Lung Viral Loads Research Articles

A novel RSV F-Fc fusion protein vaccine reduces lung injury induced by respiratory syncytial virus infection

Respiratory syncytial virus (RSV) infection causes significant disease in the lower respiratory tract of young children, and there is currently no licensed vaccine to prevent RSV infection. The F glycoprotein is considered a major antigenic target for RSV vaccine development. Recent evidence indicates that the pre-fusion F state, compared with the post-fusion F state, is a superior antigen for generation of neutralizing antibodies. In this study, we developed a novel vaccine antigen, RSV glycoprotein F fused with an IgG Fc fragment (F-Fc). The F-Fc fusion protein is predominantly a hexamer and could be recognized by the pre-fusion F-specific monoclonal antibody D25. Intranasal immunization with the F-Fc fusion protein promoted a protective Th1-biased cellular immune response relative to that promoted by immunization with the F protein. This immunization strategy significantly reduced the lung viral load in mice. Furthermore, immunization with F-Fc reduced lung pathology and the production of pro-inflammatory cytokines and chemokines in the lung after RSV infection. These results suggest that the F-Fc protein may be a safe and effective RSV vaccine candidate.

Prophylactic efficacy of a human monoclonal antibody against MERS-CoV in the common marmoset

Effective antiviral treatments for MERS-CoV are urgently needed. LCA60 is a MERS-CoV-neutralizing monoclonal antibody isolated from a convalescent MERS patient. Previously, it was shown that treatment with LCA60 resulted in reduced disease and virus titers in mouse models of MERS-CoV infection. Here, we tested the prophylactic efficacy of LCA60 in the common marmoset model of MERS-CoV infection. Intravenous administration of LCA60 one day before virus challenge resulted in high levels of MERS-CoV-neutralizing activity in circulating blood. Clinically, there was a moderate benefit of treatment with LCA60 including reduced respiratory involvement. Although viral lung loads were not reduced in LCA60-treated animals as compared to controls, there were fewer pathological changes in the lungs. Thus, prophylactic LCA60 treatment could be implemented to reduce disease burden in contacts of confirmed MERS-CoV patients.

OVX836 a recombinant nucleoprotein vaccine inducing cellular responses and protective efficacy against multiple influenza A subtypes

Inactivated influenza vaccines (IIVs) lack broad efficacy. Cellular immunity to a conserved internal antigen, the nucleoprotein (NP), has been correlated to protection against pandemic and seasonal influenza and thus could have the potential to broaden vaccine efficacy. We developed OVX836, a recombinant protein vaccine based on an oligomerized NP, which shows increased uptake by dendritic cells and immunogenicity compared with NP. Intramuscular immunization in mice with OVX836 induced strong NP-specific CD4+ and CD8+ T-cell systemic responses and established CD8+ tissue memory T cells in the lung parenchyma. Strikingly, OVX836 protected mice against viral challenge with three different influenza A subtypes, isolated several decades apart and induced a reduction in viral load. When co-administered with IIV, OVX836 was even more effective in reducing lung viral load.

Respiratory Syncytial Virus Fusion Protein-encoding DNA Vaccine Is Less Effective in Conferring Protection against Inflammatory Disease than a Virus-like Particle Platform

Formalin-inactivated respiratory syncytial virus (RSV) vaccination causes vaccine-enhanced disease (VED) after RSV infection. It is considered that vaccine platforms enabling endogenous synthesis of RSV immunogens would induce favorable immune responses than non-replicating subunit vaccines in avoiding VED. Here, we investigated the immunogenicity, protection, and disease in mice after vaccination with RSV fusion protein (F) encoding plasmid DNA (F-DNA) or virus-like particles presenting RSV F (F-VLP). F-DNA vaccination induced CD8 T cells and RSV neutralizing Abs, whereas F-VLP elicited higher levels of IgG2a isotype and neutralizing Abs, and germinal center B cells, contributing to protection by controlling lung viral loads after RSV challenge. However, mice that were immunized with F-DNA displayed weight loss and pulmonary histopathology, and induced F specific CD8 T cell responses and recruitment of monocytes and plasmacytoid dendritic cells into the lungs. These innate immune parameters, RSV disease, and pulmonary histopathology were lower in mice that were immunized with F-VLP after challenge. This study provides important insight into developing effective and safe RSV vaccines.

