Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.

Defining B cell immunodominance to viruses.

Immunodominance (ID) defines the hierarchical immune response to competing antigens in complex immunogens. Little is known regarding B cell and antibody ID despite its importance in immunity to viruses and other pathogens. We show that B cells and serum antibodies from inbred mice demonstrate a reproducible ID hierarchy to the five major antigenic sites in the influenza A virus hemagglutinin globular domain. The hierarchy changed as the immune response progressed, and it was dependent on antigen formulation and delivery. Passive antibody transfer and sequential infection experiments demonstrated 'original antigenic suppression', a phenomenon in which antibodies suppress memory responses to the priming antigenic site. Our study provides a template for attaining deeper understanding of antibody ID to viruses and other complex immunogens.

Ribosomal Proteins Regulate MHC Class I Peptide Generation for Immunosurveillance.

The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.

Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs.

Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin (HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies. Antibody-free propagation following antigenic escape rapidly selected viruses with mutations that modulated receptor binding avidity through the addition of N-linked glycans to the HA globular domain. These findings expand our understanding of the viral mechanisms that maintain fitness during antigenic evolution to include glycan addition, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms.

Correction: Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs.

[This corrects the article DOI: 10.1371/journal.ppat.1006796.].

mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry.

Variants in the IMP2 (insulin-like growth factor 2 [IGF2] mRNA-binding protein 2) gene are implicated in susceptibility to type 2 diabetes. We describe the ability of mammalian target of rapamycin (mTOR) to regulate the cap-independent translation of IGF2 mRNA through phosphorylation of IMP2, an oncofetal RNA-binding protein. IMP2 is doubly phosphorylated in a rapamycin-inhibitable, amino acid-dependent manner in cells and by mTOR in vitro. Double phosphorylation promotes IMP2 binding to the IGF2 leader 3 mRNA 5' untranslated region, and the translational initiation of this mRNA through eIF-4E- and 5' cap-independent internal ribosomal entry. Unexpectedly, the interaction of IMP2 with mTOR complex 1 occurs through mTOR itself rather than through raptor. Whereas depletion of mTOR strongly inhibits IMP2 phosphorylation in cells, comparable depletion of raptor has no effect; moreover, the ability of mTOR to phosphorylate IMP2 in vitro is unaffected by the elimination of raptor. Dual phosphorylation of IMP2 at the mTOR sites is evident in the mouse embryo, likely coupling nutrient sufficiency to IGF2 expression and fetal growth. Doubly phosphorylated IMP2 is also widely expressed in adult tissues, including islets of Langerhans.

Alternative reassortment events leading to transmissible H9N1 influenza viruses in the ferret model.

Influenza A H9N2 viruses are common poultry pathogens that occasionally infect swine and humans. It has been shown previously with H9N2 viruses that reassortment can generate novel viruses with increased transmissibility. Here, we demonstrate the modeling power of a novel transfection-based inoculation system to select reassortant viruses under in vivo selective pressure. Plasmids containing the genes from an H9N2 virus and a pandemic H1N1 (pH1N1) virus were transfected into HEK 293T cells to potentially generate the full panel of possible H9 reassortants. These cells were then used to inoculate ferrets, and the population dynamics were studied. Two respiratory-droplet-transmissible H9N1 viruses were selected by this method, indicating a selective pressure in ferrets for the novel combination of surface genes. These results show that a transfection-based inoculation system is a fast and efficient method to model reassortment and highlight the risk of reassortment between H9N2 and pH1N1 viruses.

All-in-one bacmids: an efficient reverse genetics strategy for influenza A virus vaccines.

