly selected genetic elements can play multiple roles in antigen recognition. Highlights: Isolated 11 V2 apex-targeted HIV-neutralizing lineages from 10 SHIV-infected Indian-origin rhesus macaquesCryo-EM structures of Fab-Env complexes for nine rhesus lineages reveal modes of recognition that mimic three modes of human V2 apex antibody recognitionAll SHIV-elicited V2 apex lineages, including two others previously published, derive from the same DH3-15*01 gene utilizing reading frame twoThe DH3-15*01 gene in reading frame two provides a necessary, but not sufficient, signature for V2 apex-directed broadly neutralizing antibodiesStructural roles played by DH3-15*01-encoded amino acids differed substantially in different lineages, even for those with the same recognition modePropose that the anionic, aromatic, and extended character of DH3-15*01 in reading frame two provides a selective advantage for V2 apex recognition compared to B cells derived from other D genes in the naïve rhesus repertoireDemonstrate that highly selected genetic elements can play multiple roles in antigen recognition, providing a structural means to enhance recognition diversity.

Format-tuning of in vivo-launched bispecific T cell engager enhances efficacy against renal cell carcinoma.

BACKGROUND: Advanced clear cell renal cell carcinoma (ccRCC) is a prevalent kidney cancer for which long-term survival rates are abysmal, though immunotherapies are showing potential. Not yet clinically vetted are bispecific T cell engagers (BTEs) that activate T cell-mediated cancer killing through intercellular synapsing. Multiple BTE formats exist, however, with limited cross-characterizations to help optimize new drug design. Here, we developed BTEs to treat ccRCC by targeting carbonic anhydrase 9 (CA9) while characterizing the persistent BTE (PBTE) format and comparing it to a new format, the persistent multivalent T cell engager (PMTE). These antibody therapies against ccRCC are developed as both recombinant and synthetic DNA (synDNA) medicines. METHODS: Antibody formatting effects on binding kinetics were assessed by flow cytometry and intercellular synaptic strength assays while potency was tested using T-cell activation and cytotoxicity assays. Mouse models were used to study antibody plasma and tumor pharmacokinetics, as well as antitumor efficacy as both recombinant and synDNA medicines. Specifically, three models using ccRCC cell line xenografts and human donor T cells in immunodeficient mice were used to support this study. RESULTS: Compared with a first-generation BTE, we show that the PBTE reduced avidity, intercellular synaptic strength, cytotoxic potency by as much as 33-fold, and ultimately efficacy against ccRCC tumors in vivo. However, compared with the PBTE, we demonstrate that the PMTE improved cell avidity, restored intercellular synapses, augmented cytotoxic potency by 40-fold, improved tumor distribution pharmacokinetics by 2-fold, and recovered synDNA efficacy in mouse tumor models by 20-fold. All the while, the PMTE displayed a desirable half-life of 4 days in mice compared with the conventional BTE's 2 hours. CONCLUSIONS: With impressive efficacy, the CA9-targeted PMTE is a promising new therapy for advanced ccRCC, which can be effectively delivered through synDNA. The highly potent PMTE format itself is a promising new tool for future applications in the multispecific antibody space.

Growing Glycans in Rosetta: Accurate de novo glycan modeling, density fitting, and rational sequon design.

Carbohydrates and glycoproteins modulate key biological functions. However, experimental structure determination of sugar polymers is notoriously difficult. Computational approaches can aid in carbohydrate structure prediction, structure determination, and design. In this work, we developed a glycan-modeling algorithm, GlycanTreeModeler, that computationally builds glycans layer-by-layer, using adaptive kernel density estimates (KDE) of common glycan conformations derived from data in the Protein Data Bank (PDB) and from quantum mechanics (QM) calculations. GlycanTreeModeler was benchmarked on a test set of glycan structures of varying lengths, or "trees". Structures predicted by GlycanTreeModeler agreed with native structures at high accuracy for both de novo modeling and experimental density-guided building. We employed these tools to design de novo glycan trees into a protein nanoparticle vaccine to shield regions of the scaffold from antibody recognition, and experimentally verified shielding. This work will inform glycoprotein model prediction, glycan masking, and further aid computational methods in experimental structure determination and refinement.

Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Involvement of ArlI, ArlJ, and CirA in archaeal type IV pilin-mediated motility regulation.

