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.

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.

Pathogenesis of an infectious mononucleosis-like disease induced by a murine gamma-herpesvirus: role for a viral superantigen?

The murine gamma-herpesvirus 68 has many similarities to EBV, and induces a syndrome comparable to infectious mononucleosis (IM). The frequency of activated CD8+ T cells (CD62L(lo)) in the peripheral blood increased greater than fourfold by 21 d after infection of C57BL/6J (H-2(b)) mice, and remained high for at least a further month. The spectrum of T cell receptor usage was greatly skewed, with as many as 75% of the CD8+ T cells in the blood expressing a Vbeta4+ phenotype. Interestingly, the Vbeta4 dominance was also seen, to varying extents, in H-2(k), H-2(d), H-2(u), and H-2(q) strains of mice. In addition, although CD4 depletion from day 11 had no effect on the Vbeta4 bias of the T cells, the Vbeta4+CD8+ expansion was absent in H-2IA(b)-deficient congenic mice. However, the numbers of cycling cells in the CD4 antibody-depleted mice and mice that are CD4 deficient as a consequence of the deletion of MHC class II, were generally lower. The findings suggest that the IM-like disease is driven both by cytokines provided by CD4+ T cells and by a viral superantigen presented by MHC class II glycoproteins to Vbeta4+CD8+ T cells.

Defective lymphoid development in mice lacking Jak3.

The Janus tyrosine kinases (Jaks) play a central role in signaling through cytokine receptors. Although Jak1, Jak2, and Tyk2 are widely expressed, Jak3 is predominantly expressed in hematopoietic cells and is known to associate only with the common gamma (gamma c) chain of the interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 receptors. Homozygous mutant mice in which the Jak3 gene had been disrupted were generated by gene targeting. Jak3-deficient mice had profound reductions in thymocytes and severe B cell and T cell lymphopenia similar to severe combined immunodeficiency disease (SCID), and the residual T cells and B cells were functionally deficient. Thus, Jak3 plays a critical role in gamma c signaling and lymphoid development.

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.

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.

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

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

CD8+ T-cell memory to viruses.

Recent experiments show that laboratory mice infected once with an influenza A virus or with the murine parainfluenza type 1 virus, called the Sendai virus, have enhanced numbers of cytotoxic T-lymphocyte precursors ( > 20x background) for life. Neither virus persists at the genome level, and the mice are maintained under conditions where there is no possibility of re-infection. These observations are highly relevant to any understanding of CD8+ cell memory and suggest that the clonal burst size associated with the primary challenge is a key determining factor.

Tuning into immunological dissonance: an experimental model for infectious mononucleosis.

Virus infections cause a much more profound perturbation of the lymphoid tissue than can be accounted for by the exigencies of the antigen-specific response. The extent of this 'immunological dissonance' is seen most dramatically in mice infected with a persistent gamma-herpesvirus, MHV-68. A profile of massive, continuing proliferation of both T and B cells in the lymph nodes and spleen leads to a dramatic increase in the prevalence of a CD62Llow CD8+ T cell subset in the blood, a pattern first detected two to three weeks after intranasal exposure to the inducing virus. This syndrome, which seems identical to human infectious mononucleosis (IM), persists for a further month or more. Part of the IM-like phase of MHV-68 infection reflects the selective expansion of Vbeta4+ CD8+ T cells, with the Vbeta4 effect being apparent for several different MHC class I H-2 types but not in mice that are deficient in MHC class II glycoprotein expression. Depleting CD4(+) T helper cells in MHV-68-infected mice leads to the decreased proliferation of the CD8+ T cells in the spleen and fewer CD62Llow CD8+ T lymphocytes than would be expected in peripheral blood, but fails to diminish the prominence of the V4beta+ CD8+ population. The results so far of this unique experimental mouse model of IM suggest that both cytokine-mediated effects and a viral superantigen are operating to promote the dramatic expansion and persistence of activated CD8+ T cells in the vascular compartment.

Cortisol mediated suppression of salmonid lymphocyte responses in vitro.

The suppressive activity of cortisol on the in vitro induction of coho salmon (Oncorhynchus kisutch) B cell activation was examined. Suppression was observed with splenic and pronephric (anterior kidney) derived lymphocytes. The kinetics of cortisol-induced suppression revealed distinct differences in the sensitivity of splenic and pronephric lymphocytes. Pronephric lymphocytes were only sensitive to cortisol early in the induction of the antibody response, whereas the splenic cells were sensitive to cortisol throughout the culture period. Addition of supernatants from antigen stimulated pronephric cultures completely restored the ability of pronephric lymphocytes to produce an antibody response, suggesting that this glucocorticoid-suppression may be mediated by inhibition of lymphokine production.

