An innate defense peptide BPIFA1/SPLUNC1 restricts influenza A virus infection.

This corrects the article DOI: 10.1038/mi.2017.45.

Enhancing viral vaccine production using engineered knockout vero cell lines - A second look.

The global adoption of vaccines to combat disease is hampered by the high cost of vaccine manufacturing. The work described herein follows two previous publications (van der Sanden et al., 2016; Wu et al., 2017) that report a strategy to enhance poliovirus and rotavirus vaccine production through genetic modification of the Vero cell lines used in large-scale vaccine manufacturing. CRISPR/Cas9 gene editing tools were used to knockout Vero target genes previously shown to play a role in polio- and rotavirus production. Subsequently, small-scale models of current industry manufacturing systems were developed and adopted to assess the increases in polio- and rotavirus output by multiple stable knockout cell lines. Unlike previous studies, the Vero knockout cell lines failed to achieve desired target yield increases. These findings suggest that additional research will be required before implementing the genetically engineered Vero cell lines in the manufacturing process for polio- and rotavirus vaccines to be able to supply vaccines at reduced prices.

An innate defense peptide BPIFA1/SPLUNC1 restricts influenza A virus infection.

The airway epithelium secretes proteins that function in innate defense against infection. Bactericidal/permeability-increasing fold-containing family member A1 (BPIFA1) is secreted into airways and has a protective role during bacterial infections, but it is not known whether it also has an antiviral role. To determine a role in host defense against influenza A virus (IAV) infection and to find the underlying defense mechanism, we developed transgenic mouse models that are deficient in BPIFA1 and used these, in combination with in vitro three-dimensional mouse tracheal epithelial cell (mTEC) cultures, to investigate its antiviral properties. We show that BPIFA1 has a significant role in mucosal defense against IAV infection. BPIFA1 secretion was highly modulated after IAV infection. Mice deficient in BPIFA1 lost more weight after infection, supported a higher viral load and virus reached the peripheral lung earlier, indicative of a defect in the control of infection. Further analysis using mTEC cultures showed that BPIFA1-deficient cells bound more virus particles, displayed increased nuclear import of IAV ribonucleoprotein complexes, and supported higher levels of viral replication. Our results identify a critical role of BPIFA1 in the initial phase of infection by inhibiting the binding and entry of IAV into airway epithelial cells.

Mutating the CX3C Motif in the G Protein Should Make a Live Respiratory Syncytial Virus Vaccine Safer and More Effective.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C chemokine motif (182CWAIC186) in the G protein, RSV binds to the corresponding chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log10-fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine.IMPORTANCE RSV binds to the corresponding chemokine receptor, CX3CR1, through a CX3C chemokine motif (182CWAIC186) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab')2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.

Association of adenovirus 36 infection with adiposity and inflammatory-related markers in children.

CONTEXT: Although animal studies suggest that adenovirus 36 (Ad36) infection is linked to obesity and systemic inflammation, human data are scant and equivocal. OBJECTIVE: Associations of Ad36 infection with total body adiposity and inflammatory-related markers were determined in 291 children aged 9-13 years (50% female, 49% black). DESIGN: Fasting blood samples were measured for presence of Ad36-specific antibodies and TNF-α, IL-6, vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1). Fat mass and fat-free soft tissue mass were measured by dual-energy X-ray absorptiometry. RESULTS: The overall prevalence of Ad36 seropositivity [Ad36(+)] was 42%. There was a higher percentage of Ad36(+) children in the highest tertiles of TNF-α and IL-6 compared with their respective middle and lowest tertiles (both P < .03). There was also a trend toward a higher prevalence of Ad36(+) children in the highest tertile of VEGF compared with tertiles 1 and 2 (P = .05). Multinomial logistic regression, adjusting for age, race, sex, and fat-free soft tissue mass, revealed that compared with children with the lowest TNF-α, IL-6, and VEGF levels (tertile 1), the adjusted odds ratios for Ad36(+) were 2.2 [95% confidence interval (CI) 1.2-4.0], 2.4 (95% CI 1.4-4.0), and 1.8 (95% CI 1.0-3.3), respectively, for those in the highest TNF-α, IL-6, and VEGF levels (tertile 3). No association was observed between Ad36(+) and greater levels of fat mass or MCP-1 (all P > .05). CONCLUSIONS: In children, our data suggest that Ad36(+) may be associated with biomarkers implicated in inflammation but not with greater levels of fat mass.