UNLABELLED: Vaccination is the first line of defense against influenza virus infection, yet influenza vaccine production methods are slow, antiquated, and expensive as a means to effectively reduce the virus burden during epidemic or pandemic periods. There is a great need for alternative influenza vaccines and vaccination methods with a global scale of impact. We demonstrate here a strategy to generate influenza A virus in vivo by using bacmid DNAs. Compared to the classical reverse genetics system, the "eight-in-one" bacmids (bcmd-RGFlu) showed higher efficiency of virus rescue in various cell types. Using a transfection-based inoculation (TBI) system, intranasal delivery to DBA/2J and BALB/c mice of bcmd-RGFlu plus 293T cells led to the generation of lethal PR8 virus in vivo. A prime-boost intranasal vaccination strategy using TBI in the context of a bcmd-RGFlu carrying a temperature-sensitive H1N1 virus resulted in protection of mice against lethal challenge with the PR8 strain. Taken together, these studies provide proof of principle to highlight the potential of vaccination against influenza virus by using in vivo reverse genetics. IMPORTANCE: Vaccination is the first line of defense against influenza virus infections. A major drawback in the preparation of influenza vaccines is that production relies on a heavily time-consuming process of growing the viruses in eggs. We propose a radical change in the way influenza vaccination is approached, in which a recombinant bacmid, a shuttle vector that can be propagated in both Escherichia coli and insect cells, carries an influenza virus infectious clone (bcmd-RGFlu). Using a surrogate cell system, we found that intranasal delivery of bcmd-RGFlu resulted in generation of influenza virus in mice. Furthermore, mice vaccinated with this system were protected against lethal influenza virus challenge. The study serves as a proof of principle of a potentially universal vaccine platform against influenza virus and other pathogens.

Airborne transmission of highly pathogenic H7N1 influenza virus in ferrets.

UNLABELLED: Avian H7 influenza viruses are recognized as potential pandemic viruses, as personnel often become infected during poultry outbreaks. H7 infections in humans typically cause mild conjunctivitis; however, the H7N9 outbreak in the spring of 2013 has resulted in severe respiratory disease. To date, no H7 viruses have acquired the ability for sustained transmission among humans. Airborne transmission is considered a requirement for the emergence of pandemic influenza, and advanced knowledge of the molecular changes or signature required for transmission would allow early identification of pandemic vaccine seed stocks, screening and stockpiling of antiviral compounds, and eradication efforts focused on flocks harboring threatening viruses. Thus, we sought to determine if a highly pathogenic influenza A H7N1 (A/H7N1) virus with no history of human infection could become capable of airborne transmission among ferrets. We show that after 10 serial passages, A/H7N1 developed the ability to be transmitted to cohoused and airborne contact ferrets. Four amino acid mutations (PB2 T81I, NP V284M, and M1 R95K and Q211K) in the internal genes and a minimal amino acid mutation (K/R313R) in the stalk region of the hemagglutinin protein were associated with airborne transmission. Furthermore, transmission was not associated with loss of virulence. These findings highlight the importance of the internal genes in host adaptation and suggest that natural isolates carrying these mutations be further evaluated. Our results demonstrate that a highly pathogenic avian H7 virus can become capable of airborne transmission in a mammalian host, and they support ongoing surveillance and pandemic H7 vaccine development. IMPORTANCE: The major findings of this report are that a highly pathogenic strain of H7N1 avian influenza virus can be adapted to become capable of airborne transmission in mammals without mutations altering receptor specificity. Changes in receptor specificity have been shown to play a role in the ability of avian influenza viruses to cross the species barrier, and these changes are assumed to be essential. The work reported here challenges this paradigm, at least for the influenza viruses of the H7 subtype, which have recently become the focus of major attention, as they have crossed to humans.

mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation.

Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy to treat several diseases and are compelling to consider as vehicles for delivery of biological agents. However, MSCs appear to act through a seemingly limited "hit-and-run" mode to quickly exert their therapeutic impact, mediated by several mechanisms, including a potent immunomodulatory secretome. Furthermore, MSC immunomodulatory properties are highly variable and the secretome composition following infusion is uncertain. To determine whether a transiently controlled antiinflammatory MSC secretome could be achieved at target sites of inflammation, we harnessed mRNA transfection to generate MSCs that simultaneously express functional rolling machinery (P-selectin glycoprotein ligand-1 [PSGL-1] and Sialyl-Lewis(x) [SLeX]) to rapidly target inflamed tissues and that express the potent immunosuppressive cytokine interleukin-10 (IL-10), which is not inherently produced by MSCs. Indeed, triple-transfected PSGL-1/SLeX/IL-10 MSCs transiently increased levels of IL-10 in the inflamed ear and showed a superior antiinflammatory effect in vivo, significantly reducing local inflammation following systemic administration. This was dependent on rapid localization of MSCs to the inflamed site. Overall, this study demonstrates that despite the rapid clearance of MSCs in vivo, engineered MSCs can be harnessed via a "hit-and-run" action for the targeted delivery of potent immunomodulatory factors to treat distant sites of inflammation.

