Inactivated Influenza Vaccine That Provides Rapid, Innate-Immune- System-Mediated Protection and Subsequent Long-Term Adaptive Immunity

The continual threat to global health posed by influenza has led to increased efforts to improve the effectiveness of influenza vaccines for use in epidemics and pandemics. We show in this study that formulation of a low dose of inactivated detergent-split influenza vaccine with a Toll-like receptor 2 (TLR2) agonist-based lipopeptide adjuvant (R4Pam2Cys) provides (i) immediate, antigen-independent immunity mediated by the innate immune system and (ii) significant enhancement of antigendependent immunity which exhibits an increased breadth of effector function. Intranasal administration of mice with vaccine formulated with R4Pam2Cys but not vaccine alone provides protection against both homologous and serologically distinct (heterologous) viral strains within a day of administration. Vaccination in the presence of R4Pam2Cys subsequently also induces high levels of systemic IgM, IgG1, and IgG2b antibodies and pulmonary IgA antibodies that inhibit hemagglutination (HA) and neuraminidase (NA) activities of homologous but not heterologous virus. Improved primary virus nucleoprotein (NP)-specific CD8! T cell responses are also induced by the use of R4Pam2Cys and are associated with robust recall responses to provide heterologous protection. These protective effects are demonstrated in wild-type and antibody-deficient animals but not in those depleted of CD8! T cells. Using a contact-dependent virus transmission model, we also found that heterologous virus transmission from vaccinated mice to naive mice is significantly reduced. These results demonstrate the potential of adding a TLR2 agonist to an existing seasonal influenza vaccine to improve its utility by inducing immediate short-term nonspecific antiviral protection and also antigen-specific responses to provide homologous and heterologous immunity

Using <i>Wolbachia </i>to eliminate dengue:will the virus fight back?

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here, we review the viral infection stages where selection for Wolbachia-resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures to which dengue virus might adapt in order to be effectively transmitted by Ae. aegypti that carry Wolbachia. While there are hurdles to dengue viruses developing resistance to Wolbachia, we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia-introgression biocontrol programs

Dendritic Cells Generated From Mops condylurus, a Likely Filovirus Reservoir Host, Are Susceptible to and Activated by Zaire Ebolavirus Infection

Ebola virus infection of human dendritic cells (DCs) induces atypical adaptive immune responses and thereby exacerbates Ebola virus disease (EVD). Human DCs, infected with Ebola virus aberrantly express low levels of the DC activation markers CD80, CD86, and MHC class II. The T cell responses ensuing are commonly anergic rather than protective against EVD. We hypothesize that DCs derived from potential reservoir hosts such as bats, which do not develop disease signs in response to Ebola virus infection, would exhibit features associated with activation. In this study, we have examined Zaire ebolavirus (EBOV) infection of DCs derived from the Angolan free-tailed bat species, Mops condylurus. This species was previously identified as permissive to EBOV infection in vivo, in the absence of disease signs. M. condylurus has also been recently implicated as the reservoir host for Bombali ebolavirus, a virus species that is closely related to EBOV. Due to the absence of pre-existing M. condylurus species-specific reagents, we characterized its de novo assembled transcriptome and defined its phylogenetic similarity to other mammals, which enabled the identification of cross-reactive reagents for M. condylurus bone marrow-derived DC (bat-BMDC) differentiation and immune cell phenotyping. Our results reveal that bat-BMDCs are susceptible to EBOV infection as determined by detection of EBOV specific viral RNA (vRNA). vRNA increased significantly 72 h after EBOV-infection and was detected in both cells and in culture supernatants. Bat-BMDC infection was further confirmed by the observation of GFP expression in DC cultures infected with a recombinant GFP-EBOV. Bat-BMDCs upregulated CD80 and chemokine ligand 3 (CCL3) transcripts in response to EBOV infection, which positively correlated with the expression levels of EBOV vRNA. In contrast to the aberrant responses to EBOV infection that are typical for human-DC, our findings from bat-BMDCs provide evidence for an immunological basis of asymptomatic EBOV infection outcomes.Peer Reviewe