Comparative features of infections of two Massachusetts (Mass) infectious bronchitis virus (IBV) variants isolated from Western Canadian layer flocks

BackgroundInfectious bronchitis virus (IBV) is one of the leading causes of mortality and morbidity in chickens. There are numerous serotypes and variants, which do not confer cross protection resulting in failure of currently used IBV vaccines. Although variant IBV isolates with major genetic differences have been subjected to comparative studies, it is unknown whether minor genetic differences in IBV variants within a serotype are different in terms of pathogenesis and eliciting host responses. Two Massachusetts (Mass) variant IBV isolates recovered from commercial layer flocks in the Western Canadian provinces of Alberta (AB) and Saskatchewan (SK) were compared genetically and evaluated for their pathogenicity, tissue distribution and ability to recruit and replicate in macrophages.ResultsAlthough whole genome sequencing of these two Mass IBV isolates showed low similarity with the M41 vaccinal strain, they had an identical nucleotide sequence at open reading frames (ORFs) 3a, 3b, envelop (E), matrix (M), 5a and 5b. The rest of the ORFs of these 2 IBV isolates showed 99.9% nucleotide similarity. However, upon experimental infection, we found that the IBV isolate originating from AB was different to the one that originated in SK due to higher tracheal lesion scores and lower lung viral replication and lower genome loads in cecal tonsils. Nevertheless, both IBV isolates elicited host responses characterized by significant macrophage recruitment to the respiratory tract and there was evidence that both IBV isolates replicated within tracheal and lung macrophages.ConclusionsOverall, this study shows that Mass variant IBV isolates, although possessing minor genetic variations, can lead to significant differences in pathogenicity in young chickens. Further studies are required to investigate the pathogenicity of these two Mass variant IBV isolates in laying hens.

628. Short-Term Water-Pipe (Shisha) Smoke Exposure Worsens Lung Inflammation in Mice Infected with Respiratory Syncytial Virus

BackgroundWater-pipe smoking (WPS) is becoming popular all over the world and among various populations despite the growing concern about the associated deleterious health effects. Chronic cigarette smoke exposure enhances respiratory syncytial virus (RSV) pathology in mouse airways. However, the effects of exposure to WPS on the course of RSV are not known. Here, we examined the impact of short-term WPS exposure on the course of RSV in a mouse model.MethodsBALB/c mice were exposed to nose-only mainstream WPS for 30 min/day for 4 consecutive days. Control mice were exposed with the same regimen to air only. At the end of the exposure period, WPS-exposed and control mice were intranasally inoculated with RSV A2 strain. The mice were sacrificed on selected days postinfection. Several endpoints including body weight, markers of inflammation (tumor necrosis factor-α [TNF-α] and interleukin 1β [IL1β]) and oxidative stress (superoxide dismutase [SOD]), histopathology and lung viral copies were evaluated.ResultsOn Day 3 post-infection, mice exposed to WPS and infected with RSV exhibited significant weight loss in comparison to control mice (P = 0.002), Figure 1. Lung infection with RSV was also associated with increased albumin in bronchoalvelar lavage fluid, a biomarker that distinguishes infection-induced lung injury from WPS-induced lung injury, Figure 2. Other biomarkers of inflammation (TNF-α and IL1β) and oxidative stress (SOD) increased in both types of injuries. In mice exposed to WPS and infected with RSV, the intensity of inflammatory infiltrates (mainly lymphocytes), the size of damage to the distal airway spaces and the size of involved area were clearly higher than in control mice. Despite the increased disease, lung viral load was not significantly affected by exposure to WPS. ConclusionIn this model, short-term water-pipe smoke exposure resulted in severe RSV disease (increased weight loss) and worsened pathology.Disclosures All authors: No reported disclosures.