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established; however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that the deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used ethyl methanesulfonate mutagenesis and a motility assay to identify motile suppressors of the ∆pilA[1-6] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. In trans expression of arlI and arlJ mutant constructs in the respective multi-deletion strains ∆pilA[1-6]∆arlI and ∆pilA[1-6]∆arlJ confirmed their role in suppressing the ∆pilA[1-6] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA, a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA expression in trans in wild-type cells led to decreased motility. Moreover, quantitative real-time PCR analysis revealed that in wild-type cells, higher expression levels of arlI, arlJ, and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells. Conversely, ∆cirA cells, which form rods during both early- and mid-log phases, exhibited similar expression levels of arl genes in both growth phases. Our findings contribute to a deeper understanding of the mechanisms governing archaeal motility, highlighting the involvement of ArlI, ArlJ, and CirA in pilin-mediated motility regulation.IMPORTANCEArchaea are close relatives of eukaryotes and play crucial ecological roles. Certain behaviors, such as swimming motility, are thought to be important for archaeal environmental adaptation. Archaella, the archaeal motility appendages, are evolutionarily distinct from bacterial flagella, and the regulatory mechanisms driving archaeal motility are largely unknown. Previous research has linked the loss of type IV pili subunits to archaeal motility suppression. This study reveals three Haloferax volcanii proteins involved in pilin-mediated motility regulation, offering a deeper understanding of motility regulation in this understudied domain while also paving the way for uncovering novel mechanisms that govern archaeal motility. Understanding archaeal cellular processes will help elucidate the ecological roles of archaea as well as the evolution of these processes across domains.

Heterologous prime-boost vaccination drives early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

Involvement of ArlI, ArlJ, and CirA in Archaeal Type-IV Pilin-Mediated Motility Regulation.

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established, however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used EMS mutagenesis and a motility assay to identify motile suppressors of the ΔpilA[1-6] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. Overexpression of these arlI and arlJ mutant constructs in the respective multi-deletion strains ΔpilA[1-6]ΔarlI and ΔpilA[1-6]ΔarlJ confirmed their role in suppressing the ΔpilA[1-6] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA, a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA overexpression in wild-type cells led to decreased motility. Moreover, qRT-PCR analysis revealed that in wild-type cells, higher expression levels of arlI, arlJ, and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells. Conversely, ΔcirA cells, which form rods during both early and mid-log phases, exhibited similar expression levels of arl genes in both growth phases. Our findings contribute to a deeper understanding of the mechanisms governing archaeal motility, highlighting the involvement of ArlI, ArlJ, and CirA in pilin-mediated motility regulation.

In vivo delivery of engineered synthetic DNA-encoded SARS-CoV-2 monoclonal antibodies for pre-exposure prophylaxis in non-human primates.

COVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.

Delineation of DNA and mRNA COVID-19 vaccine-induced immune responses in preclinical animal models.

Nucleic acid vaccines are designed based on genetic sequences (DNA or mRNA) of a target antigen to be expressed in vivo to drive a host immune response. In response to the COVID-19 pandemic, mRNA and DNA vaccines based on the SARS-CoV-2 Spike antigen were developed. Surprisingly, head-to-head characterizations of the immune responses elicited by each vaccine type has not been performed to date. Here, we have employed a range of preclinical animal models including the hamster, guinea pig, rabbit, and mouse to compare and delineate the immune response raised by DNA, administered intradermally (ID) with electroporation (EP) and mRNA vaccines (BNT162b2 or mRNA-1273), administered intramuscularly (IM), expressing the SARS-CoV-2 WT spike antigen. The results revealed clear differences in the quality and magnitude of the immune response between the two vaccine platforms. The DNA vaccine immune response was characterized by strong T cell responses, while the mRNA vaccine elicited robust humoral responses. The results may assist in guiding the disease target each vaccine type may be best matched against and suggest mechanisms to further enhance the breadth of each platform's immune response.

Engineered antibody cytokine chimera synergizes with DNA-launched nanoparticle vaccines to potentiate melanoma suppression in vivo.

Cancer immunotherapy has demonstrated great promise with several checkpoint inhibitors being approved as the first-line therapy for some types of cancer, and new engineered cytokines such as Neo2/15 now being evaluated in many studies. In this work, we designed antibody-cytokine chimera (ACC) scaffolding cytokine mimetics on a full-length tumor-specific antibody. We characterized the pharmacokinetic (PK) and pharmacodynamic (PD) properties of first-generation ACC TA99-Neo2/15, which synergized with DLnano-vaccines to suppress in vivo melanoma proliferation and induced significant systemic cytokine activation. A novel second-generation ACC TA99-HL2-KOA1, with retained IL-2Rß/γ binding and attenuated but preserved IL-2Rα binding, induced lower systemic cytokine activation with non-inferior protection in murine tumor studies. Transcriptomic analyses demonstrated an upregulation of Type I interferon responsive genes, particularly ISG15, in dendritic cells, macrophages and monocytes following TA99-HL2-KOA1 treatment. Characterization of additional ACCs in combination with cancer vaccines will likely be an important area of research for treating melanoma and other types of cancer.

Gene-encoded nanoparticle vaccine platforms for in vivo assembly of multimeric antigen to promote adaptive immunity.