The Thr205 phosphorylation site within respiratory syncytial virus matrix (M) protein modulates M oligomerization and virus production.

UNLABELLED: Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and the elderly worldwide; however, there is no licensed RSV vaccine or effective drug treatment available. The RSV matrix (M) protein plays key roles in virus assembly and budding, but the protein interactions that govern budding of infectious virus are not known. In this study, we focus on M protein and identify a key phosphorylation site (Thr205) in M that is critical for RSV infectious virus production. Recombinant virus with a nonphosphorylatable alanine (Ala) residue at the site was markedly attenuated, whereas virus with a phosphomimetic aspartate (Asp) resulted in a nonviable virus which could only be recovered with an additional mutation in M (serine to asparagine at position 220), strongly implying that Thr205 is critical for viral infectivity. Experiments in vitro showed that mutation of Thr205 does not affect M stability or the ability to form dimers but implicate an effect on higher-order oligomer assembly. In transfected and infected cells, Asp substitution of Thr205 appeared to impair M oligomerization; typical filamentous structures still formed at the plasma membrane, but M assembly during the ensuing elongation process seemed to be impaired, resulting in shorter and more branched filaments as observed using electron microscopy (EM). Our data thus imply for the first time that M oligomerization, regulated by a negative charge at Thr205, may be critical to production of infectious RSV. IMPORTANCE: We show here for the first time that RSV M's role in virus assembly/release is strongly dependent on threonine 205 (Thr205), a consensus site for CK2, which appears to play a key regulatory role in modulating M oligomerization and association with virus filaments. Our analysis indicates that T205 mutations do not impair M dimerization or viruslike filament formation per se but rather the ability of M to assemble in ordered fashion on the viral filaments themselves. This appears to impact in turn upon the infectivity of released virus rather than on virus production or release itself. Thus, M oligomerization would appear to be a target of interest for the development of anti-RSV agents; further, the recombinant T205-substituted mutant viruses described here would appear to be the first RSV mutants affected in viral maturation to our knowledge and hence of considerable interest for vaccine approaches in the future.

Fabrication and characterization of a multiwell array SERS chip with biological applications.

Uniform, large surface area substrates for surface-enhanced Raman spectroscopy (SERS) are fabricated by oblique angle deposition. The SERS-active substrates are patterned by a polymer-molding technique to provide a uniform array for high throughput biosensing and multiplexing. Using a conventional SERS-active molecule, 1,2-di(4-pyridyl)ethylene (BPE) >or=98%, we show that this device provides a uniform Raman signal enhancement from well to well with a detection limit of at least 10(-8)M of the BPE solution or 10(-18)mol of BPE. The SERS intensity is also demonstrated to vary logarithmically with the log of BPE concentration and the apparent sensitivity of the patterned substrate is compared to previous reports from our group on non-patterned substrates. Avian influenza is analyzed to demonstrate the utility of SERS multiwell patterned substrates for biosensing. The spectra acquired from patterned substrates show better reproducibility and less variation compared to the unpatterned substrates according to multivariate analysis. Our results highlight potential advantages of the patterned substrate.

Rapid microRNA (miRNA) detection and classification via surface-enhanced Raman spectroscopy (SERS).

microRNAs (miRNA) are recognized as regulators of gene expression during development and cell differentiation as well as biomarkers of disease. Development of rapid and sensitive miRNA profiling methods is essential for evaluating the pattern of miRNA expression that varies across normal and diseased states. The ability to identify miRNA expression patterns is limited to cumbersome assays that often lack sensitivity and specificity to distinguish between different miRNA families and members. We evaluated a surface-enhanced Raman scattering (SERS) platform for detection and classification of miRNAs. The strength of the SERS-based sensor is its sensitivity to detect extremely low levels of analyte and specificity to provide the molecular fingerprint of the analyte. We show that the SERS spectra of related and unrelated miRNAs can be detected in near-real time, that detection is sequence dependent, and that SERS spectra can be used to classify miRNA patterns with high accuracy.

Identification and classification of respiratory syncytial virus (RSV) strains by surface-enhanced Raman spectroscopy and multivariate statistical techniques.