Immune Correlates of Protection Induced by Virus-Like Particles Containing 2009 H1N1 Pandemic Influenza HA, NA or M1 Proteins

ABSTRACTInfluenza virus-like particle (VLPs) vaccines are a promising alternative to conventional egg-based vaccines. Evaluation of vaccine efficacy induced by HA-M1 VLPs, NA-M1 VLPs or M1 VLPs against virus challenge infection would provide important insight into vaccine design strategy. In this study, we generated VLPs containing hemagglutinin (HA), neuraminidase (NA) or M1 proteins derived from the A/California/04/09. Mice were immunized intramuscularly with HA-M1, NA-M1 or M1 VLPs and protective immunity was evaluated by assessing lung virus loads against low (5LD50) or high (100LD50) lethal dose of homologous virus challenges. High levels of virus-specific serum IgG antibody responses were induced in mice after HA-M1 VLPs immunization, whereas low or no IgG antibody responses were detected from immunization with NA-M1 VLPs or M1 VLPs, independently. Mice that were immunized with HA-M1 VLPs showed below the limit of detection on lung virus loads against low dose (5LD50) of challenge and significant reduction against high dose (100LD50) of challenge infection. Mice that were immunized with NA-M1 or M1 VLPs also displayed reduced lung viral loads compared to naïve control. In vitro cultures of cells from mouse spleen and bone marrow revealed that HA-M1 VLPs and NA-M1 VLPs induced higher levels of antibody-secreting cell (ASC) responses compared to naïve control, whereas M1 VLPs showed no ASC responses. HA-M1, NA-M1 or M1 VLPs immunization demonstrated varying degree of protection with respect to body weight changes and survival rates, which are consistent with the levels of antibody responses in sera and ASC responses from spleen and bone marrow.

Vaccination by microneedle patch with inactivated respiratory syncytial virus and monophosphoryl lipid A enhances the protective efficacy and diminishes inflammatory disease after challenge.

Intramuscular (IM) vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) failed in clinical trials due to vaccine-enhanced respiratory disease. To test the efficacy of skin vaccination against respiratory syncytial virus (RSV), we investigated the immunogenicity, efficacy, and inflammatory disease after microneedle (MN) patch delivery of FI-RSV vaccine (FI-RSV MN) to the mouse skin with or without an adjuvant of monophosphoryl lipid A (MPL). Compared to IM vaccination, MN patch delivery of FI-RSV was more effective in clearing lung viral loads and preventing weight loss, and in diminishing inflammation, infiltrating immune cells, and T helper type 2 (Th2) CD4 T cell responses after RSV challenge. With MPL adjuvant, MN patch delivery of FI-RSV significantly increased the immunogenicity and efficacy as well as preventing RSV disease as evidenced by lung viral clearance and avoiding pulmonary histopathology. Improved efficacy and prevention of disease by FI-RSV MN with MPL were correlated with no sign of airway resistance, lower levels of Th2 cytokines and infiltrating innate inflammatory cells, and higher levels of Th1 T cell responses into the lung. This study suggests that MN patch delivery of RSV vaccines to the skin with MPL adjuvant would be a promising vaccination method.

Co-stimulation With TLR7 Agonist Imiquimod and Inactivated Influenza Virus Particles Promotes Mouse B Cell Activation, Differentiation, and Accelerated Antigen Specific Antibody Production.

