Simultaneous enhancement of multiple functional properties using evolution-informed protein design.

A major challenge in protein design is to augment existing functional proteins with multiple property enhancements. Altering several properties likely necessitates numerous primary sequence changes, and novel methods are needed to accurately predict combinations of mutations that maintain or enhance function. Models of sequence co-variation (e.g., EVcouplings), which leverage extensive information about various protein properties and activities from homologous protein sequences, have proven effective for many applications including structure determination and mutation effect prediction. We apply EVcouplings to computationally design variants of the model protein TEM-1 ß-lactamase. Nearly all the 14 experimentally characterized designs were functional, including one with 84 mutations from the nearest natural homolog. The designs also had large increases in thermostability, increased activity on multiple substrates, and nearly identical structure to the wild type enzyme. This study highlights the efficacy of evolutionary models in guiding large sequence alterations to generate functional diversity for protein design applications.

Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline.

As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.

Simultaneous enhancement of multiple functional properties using evolution-informed protein design.

Designing optimized proteins is important for a range of practical applications. Protein design is a rapidly developing field that would benefit from approaches that enable many changes in the amino acid primary sequence, rather than a small number of mutations, while maintaining structure and enhancing function. Homologous protein sequences contain extensive information about various protein properties and activities that have emerged over billions of years of evolution. Evolutionary models of sequence co-variation, derived from a set of homologous sequences, have proven effective in a range of applications including structure determination and mutation effect prediction. In this work we apply one of these models (EVcouplings) to computationally design highly divergent variants of the model protein TEM-1 ß-lactamase, and characterize these designs experimentally using multiple biochemical and biophysical assays. Nearly all designed variants were functional, including one with 84 mutations from the nearest natural homolog. Surprisingly, all functional designs had large increases in thermostability and most had a broadening of available substrates. These property enhancements occurred while maintaining a nearly identical structure to the wild type enzyme. Collectively, this work demonstrates that evolutionary models of sequence co-variation (1) are able to capture complex epistatic interactions that successfully guide large sequence departures from natural contexts, and (2) can be applied to generate functional diversity useful for many applications in protein design.

Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2.

Influenza A virus's (IAV's) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV's genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem-loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates. A PSL2-targeting locked nucleic acid (LNA), administered 3 d after, or 14 d before, a lethal IAV inoculum provided 100% survival in mice, led to the development of strong immunity to rechallenge with a tenfold lethal inoculum, evaded attempts to select for resistance and retained full potency against neuraminidase inhibitor-resistant virus. Use of an analogous approach to target SARS-CoV-2, prophylactic administration of LNAs specific for highly conserved RNA structures in the viral genome, protected hamsters from efficient transmission of the SARS-CoV-2 USA_WA1/2020 variant. These findings highlight the potential applicability of this approach to any virus of interest via a process we term 'programmable antivirals', with implications for antiviral prophylaxis and post-exposure therapy.

Signatures of immune dysfunction in HIV and HCV infection share features with chronic inflammation in aging and persist after viral reduction or elimination.

Chronic inflammation is thought to be a major cause of morbidity and mortality in aging, but whether similar mechanisms underlie dysfunction in infection-associated chronic inflammation is unclear. Here, we profiled the immune proteome, and cellular composition and signaling states in a cohort of aging individuals versus a set of HIV patients on long-term antiretroviral therapy therapy or hepatitis C virus (HCV) patients before and after sofosbuvir treatment. We found shared alterations in aging-associated and infection-associated chronic inflammation including T cell memory inflation, up-regulation of intracellular signaling pathways of inflammation, and diminished sensitivity to cytokines in lymphocytes and myeloid cells. In the HIV cohort, these dysregulations were evident despite viral suppression for over 10 y. Viral clearance in the HCV cohort partially restored cellular sensitivity to interferon-α, but many immune system alterations persisted for at least 1 y posttreatment. Our findings indicate that in the HIV and HCV cohorts, a broad remodeling and degradation of the immune system can persist for a year or more, even after the removal or drastic reduction of the pathogen load and that this shares some features of chronic inflammation in aging.

Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition.