Nanoparticle vaccines are a diverse category of vaccines for the prophylaxis or treatment of various diseases. Several strategies have been employed for their optimization, especially to enhance vaccine immunogenicity and generate potent B-cell responses. Two major modalities utilized for particulate antigen vaccines include using nanoscale structures for antigen delivery and nanoparticles that are themselves vaccines due to antigen display or scaffolding-the latter of which we will define as "nanovaccines." Multimeric antigen display has a variety of immunological benefits compared to monomeric vaccines mediated through potentiating antigen-presenting cell presentation and enhancing antigen-specific B-cell responses through B-cell activation. The majority of nanovaccine assembly is done in vitro using cell lines. However, in vivo assembly of scaffolded vaccines potentiated using nucleic acids or viral vectors is a burgeoning modality of nanovaccine delivery. Several advantages to in vivo assembly exist, including lower costs of production, fewer production barriers, as well as more rapid development of novel vaccine candidates for emerging diseases such as SARS-CoV-2. This review will characterize the methods for de novo assembly of nanovaccines in the host using methods of gene delivery including nucleic acid and viral vectored vaccines. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies.

A mAb against surface-expressed FSHR engineered to engage adaptive immunity for ovarian cancer immunotherapy.

Despite advances in ovarian cancer (OC) therapy, recurrent OC remains a poor-prognosis disease. Because of the close interaction between OC cells and the tumor microenvironment (TME), it is important to develop strategies that target tumor cells and engage components of the TME. A major obstacle in the development of OC therapies is the identification of targets with expression limited to tumor surface to avoid off-target interactions. The follicle-stimulating hormone receptor (FSHR) has selective expression on ovarian granulosa cells and is expressed on 50%-70% of serous OCs. We generated mAbs targeting the external domain of FSHR using in vivo-expressed FSHR vector. By high-throughput flow analysis, we identified multiple clones and downselected D2AP11, a potent FSHR surface-targeted mAb. D2AP11 identifies important OC cell lines derived from tumors with different mutations, including BRCA1/2, and lines resistant to a wide range of therapies. We used D2AP11 to develop a bispecific T cell engager. In vitro addition of PBMCs and T cells to D2AP11-TCE induced specific and potent killing of different genetic and immune escape OC lines, with EC50s in the ng/ml range, and attenuated tumor burden in OC-challenged mouse models. These studies demonstrate the potential utility of biologics targeting FSHR for OC and perhaps other FSHR-positive cancers.

Mucosal chemokine adjuvant enhances synDNA vaccine-mediated responses to SARS-CoV-2 and provides heterologous protection in vivo.

The global coronavirus disease 2019 (COVID-19) pandemic has claimed more than 5 million lives. Emerging variants of concern (VOCs) continually challenge viral control. Directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces may enhance vaccine efficacy. Here we investigate the immunogenicity and protective efficacy of optimized synthetic DNA plasmids encoding wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (pS) co-formulated with the plasmid-encoded mucosal chemokine cutaneous T cell-attracting chemokine (pCTACK; CCL27). pCTACK-co-immunized animals exhibit increased spike-specific antibodies at the mucosal surface and increased frequencies of interferon gamma (IFNγ)+ CD8+ T cells in the respiratory mucosa. pCTACK co-immunization confers 100% protection from heterologous Delta VOC challenge. This study shows that mucosal chemokine adjuvants can direct vaccine-induced responses to specific immunological sites and have significant effects on heterologous challenge. Further study of this unique chemokine-adjuvanted vaccine approach in the context of SARS-CoV-2 vaccines is likely important.

Induction of tier-2 neutralizing antibodies in mice with a DNA-encoded HIV envelope native like trimer.

HIV Envelope (Env) is the main vaccine target for induction of neutralizing antibodies. Stabilizing Env into native-like trimer (NLT) conformations is required for recombinant protein immunogens to induce autologous neutralizing antibodies(nAbs) against difficult to neutralize HIV strains (tier-2) in rabbits and non-human primates. Immunizations of mice with NLTs have generally failed to induce tier-2 nAbs. Here, we show that DNA-encoded NLTs fold properly in vivo and induce autologous tier-2 nAbs in mice. DNA-encoded NLTs also uniquely induce both CD4 + and CD8 + T-cell responses as compared to corresponding protein immunizations. Murine neutralizing antibodies are identified with an advanced sequencing technology. The structure of an Env-Ab (C05) complex, as determined by cryo-EM, identifies a previously undescribed neutralizing Env C3/V5 epitope. Beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.

Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drives rapid and potent immunogenicity capable of single-dose protection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may target epitopes that reduce durability or increase the potential for escape from vaccine-induced immunity. Using synthetic vaccinology, we have developed rationally immune-focused SARS-CoV-2 Spike-based vaccines. Glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and in vitro screening, we have incorporated glycans into the receptor-binding domain (RBD) and assessed antigenic profiles. We demonstrate that glycan-coated RBD immunogens elicit stronger neutralizing antibodies and have engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicits potent neutralizing antibodies in guinea pigs, hamsters, and multiple mouse models, including human ACE2 and human antibody repertoire transgenics. RBD nanoparticles induce high levels of cross-neutralizing antibodies against variants of concern with durable titers beyond 6 months. Single, low-dose immunization protects against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of potent and durable coronavirus vaccines.

Intradermal-delivered DNA vaccine induces durable immunity mediating a reduction in viral load in a rhesus macaque SARS-CoV-2 challenge model.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health and social and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (i.d.)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system, which are likely important for providing durable protection against COVID-19 disease.

Identification of Novel Neutralizing Monoclonal Antibodies against SARS-CoV-2 Spike Glycoprotein.

Coronavirus disease 2019 (COVID-19) is caused by the newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the highly contagious nature of SARS-CoV-2, it has infected more than 137 million individuals and caused more than 2.9 million deaths globally as of April 13, 2021. There is an urgent need to develop effective novel therapeutic strategies to treat or prevent this infection. Toward this goal, we focused on the development of monoclonal antibodies (mAbs) directed against the SARS-CoV-2 spike glycoprotein (SARS-CoV-2 Spike) present on the surface of virus particles as well as virus-infected cells. We isolated anti-SARS-CoV-2 Spike mAbs from animals immunized with a DNA vaccine. We then selected a highly potent set of mAbs against SARS-CoV-2 Spike protein and evaluated each candidate for their expression, target binding affinity, and neutralization potential using complementary ACE2-blocking and pseudovirus neutralization assays. We identified a total of 10 antibodies, which specifically and strongly bound to SARS-CoV-2 Spike, blocked the receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) interaction, and neutralized SARS-CoV-2. Furthermore, the glycomic profile of the antibodies suggested that they have high Fc-mediated effector functions. These antibodies should be further investigated for elucidating the neutralizing epitopes on Spike for the design of next-generation vaccines and for their potential in diagnostic as well as therapeutic utilities against SARS-CoV-2.

PyRosetta Jupyter Notebooks Teach Biomolecular Structure Prediction and Design.

Biomolecular structure drives function, and computational capabilities have progressed such that the prediction and computational design of biomolecular structures is increasingly feasible. Because computational biophysics attracts students from many different backgrounds and with different levels of resources, teaching the subject can be challenging. One strategy to teach diverse learners is with interactive multimedia material that promotes self-paced, active learning. We have created a hands-on education strategy with a set of sixteen modules that teach topics in biomolecular structure and design, from fundamentals of conformational sampling and energy evaluation to applications like protein docking, antibody design, and RNA structure prediction. Our modules are based on PyRosetta, a Python library that encapsulates all computational modules and methods in the Rosetta software package. The workshop-style modules are implemented as Jupyter Notebooks that can be executed in the Google Colaboratory, allowing learners access with just a web browser. The digital format of Jupyter Notebooks allows us to embed images, molecular visualization movies, and interactive coding exercises. This multimodal approach may better reach students from different disciplines and experience levels as well as attract more researchers from smaller labs and cognate backgrounds to leverage PyRosetta in their science and engineering research. All materials are freely available at https://github.com/RosettaCommons/PyRosetta.notebooks.

A DNA-Launched Nanoparticle Vaccine Elicits CD8+ T-cell Immunity to Promote In Vivo Tumor Control.

Cytolytic T cells (CTL) play a pivotal role in surveillance against tumors. Induction of CTL responses by vaccination may be challenging, as it requires direct transduction of target cells or special adjuvants to promote cross-presentation. Here, we observed induction of robust CTL responses through electroporation-facilitated, DNA-launched nanoparticle vaccination (DLnano-vaccines). Electroporation was observed to mediate transient tissue apoptosis and macrophage infiltration, which were deemed essential to the induction of CTLs by DLnano-vaccines through a systemic macrophage depletion study. Bolus delivery of protein nano-vaccines followed by electroporation, however, failed to induce CTLs, suggesting direct in vivo production of nano-vaccines may be required. Following these observations, new DLnano-vaccines scaffolding immunodominant melanoma Gp100 and Trp2 epitopes were designed and shown to induce more potent and consistent epitope-specific CTL responses than the corresponding DNA monomeric vaccines or CpG-adjuvanted peptide vaccines. DNA, but not recombinant protein, nano-vaccinations induced CTL responses to these epitopes and suppressed melanoma tumor growth in mouse models in a CD8+ T-cell-dependent fashion. Further studies to explore the use of DLnano-vaccines against other cancer targets and the biology with which they induce CTLs are important.