Current influenza vaccines have relatively low effectiveness, especially against antigenically drifted strains, the effectiveness is even lower in the elderly and immunosuppressed individuals. We have previously shown in a randomized clinical trial that the topical application of a toll-like receptor 7 agonist, imiquimod, just before intradermal influenza vaccine could expedite and augment antibody response, including to antigenically-drifted strains. However, the mechanism of this vaccine and imiquimod combination approach is poorly understood. Here, we demonstrated that imiquimod alone directly activated purified mouse peritoneal B cells. When combined with inactivated H1N1/415742Md influenza virus particle (VP) as vaccine, co-stimulation of mouse peritoneal B cells in vitro induced stronger activation, proliferation, and production of virus-antigen specific IgM and IgG. Intraperitoneal injection of a combination of VP and imiquimod (VCI) was associated with an increased number of activated B cells with enhanced expression of CD86 in the mesenteric draining lymph nodes (mesLN) and the spleen at 18 h after injection. Three days after immunization with VCI, mouse spleen showed significantly more IgM and IgG secreting cells upon in vitro re-stimulation with inactivated virus, mouse sera were detected with viral neutralizing antibody. Transfer of these spleen B cells to naïve mice improved survival after lethal dose of H1N1/415742Md challenge. More importantly, the functional response of VCI-induced B cell activation was demonstrated by early challenge with a lethal dose of H1N1/415742Md influenza virus at 3 days after immunization. The spleen and mediastinal lymph nodes (mdLN) in mice immunized with VCI had germinal center formation, and significantly higher number of plasmablasts, plasma cells, and virus-antigen specific IgM and IgG secreting cells at only 3–4 days post virus challenge, compared with those of mice that have received imiquimod, inactivated virus alone or PBS. Serum virus-specific IgG2a, IgG2b, and IgG1 and bronchoalveolar lavage fluid (BALF) virus-specific IgA at 3 or 4 days post challenge were significantly higher in mice immunized with VCI, which had significantly reduced lung viral load and 100% survival. These findings suggested that imiquimod accelerates the vaccine-induced antibody production via inducing rapid differentiation of naïve B cells into antigen-specific antibody producing cells.

Salmonella Gallinarum delivering M2eCD40L in protein and DNA formats acts as a bivalent vaccine against fowl typhoid and H9N2 infection in chickens

Fowl typhoid (FT), a septicemic disease caused by Salmonella Gallinarum (SG), and H9N2 influenza infection are two economically important diseases that affect poultry industry worldwide. Herein, we exploited a live attenuated SG mutant (JOL967) to deliver highly conserved extracellular domains of H9N2 M2 (M2e) to induce protective immunity against both H9N2 infection and FT. To increase the immunogenicity of M2e, we physically linked it with CD40L and cloned the fusion gene into either prokaryotic constitutive expression vector pJHL65 or mammalian expression vector pcDNA3.1+. Then pJHL65-M2eCD40L or pcDNA-M2eCD40L recombinant plasmid was electroporated into JOL967 strain and the resultant clones were designated as JOL2074 and JOL2076, respectively. We demonstrated that the chickens vaccinated once orally with a co-mix of JOL2074 and JOL2076 strains elicited significantly (p < 0.05) higher M2e-specific humoral and cell-mediated immunity compared to JOL2074 alone vaccinated group. However, SG-specific immune responses were comparable in both the vaccination groups. On challenge with the virulent H9N2 virus (105 TCID50) at 28th day post-vaccination, chickens that received a co-mix of JOL2074 plus JOL2076 strains exhibited significantly (p < 0.05) lower lung inflammation and viral load in both lungs and cloacal samples than JOL2074 alone vaccinated group. Against challenge with the lethal wild-type SG, both the vaccination groups exhibited only 12.5% mortality compared to 75% mortality observed in the control group. In conclusion, we show that SG delivering M2eCD40L can act as a bivalent vaccine against FT and H9N2 infection and further studies are warranted to develop this SG-M2eCD40L vaccine as a broadly protective vaccine against avian influenza virus subtypes.

Recombinant Porcine Reproductive and Respiratory Syndrome Virus Expressing Membrane-Bound Interleukin-15 as an Immunomodulatory Adjuvant Enhances NK and γδ T Cell Responses and Confers Heterologous Protection.