Serum Amyloid P Is a Sialylated Glycoprotein Inhibitor of Influenza A Viruses

<div><p>Members of the pentraxin family, including PTX3 and serum amyloid P component (SAP), have been reported to play a role in innate host defence against a range of microbial pathogens, yet little is known regarding their antiviral activities. In this study, we demonstrate that human SAP binds to human influenza A virus (IAV) strains and mediates a range of antiviral activities, including inhibition of IAV-induced hemagglutination (HA), neutralization of virus infectivity and inhibition of the enzymatic activity of the viral neuraminidase (NA). Characterization of the anti-IAV activity of SAP after periodate or bacterial sialidase treatment demonstrated that α(2,6)-linked sialic acid residues on the glycosidic moiety of SAP are critical for recognition by the HA of susceptible IAV strains. Other proteins of the innate immune system, namely human surfactant protein A and porcine surfactant protein D, have been reported to express sialylated glycans which facilitate inhibition of particular IAV strains, yet the specific viral determinants for recognition of these inhibitors have not been defined. Herein, we have selected virus mutants in the presence of human SAP and identified specific residues in the receptor-binding pocket of the viral HA which are critical for recognition and therefore susceptibility to the antiviral activities of SAP. Given the widespread expression of α(2,6)-linked sialic acid in the human respiratory tract, we propose that SAP may act as an effective receptor mimic to limit IAV infection of airway epithelial cells.</p> </div

Sialic acids expressed by SAP are resistant to hydrolysis by the viral NA.

<p>(<b>A</b>) Wells coated with 1 µg/ml of HKx31 were incubated with increasing concentrations of fetuin or asialofetuin (ASF) for 1 hr before addition of 5 µg/ml biotin-labelled SAP. Binding of biotin-labelled SAP to HKx31 was then determined by ELISA. All incubations and washes were performed at 4°C using chilled buffers to inhibit the enzymatic activity of the viral NA. Data show a single titration (left panel) as well as triplicate wells at a specific concentration of fetuin/ASF (right panel). The absorbance of biotin-labelled SAP binding in the absence of fetuin/ASF was 1.558 (±0.105). ***p<0.001, Student’s t-test for equivalent concentrations of fetuin compared to ASF. (<b>B</b>) Purified PTX3 and SAP were subjected to 12% SDS-PAGE under reducing conditions followed by lectin blot using DIG-conjugated MAA (panel i) or SNA (panel ii) for detection of α(2,3)- or α(2,6)-linked SA, respectively. Fetuin was included as a positive control for each SA linkage. (<b>C</b>) SAP (2.5 µg) was incubated for 1 hr at 37°C with sialidase from <i>V. cholerae</i> (25 mU), HKx31 (1/20 dilution of allantoic fluid) or with buffer alone and then analysed by SDS-PAGE and DIG-lectin blot using DIG-SNA (panel i). Ponceau S stain confirmed similar amounts of SAP were transferred to the PVDF membrane prior to the lectin blot (panel ii). (<b>D</b>) ELISA plates coated with (i) fetuin (20 µg/ml), or (ii) SAP (40 µg/ml) were incubated with HKx31 (1/10 dilution of allanotic fluid) or sialidase from <i>V. cholerae</i> (0.5 mU) for 30 min at 37°C and removal of SA was determined using NA assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059623#s2" target="_blank">Material and Methods</a>. Uninfected allantoic fluid (AF) was included as a negative control. Data shows the mean absorbance (±1 SD) of triplicate wells and is representative of at least two independent experiments. ***p<0.001, One-way ANOVA compared to uninfected allantoic fluid control or buffer alone control.</p

HA receptor specificity of H3 subtype IAV strains correlates with sensitivity to SAP and PTX3.