There is a critical need for a rapid and sensitive means of detecting viruses. Recent reports from our laboratory have shown that surface-enhanced Raman spectroscopy (SERS) can meet these needs. In this study, SERS was used to obtain the Raman spectra of respiratory syncytial virus (RSV) strains A/Long, B1, and A2. SERS-active substrates composed of silver nanorods were fabricated using an oblique angle vapor deposition method. The SERS spectra obtained for each virus were shown to possess a high degree of reproducibility. Based on their intrinsic SERS spectra, the four virus strains were readily detected and classified using the multivariate statistical methods principal component analysis (PCA) and hierarchical cluster analysis (HCA). The chemometric results show that PCA is able to separate the three virus strains unambiguously, whereas the HCA method was able to readily distinguish an A2 strain-related G gene mutant virus (DeltaG) from the A2 strain. The results described here demonstrate that SERS, in combination with multivariate statistical methods, can be utilized as a highly sensitive and rapid viral identification and classification method.

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.

CX3C chemokine mimicry by respiratory syncytial virus G glycoprotein.

Chemokines are chemoattractant proteins that are divided into subfamilies based upon cysteine signature motifs termed C, CC, CXC and CX3C. Chemokines have roles in immunity and inflammation that affect cell trafficking and activation of T cells as well as cells of the innate immune system. We report here CX3C chemokine mimicry for the G glycoprotein of respiratory syncytial virus (RSV) and show binding to CX3CR1--the specific receptor for the CX3C chemokine fractalkine--and induction of leukocyte chemotaxis. We also show that CX3CR1 facilitates RSV infection of cells. Thus, G glycoprotein interaction with CX3CR1 probably plays a key role in the biology of RSV infection.

CD4(+) T cell frequencies and Th1/Th2 cytokine patterns expressed in the acute and memory response to respiratory syncytial virus I-E(d)-restricted peptides.

The respiratory syncytial virus (RSV)-specific frequencies and cytokine expression patterns of acute and memory CD4(+) T cells from RSV strain-A- and strain-B-infected BALB/c mice were determined following restimulation with a panel of 14 predicted RSV I-E(d) peptides from NSP-2, M, SH, F, and L proteins. Ten of fourteen peptides stimulated intracellular Th1 and/or Th2 cytokines in CD4(+) T cells from the mediastinal lymph nodes (MLN) and spleens of RSV strain-A- or strain-B-immune BALB/c mice. Spleen cells exhibited a predominant Th2 cytokine expression pattern after peptide stimulation, whereas MLN cells exhibited a mixed Th1/Th2 cytokine pattern. For a few peptides, there were differences in the Th1/Th2 cytokine response to peptides from the homologous versus heterologous RSV group. None of the 10 peptides induced both Th1 and Th2 cytokines in cells from similarly immunized mice. The frequency and breadth of cytokine expression by I-E(d)-restricted CD4(+) T cells to peptide stimulation was diminished in the memory response.

Respiratory syncytial virus G and/or SH glycoproteins modify CC and CXC chemokine mRNA expression in the BALB/c mouse.

Chemokine mRNA expression by pulmonary leukocytes following infection of BALB/c mice with two strains of respiratory syncytial virus (RSV) and one strain of parainfluenza virus type 3 (PIV-3) was determined. The results suggest that RSV G and/or SH proteins inhibit early MIP-1alpha, MIP-1beta, MIP-2, MCP-1, and IP-10 mRNA expression. TCA-3 mRNA expression was found to be increased during PIV-3 infection.

TH(1)- and TH(2)-TYPE cytokine expression by activated t lymphocytes from the lung and spleen during the inflammatory response to respiratory syncytial virus.

RSV is an important cause of lower respiratory tract illness in infants and the elderly worldwide. The components involved in immunity and those that contribute to inflammation of RSV-induced disease are not clearly understood. To address the relationship between activation antigen and cytokine expression, intracellular levels of IL-2, IL-4, IL-5 and IFN-gamma were determined for CD3, CD44, CD49d, CD54, CD62L and CD102 lymphocytes from the bronchoalveolar lavage and spleen. To examine activation at the DNA level, lymphocytes expressing IL-2, IL-4, IL-5 or IFN-gamma were analysed for G2+M DNA content or phosphatidylserine expression (apoptosis). Trafficking of lymphocytes to the BAL was detected at day 5 p.i., peaked day 7 p.i., and predominately involved CD54(+)and CD102(+)lymphocytes expressing high levels of IL-2, IL-4, IL-5 and IFN-gamma. Lymphocytes expressing CD44(+), CD49d(+)and CD62L(lo)were also observed, however they expressed these cytokines to a lesser extent. DNA analysis of lymphocytes expressing IL-2 or IFN-gamma revealed higher G2'M levels compared to lymphocytes expressing IL-4 or IL-5, suggesting greater activation of Th(1)-type lymphocytes in the lung. These data demonstrate that RSV-induced pulmonary inflammation involves extensive cellular activation and cytokine expression, particularly by CD54(+)and CD102(+)lymphocytes in the lung.