Cytokines are often used as adjuvants to improve vaccine immunogenicity, since they are important in initiating and shaping the immune response. The available commercial modified live-attenuated vaccines (MLVs) against porcine reproductive and respiratory syndrome virus (PRRSV) are unable to mount sufficient heterologous protection, as they typically induce weak innate and inadequate T cell responses. In this study, we investigated the immunogenicity and vaccine efficacy of recombinant PRRSV MLVs incorporated with the porcine cytokine interleukin-15 (IL-15) or IL-18 gene fused to a glycosylphosphatidylinositol (GPI) modification signal that can anchor the cytokines to the cell membrane. We demonstrated that both cytokines were successfully expressed on the cell membrane of porcine alveolar macrophages after infection with recombinant MLVs. Pigs vaccinated with recombinant MLVs or the parental Suvaxyn MLV had significantly reduced lung lesions and viral RNA loads in the lungs after heterologous challenge with the PRRSV NADC20 strain. The recombinant MLVs SUV-IL-15 and SUV-IL-18 recovered the inhibition of the NK cell response seen with Suvaxyn MLV. The recombinant MLV SUV-IL-15 significantly increased the numbers of gamma interferon (IFN-γ)-producing cells in circulation at 49 days postvaccination (dpv), especially for IFN-γ-producing CD4- CD8+ T cells and γδ T cells, compared to the Suvaxyn MLV and SUV-IL-18. Additionally, MLV SUV-IL-15-vaccinated pigs also had elevated levels of γδ T cell responses observed at 7 dpv, 49 dpv, and 7 days postchallenge. These data demonstrate that the recombinant MLV expressing membrane-bound IL-15 enhances NK and T cell immune responses after vaccination and confers improved heterologous protection, although this was not statistically significant compared to the parental MLV.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) has arguably been the most economically important global swine disease, causing immense economic losses worldwide. The available commercial modified live-attenuated vaccines (MLVs) against PRRS virus (PRRSV) are generally effective against only homologous or closely related virus strains but are ineffective against heterologous strains, partially due to the insufficient immune response induced by the vaccine virus. To improve the immunogenicity of MLVs, in this study, we present a novel approach of using porcine IL-15 or IL-18 as an adjuvant by directly incorporating its encoding gene into a PRRSV MLV and expressing it as an adjuvant. Importantly, we directed the expression of the incorporated cytokines to the cell membrane surface by fusing the genes with a membrane-targeting signal from CD59. The recombinant MLV virus expressing the membrane-bound IL-15 cytokine greatly enhanced NK cell and γδ T cell responses and also conferred improved protection against heterologous challenge with the PRRSV NADC20 strain.

A viral-vectored RSV vaccine induces long-lived humoral immunity in cotton rats

Human respiratory syncytial virus (RSV) is the leading cause of lower airway disease in infants worldwide and repeatedly infects immunocompetent individuals throughout life. Severe lower airway RSV infection during infancy can be life-threatening, but is also associated with important sequelae including development of asthma and recurrent wheezing in later childhood. The basis for the inadequate, short-lived adaptive immune response to RSV infection is poorly understood, but it is widely recognized that RSV actively antagonizes Type I interferon (IFN) production. In addition to the induction of the anti-viral state, IFN production during viral infection is critical for downstream development of robust, long-lived immunity. Based on the hypothesis that a vaccine that induced robust IFN production would be protective, we previously constructed a Newcastle disease virus-vectored vaccine that expresses the F glycoprotein of RSV (NDV-F) and demonstrated that vaccinated mice had reduced lung viral loads and an enhanced IFN-γ response after RSV challenge. Here we show that vaccination also protected cotton rats from RSV challenge and induced long-lived neutralizing antibody production, even in RSV immune animals. Finally, pulmonary eosinophilia induced by RSV infection of unvaccinated cotton rats was prevented by vaccination. Overall, these data demonstrate enhanced protective immunity to RSV F when this protein is presented in the context of an abortive NDV infection.