<p>(<b>A</b>) Human erythrocytes were desialylated and subsequently resialylated with α(2,3)- or α(2,6)-sialyltransferases and used in hemagglutination assays. All viruses were adjusted to 64 HAU on native erythrocytes and showed no hemagglutinating activity (<1 HAU) against desialylated erythrocytes (data not shown). (<b>B</b>) Inhibition of HA activity of H3 subtype IAV by pentraxins. SAP or PTX3 were diluted in TBS containing 10 mM Ca²⁺ and used in a standard HI assay. Bars indicate the minimum concentration of SAP or PTX3 required to inhibit 4 HAU of each virus tested. Dashed line represents the highest concentration of inhibitor tested (10 µg/ml). ‘+’ indicates a value of >10 µg/ml. Data are representative of 2 or more independent experiments.</p

Antiviral activities of SAP and PTX3 against IAV and IAV-infected cells.

<p>(<b>A</b>) Inhibition of NA activity by SAP and PTX3. A dilution of HKx31 was mixed with a final concentration of 10 or 1 µg/ml of SAP (white bars) or PTX3 (black bars) in BSA₅-TBS-Ca²⁺ and assayed for NA activity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059623#s2" target="_blank">Materials and Methods</a>. Results are expressed as the percent of NA activity compared to a virus only control. Data shown are the mean (±1 SD) from three independent experiments. *p<0.05, Students <i>t</i>-test, significant difference in levels of NA activity in the presence of SAP compared to an equivalent concentration of PTX3. (<b>B</b>) Neutralization of IAV by SAP and PTX3. Pentraxins were treated with sialidase from <i>Vibrio cholerae</i> (50 mU) in TBS containing 10 mM Ca²⁺ or mock-treated (M) in buffer alone for 30 min at 37°C, and then heated to 56°C for 30 min to inactivate sialidase. Sialidase- (S) or mock- (M) treated pentraxins were mixed with a dilution of HKx31 to give a final concentration of 10 or 1 µg/ml of PTX3 or SAP, and the amount of infectious virus remaining determined by fluorescent-focus reduction assay. Results are expressed as a percent of the number of fluorescent foci in the virus only control. Data show the mean (±1 SD) from three independent experiments. *p<0.05, ***p<0.001, Student’s <i>t</i>-test, significant difference between sialidase-treated sample compared to appropriate mock-treated control. (<b>C</b>) Binding of SAP and PTX3 to IAV-infected MDCK cells. Binding of SAP and PTX3 to uninfected (i, iii) or HKx31-infected MDCK cells (ii, iv) was determined at 6 hrs post-infection. Aliquots of 10⁶ cells were incubated with biotin-labelled SAP (10 µg/ml) or PTX3 (2 µg/ml) in BSA₅-TBS-Ca²⁺ (white histograms) or BSA₅-TBS-EDTA (grey histograms). Binding of biotin-labelled pentraxins was determined by flow-cytometry. Histograms are representative of two independent experiments.</p

SAP enhances the neutralizing activity of SP-D and MBL against WT but not SAP^R Mem/71-Bel.

<p>Dilutions of (i) SAP, SP-D and MBL were prepared in TBS supplemented with 10 mM Ca²⁺ alone, or (ii) SP-D and MBL were prepared in TBS supplemented with 10 mM Ca²⁺ and 0.60 µg/ml SAP, and incubated at 37°C for 15 mins to allow for interactions between different proteins. After this time, an equivalent volume of WT or SAP^R Mem71-Bel was added and incubated at 37°C for 30 mins and the amount of infectious virus remaining was determined by fluorescent-focus reduction assay. Final concentrations after incubation with virus were SAP alone (0.6 µg/ml), SP-D alone (0.3 µg/ml), MBL alone (2.5 µg/ml), SAP+SP-D (0.6 µg/ml +0.3 µg/ml) and SAP+MBL (0.6 µg/ml +2.5 µg/ml final). Neutralization data for WT (left panel) or SAP^R Mem/71-Bel (right panel) are expressed as the mean (±1 SD) percent of the number of fluorescent foci in each virus only control and are pooled data from two independent experiments, each performed in triplicate. **p<0.01, ***p<0.001, One-way ANOVA, Tukey’s post hoc.</p

SAP^R mutants of H3 subtype IAV remain sensitive to inhibition by PTX3.