Stress alters immune function and disease resistance in chinook salmon (Oncorhynchus tshawytscha).

We examined the effects of acute stress on the immune system and disease resistance of juvenile chinook salmon (Oncorhynchus tshawytscha) in laboratory and clinical trials. Immune function, as measured by the ability of lymphocytes from the anterior kidney to generate specific antibody-producing cells (APC) in vitro, was depressed 4 h after stress, when plasma cortisol levels were highest. At the same time, resistance to the fish pathogen, Vibrio anguillarum, was also depressed. Compared with controls, plasma cortisol and APC of stressed fish were unchanged after 24 h, and disease resistance was enhanced as evidenced by higher survival rate and longer mean time to death of mortalities. After 7 days, even though numbers of APC were depressed, plasma cortisol concentration and disease resistance did not differ from controls. This pattern was generally the same, independent of the type of stress applied: i.e. being held out of water in a dipnet for 30 s, manipulation during hatchery operations for 4 h, or transportation for 9 h. These and earlier findings suggest that similar endocrine-immune interactions operate in the mammalian and salmonid systems during acute stress.

Primary in vitro stimulation of antibody production by rainbow trout lymphocytes.

Trinitrophenylated (TNP) forms of E. coli lipopolysaccharide (LPS) and keyhole limpet hemocyanin (KLH) were used to produce antigen specific plaque-forming cell (PFC) responses with rainbow trout (Salmo gairdneri) splenocytes from unprimed fish in vitro. The culture system that was developed is described and characterized with respect to the kinetics and dose responses for both the haptenated and unhaptenated forms of the carriers. The induction of the PFC response to TNP-LPS was inhibited with TNP-lysine. Exposure to graded levels of gamma-radiation demonstrated a low dose augmentation of the PFC response with both antigens. Antigen addition experiments reveal that both antigens appear to stimulate the same population of antibody-producing B lymphocytes.

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.

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.

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.

Characteristics of the influenza virus-specific CD8+ T cell response in mice homozygous for disruption of the H-2lAb gene.

The development of influenza virus-specific CD8+ cytotoxic T lymphocyte precursors (CTLp) is diminished in H-2b mice that are homozygous (-/-) for disruption of the H-2lAb gene. Virus clearance was not obviously delayed when compared with the congenic H-2lAb (+/+) controls, and evidence of CTL activity was apparent for inflammatory cells recovered from the respiratory tract in both cases. However, the virus-specific CTLp that are normally present in the regional lymph nodes and in the infected lung were evidently being consumed at the peak of the host response to give the terminally differentiated CTL effectors. Even so, any exhaustion of the CTLp pool was apparently transitory with this localized infection as, though the frequencies were consistently lower than those found for the (+/+) controls, CTLp could be detected reproducibly in both lymph nodes and spleen through 14 mo after infection. Analysis of cytokine profiles during the acute response showed a substantial defect in the (-/-) mice, indicating that cytokine production is largely dependent on the influenza-specific CD4+ set.

Temporal loss of the activated L-selectin-low phenotype for virus-specific CD8+ memory T cells.

Whether the L-selectin-low (L-sel-lo) phenotype of acutely stimulated CD8+ T cells is a permanent characteristic of long-term memory CTL precursors (p) is addressed for mice primed with an influenza A virus or the murine parainfluenza type 1 virus, Sendai virus. In both cases, many of the splenic CD8+ CTLp gradually lose the predominantly L-sel-lo profile associated with recently generated CTLp populations. The influenza-specific CTLp also tend to revert from the activated alpha 4-integrin-high to the resting alpha 4-integrin-low form. The kinetics of the switch back to the "naive" L-sel-hi phenotype differs for the influenza and Sendai virus models, perhaps reflecting events occurring during the acute phases of these responses. The return to being L-sel-hi is not due to irreversible lymphocyte senescence, because restimulation of this set with the inducing virus in vitro causes most of the cells to become L-sel-lo. Also, despite the time-related drift of these particular memory CTLp to the L-sel-hi state, the size of the total pool of L-sel-lo CD8+ T cells increases with age.

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.