CD40 ligand (CD154) enhances the Th1 and antibody responses to respiratory syncytial virus in the BALB/c mouse.

CD40 ligand (CD40L) is a cell surface costimulatory molecule expressed mainly by activated T cells. CD40L is critically important for T-B cell and T cell-dendritic cell interactions. CD40L expression promotes Th1 cytokine responses to protein Ags and is responsible for Ig isotype switching in B cells. Respiratory syncytial virus (RSV) is an important pathogen of young children and the elderly, which causes bronchiolitis and pneumonia. Studies of mice infected with RSV suggest that a Th2 cytokine response may be responsible for enhanced pulmonary disease. To investigate the effect CD40L has on RSV immunity, mice were infected simultaneously with RSV and either an empty control adenovirus vector or one expressing CD40L or were coimmunized with plasmid DNA vectors expressing CD40L and RSV F and/or G proteins and subsequently challenged with RSV. The kinetics of the intracellular and secreted cytokine responses, the cytotoxic T lymphocyte precursor frequency, NO levels in lung lavage, rates of virus clearance, and anti-RSV Ab titers were determined. These studies show that coincident expression of CD40L enhances the Th1 (IL-2 and IFN-gamma) cytokine responses, increases the expression of TNF-alpha and NO, accelerates virus clearance, and increases the anti-F and anti-G Ab responses. These data suggest that CD40L may have the adjuvant properties needed to optimize the safety and efficacy of RSV vaccines.

Respiratory syncytial virus infection and G and/or SH protein expression contribute to substance P, which mediates inflammation and enhanced pulmonary disease in BALB/c mice.

A distinct clinical presentation of respiratory syncytial virus (RSV) infection of humans is bronchiolitis, which has clinical features similar to those of asthma. Substance P (SP), a tachykinin neuropeptide, has been associated with neurogenic inflammation and asthma; therefore, we chose to examine SP-induced inflammation with RSV infection. In this study, we examined the production of pulmonary SP associated with RSV infection of BALB/c mice and the effect of anti-SP F(ab)(2) antibodies on the pulmonary inflammatory response. The peak production of pulmonary SP occurred between days 3 and 5 following primary RSV infection and day 1 after secondary infection. Treatment of RSV-infected mice with anti-SP F(ab)(2) antibodies suggested that SP may alter the natural killer cell response to primary and secondary infection. In mice challenged after formalin-inactivated RSV vaccination, SP appears to markedly enhance pulmonary eosinophilia as well as increase polymorphonuclear cell trafficking to the lung. Based on studies with a strain of RSV that lacks the G and SH genes, the SP response to RSV infection appears to be associated with G and/or SH protein expression. These data suggest that SP may be an important contributor to the inflammatory response to RSV infection and that anti-SP F(ab)(2) antibodies might be used to ameliorate RSV-associated disease.

Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus.

The innate immune system contributes to the earliest phase of the host defense against foreign organisms and has both soluble and cellular pattern recognition receptors for microbial products. Two important members of this receptor group, CD14 and the Toll-like receptor (TLR) pattern recognition receptors, are essential for the innate immune response to components of Gram-negative and Gram-positive bacteria, mycobacteria, spirochetes and yeast. We now find that these receptors function in an antiviral response as well. The innate immune response to the fusion protein of an important respiratory pathogen of humans, respiratory syncytial virus (RSV), was mediated by TLR4 and CD14. RSV persisted longer in the lungs of infected TLR4-deficient mice compared to normal mice. Thus, a common receptor activation pathway can initiate innate immune responses to both bacterial and viral pathogens.

Reconstitution of early lymphoid proliferation and immune function in Jak3-deficient mice by interleukin-3.