Long duration of immunity against a type 1 heterologous PRRS virus challenge in pigs immunised with a novel PRRS MLV vaccine: a randomised controlled study

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) is widespread in commercial pig farms worldwide, and has a significant cost to the swine industry. Herd owners need a vaccine that will confer long-lasting immunity to prevent PRRSV infection and transmission. The studies described here evaluated duration of immunity conferred by a European-derived PRRS (isolate 94,881) modified live virus (MLV) vaccine, Ingelvac PRRSFLEX® EU, at 20, 24, and 26 weeks post-vaccination. Primary endpoints were the assessment of gross and histological lung lesions and viral RNA load in lung tissue 10 days following heterologous PRRSV challenge. Secondary endpoints included clinical observations, average daily weight gain (ADWG) and viral RNA load in serum 10 days post-challenge. Three blinded, vaccination-challenge efficacy studies were performed using separate cohorts of pigs (n = 56 per study). Pigs received either Ingelvac PRRSFLEX® EU (Group 1) or placebo (Groups 2 and 3). Groups 1 and 2 were subsequently challenged with heterologous European PRRSV isolate 205,817 at 20, 24 or 26 weeks post-vaccination.ResultsMean gross lung lesion scores were significantly lower in Group 1 than in Group 2 at 24 and 26 weeks (p < 0.0001), but not at 20 weeks (p = 0.299). Significantly lower mean histological lung lesion scores were observed in Group 1 versus Group 2 at 20 (p = 0.0065), 24 (p < 0.0001) and 26 weeks (p < 0.0001). Mean viral RNA load in lung tissue was significantly lower in Group 1 than in Group 2 (p < 0.0001) at 20 (p < 0.0001), 24 (p < 0.0001) and 26 weeks (p < 0.0001). Cumulative viral RNA loads in serum during days 1–10 post-challenge were significantly lower in Group 1 than in Group 2 (p < 0.0001) in all studies. A significant increase in ADWG was observed in Group 1 compared with Group 2 at 20 weeks (p = 0.0027) and 24 weeks (p = 0.0004), but not at 26 weeks (p = 0.1041). There were no significant differences in clinical signs post-challenge in any study.ConclusionThese results suggest that Ingelvac PRRSFLEX® EU confers long-term immunity to European heterologous PRRSV, which is maintained up to 26 weeks after vaccination, corresponding to the expected lifespan of commercial pigs.

Sheng Jiang San, a traditional multi-herb formulation, exerts anti-influenza effects in vitro and in vivo via neuraminidase inhibition and immune regulation

BackgroundSheng Jiang San (SJS), a multi-herb formulation, is used in treating high fever, thirsty and anxiety in ancient China and it is sometimes used to treat seasonal influenza nowadays. However, there is no evidence-based investigation and mechanism research to support the anti-influenza efficacy of SJS. This study aims at evaluating the anti-influenza effect of SJS and investigating its possible mechanism.MethodsThe inhibitory effect of SJS against different influenza virus strains on MDCK cells was examined. Influenza virus infected BALB/c mice were employed to evaluate the efficacy as in vivo model. Mice challenged with A/PR/8/34 (H1N1) were orally administrated 1 g/kg/day of SJS for seven days and monitored for 14 days. The survival rate, body weight changes, lung index, lung viral load, histopathologic changes and immune regulation of the mice were measured. The underlying anti-influenza virus mechanism of SJS was studied by a series of biological assays to determine if hemagglutinin, ribonucleoprotein complex or neuraminidase were targets of SJS.ResultsResults showed SJS exerted a broad-spectrum of inhibitory effects on multiple influenza strains in a dose-dependent manner. IC50 of SJS against A/WSN/33 (H1N1) was lower than 35 μg/ml. SJS also protected 50% of mice from A/PR/8/34 (H1N1) infection. The lung index and the lung viral load of SJS treated mice were significantly decreased compared with untreated mice. Meanwhile, SJS targeted on neuraminidase of influenza virus as SJS at 2 mg/ml inhibited 80% of neuraminidase enzymatic activity. SJS also significantly down-regulated TNF-α and up-regulated IL-2 of influenza virus induced mice.ConclusionsThus, SJS is a useful formulation for treating influenza virus infection.