In vivo selection of H1N2 influenza virus reassortants in the ferret model.

Although the ferret model has been extensively used to study pathogenesis and transmission of influenza viruses, little has been done to determine whether ferrets are a good surrogate animal model to study influenza virus reassortment. It has been previously shown that the pandemic 2009 H1N1 (H1N1pdm) virus was able to transmit efficiently in ferrets. In coinfection studies with either seasonal H1N1 or H3N2 strains (H1N1s or H3N2s, respectively), the H1N1pdm virus was able to outcompete these strains and become the dominant transmissible virus. However, lack of reassortment could have been the result of differences in the cell or tissue tropism of these viruses in the ferret. To address this issue, we performed coinfection studies with recombinant influenza viruses carrying the surface genes of a seasonal H3N2 strain in the background of an H1N1pdm strain and vice versa. After serial passages in ferrets, a dominant H1N2 virus population was obtained with a constellation of gene segments, most of which, except for the neuraminidase (NA) and PB1 segments, were from the H1N1pdm strain. Our studies suggest that ferrets recapitulate influenza virus reassortment events. The H1N2 virus generated through this process resembles similar viruses that are emerging in nature, particularly in pigs.

Influenza viruses with rearranged genomes as live-attenuated vaccines.

H5N1 and H9N2 avian influenza virus subtypes top the World Health Organization's list for the greatest pandemic potential. Inactivated H5N1 vaccines induce limited immune responses and, in the case of live-attenuated influenza virus vaccines (LAIV), there are safety concerns regarding the possibility of reassortment between the H5 gene segment and circulating influenza viruses. In order to overcome these drawbacks, we rearranged the genome of an avian H9N2 influenza virus and expressed the entire H5 hemagglutinin open reading frame (ORF) from the segment 8 viral RNA. These vectors had reduced polymerase activities as well as viral replication in vitro and excellent safety profiles in vivo. Immunization with the dual H9-H5 influenza virus resulted in protection against lethal H5N1 challenge in mice and ferrets, and also against a potentially pandemic H9 virus. Our studies demonstrate that rearranging the influenza virus genome has great potential for the development of improved vaccines against influenza virus as well as other pathogens.

Receptor characterization and susceptibility of cotton rats to avian and 2009 pandemic influenza virus strains.

Animal influenza viruses (AIVs) are a major threat to human health and the source of pandemic influenza. A reliable small-mammal model to study the pathogenesis of infection and for testing vaccines and therapeutics against multiple strains of influenza virus is highly desirable. We show that cotton rats (Sigmodon hispidus) are susceptible to avian and swine influenza viruses. Cotton rats express α2,3-linked sialic acid (SA) and α2,6-linked SA residues in the trachea and α2,6-linked SA residues in the lung parenchyma. Prototypic avian influenza viruses (H3N2, H9N2, and H5N1) and swine-origin 2009 pandemic H1N1 viruses replicated in the nose and in the respiratory tract of cotton rats without prior adaptation and produced strong lung pathology that was characterized by early lung neutrophilia, followed by subsequent pneumonia. Consistent with other natural and animal models of influenza, only the H5N1 virus was lethal for cotton rats. More importantly, we show that the different avian and pandemic H1N1 strains tested are strong activators of the type I interferon (IFN)-inducible MX-1 gene both locally and systemically. Our data indicate that the cotton rat is a suitable small-mammal model to study the infection of animal influenza viruses and for validation of vaccines and therapeutics against these viruses.

Paradoxical imbalance between activated lymphocyte protein synthesis capacity and rapid division rate.

Rapid lymphocyte cell division places enormous demands on the protein synthesis machinery. Flow cytometric measurement of puromycylated ribosome-associated nascent chains after treating cells or mice with translation initiation inhibitors reveals that ribosomes in resting lymphocytes in vitro and in vivo elongate at typical rates for mammalian cells. Intriguingly, elongation rates can be increased up to 30% by activation in vivo or fever temperature in vitro. Resting and activated lymphocytes possess abundant monosome populations, most of which actively translate in vivo, while in vitro, nearly all can be stalled prior to activation. Quantitating lymphocyte protein mass and ribosome count reveals a paradoxically high ratio of cellular protein to ribosomes insufficient to support their rapid in vivo division, suggesting that the activated lymphocyte proteome in vivo may be generated in an unusual manner. Our findings demonstrate the importance of a global understanding of protein synthesis in lymphocytes and other rapidly dividing immune cells.