Expansion of early lymphoid progenitors requires interleukin-7 (IL-7), which functions through gamma(c)-mediated receptor activation of Jak3. Jak3 deficiency is a cause of severe combined immunodeficiency (SCID) in humans and mice. IL-3 activates many of the same signaling pathways as IL-7, such as Stat5, but achieves this effect through the activation of Jak2 rather than Jak3. We hypothesized that expansion of an IL-7-responsive precursor population through a Jak3-independent pathway using IL-3 may stimulate early lymphoid progenitors and restore lymphopoiesis in Jak3(-/-) mice. Newborn Jak3(-/-) mice that were injected with IL-3 demonstrated thymic enlargement, a 2- to 20-fold increase in thymocyte numbers, and up to a 10-fold expansion in the number of CD4(+), CD8(+), and B220(+)/IgM(+) splenic lymphocytes, consistent with an effect upon an early lymphoid progenitor population. In contrast to control mice, IL-3-treated Jak3(-/-) mice challenged with the allogeneic major histocompatibility complex (MHC) class I-bearing tumor P815 developed a specific CD8-dependent cytotoxic T lymphocyte (CTL) response. IL-3-treated mice also mounted influenza-specific CTL responses and survival was prolonged. The beneficial effects of IL-3 are proposed to be produced by stimulation of a lymphoid precursor population of IL-7Ralpha(+)/IL-3Ralpha(+) cells that we identified in wild-type bone marrow. In vitro, we show that an early IL-7R(+) lymphoid progenitor population expresses IL-3R and proliferates in response to IL-3 and that IL-3 activates Stat5 comparably to IL-7. Clinically, IL-3 may therefore be useful treatment for X-linked and Jak3-deficient SCID patients who lack bone marrow donors.

Respiratory syncytial virus G and/or SH protein alters Th1 cytokines, natural killer cells, and neutrophils responding to pulmonary infection in BALB/c mice.

BALB/c mice sensitized to vaccinia virus expressed G protein of respiratory syncytial virus (RSV) develop a Th2-type cytokine response and pulmonary eosinophilia when challenged with live RSV. In this study, BALB/c mice were immunized or challenged with an RSV mutant lacking the G and SH proteins or with DNA vaccines coding for RSV G or F protein. F or G protein DNA vaccines were capable of sensitizing for pulmonary eosinophilia. The absence of the G and/or SH protein in the infecting virus resulted in a consistent increase both in pulmonary natural killer cells and in gamma interferon and tumor necrosis factor expression, as well as, with primary infection, a variable increase in neutrophils and CD11b(+) cells. The development of pulmonary eosinophilia in formalin-inactivated RSV-vaccinated mice required the presence of the G and/or SH protein in the challenge virus. These data show that G and/or SH protein has a marked impact on the inflammatory and innate immune response to RSV infection.

Cytotoxic T-lymphocyte precursor frequencies in BALB/c mice after acute respiratory syncytial virus (RSV) infection or immunization with a formalin-inactivated RSV vaccine.

A better understanding of the immune response to live and formalin-inactivated respiratory syncytial virus (RSV) is important for developing nonlive vaccines. In this study, major histocompatibility complex (MHC) class I- and II-restricted, RSV-specific cytotoxic T-lymphocyte precursor (CTLp) frequencies were determined in bronchoalveolar lavage (BAL) samples and spleen lymphocytes of BALB/c mice intranasally infected with live RSV or intramuscularly inoculated with formalin-inactivated RSV (FI-RSV). After RSV infection, both class I- and class II-restricted CTLps were detected by day 4 or 5 postinfection (p.i.). Peak CTLp frequencies were detected by day 7 p.i. The class II-restricted CTLp frequencies in the BAL following RSV infection were less than class I-restricted CTLp frequencies through day 14 p.i., during which class I-restricted CTLp frequencies remained elevated, but then declined by 48 days p.i. The frequencies of class II-restricted CTLps in the BAL were 2- to 10-fold less than those of class I-restricted CTLps. For spleen cells, frequencies of both MHC class I- and II-restricted CTLps to live RSV were similar. In contrast, class II-restricted CTLps predominated in FI-RSV-vaccinated mice. RSV challenge of vaccinated mice resulted in an increase in the frequency of class I-restricted CTLps at day 3 p.i. but did not enhance class II-restricted CTLp frequencies. These studies demonstrate differences in the CTLp response to live RSV infection compared with FI-RSV immunization and help define possible mechanisms of enhanced disease after FI-RSV immunization. In addition, these studies provide a quantitative means to address potential vaccine candidates by examining both MHC class I- and II-restricted CTLp frequencies.