<p>(<b>A</b>) Binding of SAP or PTX3 to purified Mem/71-Bel WT or Mem/71-Bel SAP^R. Biotin-labelled (i) SAP (2 µg/ml) and (ii) PTX3 (2 µg/ml) diluted in BSA₅-TBST-Ca²⁺ were applied to wells coated with 1 µg/ml or 0.1 µg/ml (as indicated) of purified virus and binding of SAP or PTX3 was determined by ELISA. Equivalent coating levels of purified viruses was confirmed using mAb 165 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059623#s2" target="_blank">Materials and Methods</a> (data not shown). Data represent the mean of three independent experiments (±1 SD). **p<0.01, ***p<0.001, Student’s t-test, comparing SAP^R virus to WT virus for each pentraxin. (<b>B</b>) Neutralization of Mem/71-Bel WT and Mem/71-Bel SAP^R by SAP or PTX3. Viruses were mixed with an equal volume of PTX3 or SAP prepared in TBS/Ca (to a final concentration of 10 or 1 µg/ml of each pentraxin, as indicated), incubated for 30 min at 37°C and the amount of infectious virus remaining was determined by fluorescent-focus reduction assay. Results are expressed as a percent of the number of fluorescent foci in the virus only control and represent the mean (±1 SD) from three independent experiments. ***p<0.001, Student’s t-test, comparing SAP^R to WT virus for each pentraxin. (<b>C</b>) Inhibition of virus-induced hemagglutination by SAP and PTX3. Dilutions of PTX3 or SAP in TBS +10 mM Ca²⁺ were tested for their ability to inhibit hemagglutination by Mem/71-Bel WT (black bars) or Mem/71-Bel SAP^R (white bars). Results show the minimum inhibitory concentration (MIC) of SAP or PTX3 required to inhibit 4 HAU of virus. Dashed line represents the highest concentration of inhibitor tested (10 µg/ml); values above this line designated >10 µg/ml are indicated by a ‘+’. Data represent mean (±1 SD) from three independent experiments. (<b>D</b>) Mem/71-Bel SAP^R virus does not agglutinate erythrocytes treated with <i>S. pneumoniae</i> sialidase. Chicken erythrocytes were treated with 160 mU of <i>S. pneumoniae</i> sialidase for 1 hour at 37°C. Mem/71-Bel (WT, black bars) or Mem/71-Bel SAP^R (SAP^R, white bars) were adjusted to 64 HAU on untreated chicken erythrocytes and assayed for their ability to agglutinate sialidase-treated erythrocytes. Data represent mean (±1 SD) from three independent experiments.</p

Neutralization of RG viruses by SAP or horse serum.

<p>Dilutions of WT or SAP^R PR8-PC/73 HA and PR8-Ud/72 HA viruses in TBS were incubated for 30 min at 37°C with an equivalent volume of (<b>A</b>) SAP, at a final concentration of 20 µg/ml or 2 µg/ml in TBS/Ca, or (<b>B</b>) horse serum (HS), at a final dilution factor (DF) of 100 or 1000 in TBS/Ca, and the amount of infectious virus remaining was determined by fluorescent-focus reduction assay. Data are expressed as a percent of the number of fluorescent foci in the virus only control. Data show the mean of three independent experiments (±1 SD). *p<0.05, **p<0.01, ***p<0.001, Student’s <i>t</i>-test, comparing each SAP^R virus to its appropriate WT control.</p