Step-dose IL-7 treatment promotes systemic expansion of T cells and alters immune cell landscape in blood and lymph nodes.

We employed a dose-escalation regimen in rhesus macaques to deliver glycosylated IL-7, a cytokine critical for development and maintenance of T lymphocytes. IL-7 increased proliferation and survival of T cells and triggered several chemokines and cytokines. Induction of CXCL13 in lymph nodes (LNs) led to a remarkable increase of B cells in the LNs, proliferation of germinal center follicular T helper cells and elevated IL-21 levels suggesting an increase in follicle activity. Transcriptomics analysis showed induction of IRF-7 and Flt3L, which was linked to increased frequency of circulating plasmacytoid dendritic cells (pDCs) on IL-7 treatment. These pDCs expressed higher levels of CCR7, homed to LNs, and were associated with upregulation of type-1 interferon gene signature and increased production of IFN-α2a on TLR stimulation. Superior effects and dose-sparing advantage was observed by the step-dose regimen. Thus, IL-7 treatment leads to systemic effects involving both lymphoid and myeloid compartments.

Tumor eradication by hetIL-15 locoregional therapy correlates with an induced intratumoral CD103intCD11b+ dendritic cell population.

Locoregional monotherapy with heterodimeric interleukin (IL)-15 (hetIL-15) in a triple-negative breast cancer (TNBC) orthotopic mouse model resulted in tumor eradication in 40% of treated mice, reduction of metastasis, and induction of immunological memory against breast cancer cells. hetIL-15 re-shaped the tumor microenvironment by promoting the intratumoral accumulation of cytotoxic lymphocytes, conventional type 1 dendritic cells (cDC1s), and a dendritic cell (DC) population expressing both CD103 and CD11b markers. These CD103intCD11b+DCs share phenotypic and gene expression characteristics with both cDC1s and cDC2s, have transcriptomic profiles more similar to monocyte-derived DCs (moDCs), and correlate with tumor regression. Therefore, hetIL-15, a cytokine directly affecting lymphocytes and inducing cytotoxic cells, also has an indirect rapid and significant effect on the recruitment of myeloid cells, initiating a cascade for tumor elimination through innate and adoptive immune mechanisms. The intratumoral CD103intCD11b+DC population induced by hetIL-15 may be targeted for the development of additional cancer immunotherapy approaches.

Modifications in the polymerase genes of a swine-like triple-reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 viruses.

On 11 June 2009, the World Health Organization (WHO) declared that the outbreaks caused by novel swine-origin influenza A (H1N1) virus had reached pandemic proportions. The pandemic H1N1 (H1N1pdm) virus is the predominant influenza virus strain in the human population. It has also crossed the species barriers and infected turkeys and swine in several countries. Thus, the development of a vaccine that is effective in multiple animal species is urgently needed. We have previously demonstrated that the introduction of temperature-sensitive mutations into the PB2 and PB1 genes of an avian H9N2 virus, combined with the insertion of a hemagglutinin (HA) tag in PB1, resulted in an attenuated (att) vaccine backbone for both chickens and mice. Because the new pandemic strain is a triple-reassortant (TR) virus, we chose to introduce the double attenuating modifications into a swine-like TR virus isolate, A/turkey/OH/313053/04 (H3N2) (ty/04), with the goal of producing live attenuated influenza vaccines (LAIV). This genetically modified backbone had impaired polymerase activity and restricted virus growth at elevated temperatures. In vivo characterization of two H1N1 vaccine candidates generated using the ty/04 att backbone demonstrated that this vaccine is highly attenuated in mice, as indicated by the absence of signs of disease, limited replication, and minimum histopathological alterations in the respiratory tract. A single immunization with the ty/04 att-based vaccines conferred complete protection against a lethal H1N1pdm virus infection in mice. More importantly, vaccination of pigs with a ty/04 att-H1N1 vaccine candidate resulted in sterilizing immunity upon an aggressive intratracheal challenge with the 2009 H1N1 pandemic virus. Our studies highlight the safety of the ty/04 att vaccine platform and its potential as a master donor strain for the generation of live attenuated vaccines for humans and livestock.