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

A functional subset of CD8+ T cells during chronic exhaustion is defined by SIRPα expression.

Prolonged exposure of CD8+ T cells to antigenic stimulation, as in chronic viral infections, leads to a state of diminished function termed exhaustion. We now demonstrate that even during exhaustion there is a subset of functional CD8+ T cells defined by surface expression of SIRPα, a protein not previously reported on lymphocytes. On SIRPα+ CD8+ T cells, expression of co-inhibitory receptors is counterbalanced by expression of co-stimulatory receptors and it is only SIRPα+ cells that actively proliferate, transcribe IFNγ and show cytolytic activity. Furthermore, target cells that express the ligand for SIRPα, CD47, are more susceptible to CD8+ T cell-killing in vivo. SIRPα+ CD8+ T cells are evident in mice infected with Friend retrovirus, LCMV Clone 13, and in patients with chronic HCV infections. Furthermore, therapeutic blockade of PD-L1 to reinvigorate CD8+ T cells during chronic infection expands the cytotoxic subset of SIRPα+ CD8+ T cells.

The balance of type 1 and type 2 immune responses in the contexts of hepatitis B infection and hepatitis D infection.

BACKGROUND AND AIM: Hepatitis delta virus (HDV) infection is the most rapidly progressive chronic viral hepatitis. Little is understood about the immune responses to HDV. This study aims to characterize the systemic immune environments of hepatitis B virus (HBV) and HDV patients at various disease stages. METHODS: A total of 129 subjects were evaluated: 53 HBV, 43 HDV, and 33 healthy controls. HBV and HDV subjects were categorized by aspartate aminotransferase to platelet ratio index (APRI) into mild (APRI < 0.5), moderate, and severe (APRI > 1.0). Serum cytokines and immune markers were assessed at a single treatment-naïve time-point. RESULTS: Type 1 cytokines are elevated in both HBV and HDV. Both groups show higher tumor necrosis factor-α (TNF-α), interleukin (IL)-12p40, and C-X-C motif chemokine ligand 9 when compared with controls (all P < 0.05). However, only HBV group displayed elevated γ-interferon compared with controls. Type 2 cytokines are elevated in HBV. HBV group shows higher IL-4, IL-13, and C-C motif chemokine ligand (CCL) 26 compared with healthy controls and HDV. Chemokines CCL2 and CCL13 are lower in HDV. When assessing ratios, HDV displays higher γ-interferon/IL-4, TNF-α/IL-4, and TNF-α/IL-13 ratios than HBV and controls. CONCLUSION: Hepatitis B virus and HDV subjects show similarly elevated type 1 cytokines. HDV subjects display relatively lower type 2 cytokines. These differences in the systemic immune environments, particularly the predominance of type 1 responses, may contribute to the comparatively rapid progression of HDV disease. Characterization of the imbalance in type 1 and type 2 immunity unique HDV has the potential to provide immunological insights for designing therapeutic targets in HDV-associated disease progression.

Hepatitis D Viremia Among Injection Drug Users in San Francisco.

People who inject drugs (PWID) are commonly exposed to hepatitis B virus (HBV) and hepatitis D virus (HDV). We evaluated the prevalence of HDV viremia among hepatitis B surface antigen (HBsAg)-positive PWID (n = 73) using a new quantitative microarray antibody capture (Q-MAC) assay, HDV western blot, and HDV RNA. HDV Q-MAC performed well in this cohort: anti-HDV, 100% sensitivity and specificity; HDV viremia, 61.5% sensitivity and 100% specificity. Hepatitis D viremia was present in 35.6% of HBsAg-positive participants and was more common in those with resolved compared to chronic hepatitis C (5.1% vs 0.6%; adjusted odds ratio, 9.80; P < .0001).

A novel quantitative microarray antibody capture assay identifies an extremely high hepatitis delta virus prevalence among hepatitis B virus-infected mongolians.