Variations in the hemagglutinin of the 2009 H1N1 pandemic virus: potential for strains with altered virulence phenotype?

A novel, swine-origin influenza H1N1 virus (H1N1pdm) caused the first pandemic of the 21st century. This pandemic, although efficient in transmission, is mild in virulence. This atypical mild pandemic season has raised concerns regarding the potential of this virus to acquire additional virulence markers either through further adaptation or possibly by immune pressure in the human host. Using the mouse model we generated, within a single round of infection with A/California/04/09/H1N1 (Ca/04), a virus lethal in mice--herein referred to as mouse-adapted Ca/04 (ma-Ca/04). Five amino acid substitutions were found in the genome of ma-Ca/04: 3 in HA (D131E, S186P and A198E), 1 in PA (E298K) and 1 in NP (D101G). Reverse genetics analyses of these mutations indicate that all five mutations from ma-Ca/04 contributed to the lethal phenotype; however, the D131E and S186P mutations--which are also found in the 1918 and seasonal H1N1 viruses-in HA alone were sufficient to confer virulence of Ca/04 in mice. HI assays against H1N1pdm demonstrate that the D131E and S186P mutations caused minor antigenic changes and, likely, affected receptor binding. The rapid selection of ma-Ca/04 in mice suggests that a virus containing this constellation of amino acids might have already been present in Ca/04, likely as minor quasispecies.

The Role of Acetate Kinase in the Human Parasite Entamoeba histolytica.

The human parasite Entamoeba histolytica, which causes approximately 100 million cases of amoebic dysentery each year, relies on glycolysis as the major source of ATP production from glucose as it lacks a citric acid cycle and oxidative phosphorylation. Ethanol and acetate, the two major glycolytic end products for E. histolytica, are produced at a ratio of 2:1 under anaerobic conditions, creating an imbalance between NADH production and utilization. In this study we investigated the role of acetate kinase (ACK) in acetate production during glycolysis in E. histolytica metabolism. Analysis of intracellular and extracellular metabolites demonstrated that acetate levels were unaffected in an ACK RNAi cell line, but acetyl-CoA levels and the NAD+/NADH ratio were significantly elevated. Moreover, we demonstrated that glyceraldehyde 3-phosphate dehydrogenase catalyzes the ACK-dependent conversion of acetaldehyde to acetyl phosphate in E. histolytica. We propose that ACK is not a major contributor to acetate production, but instead provides a mechanism for maintaining the NAD+/NADH balance during ethanol production in the extended glycolytic pathway.

Comparative immunogenicity of an mRNA/LNP and a DNA vaccine targeting HIV gag conserved elements in macaques.

Immunogenicity of HIV-1 mRNA vaccine regimens was analyzed in a non-human primate animal model. Rhesus macaques immunized with mRNA in lipid nanoparticle (mRNA/LNP) formulation expressing HIV-1 Gag and Gag conserved regions (CE) as immunogens developed robust, durable antibody responses but low adaptive T-cell responses. Augmentation of the dose resulted in modest increases in vaccine-induced cellular immunity, with no difference in humoral responses. The gag mRNA/lipid nanoparticle (LNP) vaccine provided suboptimal priming of T cell responses for a heterologous DNA booster vaccination regimen. In contrast, a single immunization with gag mRNA/LNP efficiently boosted both humoral and cellular responses in macaques previously primed by a gag DNA-based vaccine. These anamnestic cellular responses were mediated by activated CD8+ T cells with a phenotype of differentiated T-bet+ cytotoxic memory T lymphocytes. The heterologous prime/boost regimens combining DNA and mRNA/LNP vaccine modalities maximized vaccine-induced cellular and humoral immune responses. Analysis of cytokine responses revealed a transient systemic signature characterized by the release of type I interferon, IL-15 and IFN-related chemokines. The pro-inflammatory status induced by the mRNA/LNP vaccine was also characterized by IL-23 and IL-6, concomitant with the release of IL-17 family of cytokines. Overall, the strong boost of cellular and humoral immunity induced by the mRNA/LNP vaccine suggests that it could be useful as a prophylactic vaccine in heterologous prime/boost modality and in immune therapeutic interventions against HIV infection or other chronic human diseases.