Hepatitis delta virus (HDV) causes the most severe form of human viral hepatitis. HDV requires a hepatitis B virus (HBV) coinfection to provide HDV with HBV surface antigen envelope proteins. The net effect of HDV is to make the underlying HBV disease worse, including higher rates of hepatocellular carcinoma. Accurate assessments of current HDV prevalence have been hampered by the lack of readily available and reliable quantitative assays, combined with the absence of a Food and Drug Administration-approved therapy. We sought to develop a convenient assay for accurately screening populations and to use this assay to determine HDV prevalence in a population with abnormally high rates of hepatocellular carcinoma. We developed a high-throughput quantitative microarray antibody capture assay for anti-HDV immunoglobulin G wherein recombinant HDV delta antigen is printed by microarray on slides coated with a noncontinuous, nanostructured plasmonic gold film, enabling quantitative fluorescent detection of anti-HDV antibody in small aliquots of patient serum. This assay was then used to screen all HBV-infected patients identified in a large randomly selected cohort designed to represent the Mongolian population. We identified two quantitative thresholds of captured antibody that were 100% predictive of the sample either being positive on standard western blot or harboring HDV RNA detectable by real-time quantitative PCR. Subsequent screening of the HBV+ cohort revealed that a remarkable 57% were RNA+ and an additional 4% were positive on western blot alone. CONCLUSION: The quantitative microarray antibody capture assay's unique performance characteristics make it ideal for population screening; its application to the Mongolian HBV surface antigen-positive population reveals an apparent ∼60% prevalence of HDV coinfection among these HBV-infected Mongolian subjects, which may help explain the extraordinarily high rate of hepatocellular carcinoma in Mongolia. (Hepatology 2017;66:1739-1749).

Future Therapy for Hepatitis B Virus: Role of Immunomodulators.

Although currently available therapies for chronic hepatitis B virus infection can suppress viremia and provide long-term benefits for patients, they do not lead to a functional cure for most patients. Advances in our understanding of the virus-host interaction and the recent remarkable success of immunotherapy in cancer offer new and promising strategies for developing immune modulators that may become important components of a total therapeutic approach to hepatitis B, some of which are now in clinical development. Among the immunomodulatory agents currently being investigated to combat chronic HBV are toll-like receptor agonists, immune checkpoint inhibitors, therapeutic vaccines, and engineered T cells. The efficacy of some immune modulatory therapies is compromised by high viral antigen levels. Cutting edge strategies, including RNA interference and CRISPR/Cas9, are now being studied that may ultimately be shown to have the capacity to lower viral antigen levels sufficiently to substantially increase the efficacy of these agents. The current advances in therapies for chronic hepatitis B are leading us toward the possibility of a functional cure.

Phosphatidylinositol 4,5-bisphosphate is an HCV NS5A ligand and mediates replication of the viral genome.

BACKGROUND & AIMS: Phosphoinositides (PIs) bind and regulate localization of proteins via a variety of structural motifs. PI 4,5-bisphosphate (PI[4,5]P2) interacts with and modulates the function of several proteins involved in intracellular vesicular membrane trafficking. We investigated interactions between PI(4,5)P2 and hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and effects on the viral life cycle. METHODS: We used a combination of quartz crystal microbalance, circular dichroism, molecular genetics, and immunofluorescence to study specific binding of PI(4,5)P2 by the HCV NS5A protein. We evaluated the effects of PI(4,5)P2 on the function of NS5A by expressing wild-type or mutant forms of Bart79I or FL-J6/JFH-5'C19Rluc2AUbi21 RNA in Huh7 cells. We also studied the effects of strategies designed to inhibit PI(4,5)P2 on HCV replication in these cells. RESULTS: The N-terminal amphipathic helix of NS5A bound specifically to PI(4,5)P2, inducing a conformational change that stabilized the interaction between NS5A and TBC1D20, which is required for HCV replication. A pair of positively charged residues within the amphipathic helix (the basic amino acid PI(4,5)P2 pincer domain) was required for PI(4,5)P2 binding and replication of the HCV-RNA genome. A similar motif was found to be conserved across all HCV isolates, as well as amphipathic helices of many pathogens and apolipoproteins. CONCLUSIONS: PI(4,5)P2 binds to HCV NS5A to promote replication of the viral RNA genome in hepatocytes. Strategies to disrupt this interaction might be developed to inhibit replication of HCV and other viruses.