A unique adjuvant combination modulates immune responses preventing vaccine-enhanced pulmonary histopathology after vaccination with fusion protein and challenge with respiratory syncytial virus

Abstract Vaccination of young children with alum-adjuvanted formalin-inactivated respiratory syncytial virus (FI-RSV) induced vaccine-enhanced disease (VED) after natural infection during epidemic season, resulting in hospitalizations and two deaths. There is no licensed RSV vaccine. VED was also observed in previous reports with fusion (F) protein RSV vaccines and adjuvants including oil-in-water emulsion, delta-inulin, and natural killer T cell agonist α-GalCer. In this study to search for adjuvants preventing RSV VED, we investigated a novel combination of monophosphoryl lipid A (MPL TLR4 agonist) and oligodeoxynucleotide CpG (CpG TLR9 agonist) in the RSV F protein vaccination. RSV specific IgG antibodies and neutralizing titers, and controlling RSV lung viral loads after challenge were similarly observed in the alum and MPL+CpG adjuvant groups of mice that received a single dose prime immunization with RSV F protein vaccine. Analysis of IgG isotypes and cytokines suggested the induction of T helper type 1 (Th1) immune responses by inclusion of MPL+CpG adjuvant in contrast to alum adjuvant biasing Th2 type immune responses. Most importantly, lung histopathology and infiltration of inflammatory innate immune cells (eosinophils, neutrophils) were not observed after F protein vaccination with MPL+CpG adjuvant whereas severe lung histopathology was induced after alum adjuvanted F protein vaccination and RSV challenge. The results in this study suggest a novel adjuvant approach to improve efficacy and safety of RSV subunit vaccines.

Engystol reduces onset of experimental respiratory syncytial virus-induced respiratory inflammation in mice by modulating macrophage phagocytic capacity

BackgroundRespiratory viruses such as respiratory syncytial virus (RSV) or rhinovirus are one of the major causes for respiratory tract infections causing common cold disease. Respiratory viral infections range from mild symptoms in adults to serious illness especially in the very young or elderly as well as patients suffering from lung diseases or being immunocompromised due to other reasons. Engystol (EGY-2) is a multicomponent, multitarget preparation consisting of Vincetoxicum hirundinaria and Sulfur in various dilutions. The study objective was to test the effect of EGY-2 on the innate immune response during the early onset of respiratory viral infection in vivo as exemplified in a mouse model of RSV-induced respiratory inflammation.MethodsNaïve BALB/c mice were infected with 1x106 infectious units RSV A2 intranasally to cause a mild respiratory infection. EGY-2 was administered daily per oral gavage starting seven days prior to RSV infection at doses of 0.4 to 5.1 tablets/kg. Control groups received placebo treatment. Animals were sacrificed 1 to 3 days post infection (p.i.) to analyse the infection and induced immune response in the lung. Viral load in bronchoalveolar lavage fluid (BALF) and lung homogenate was determined by TCID50 assay as well as immunofluorescence staining of BALF cells using anti-RSV antibody and microscopic analysis. The RSV induced immune response was assessed by evaluation of BALF differential cell count, BALF cytokine secretion and analysis of the phagocytic capacity of alveolar macrophages.ResultsEGY-2 significantly reduced the RSV induced neutrophil and early lymphocyte influx on day 1 p.i. in BALF. EGY-2 treatment significantly diminished the RSV induced secretion of pro-inflammatory cytokines such as IFN-γ, IL-1β, IL-6, KC and TNF-α at day 1. EGY-2 treatment was not protective for RSV infection per se, as no alteration in the viral load in lung and BALF was detected. Enhanced numbers of phagocytic-active macrophages were observed in EGY-2 treated animals on day 1 and this macrophage population showed strongly enhanced phagocytic activity on day 1 and day 3.ConclusionThe data suggest a beneficial immunomodulatory effect of EGY-2 during early onset of respiratory viral infection in vivo, mediated by stimulation of macrophage phagocytosis, resulting in a reduced innate inflammatory response in terms of neutrophil and early lymphocyte infiltration as well as reduced inflammatory cytokine secretion.

A recombinant trivalent vaccine candidate against human adenovirus types 3, 7, and 55

Human adenoviruses types 3 (HAdV-3), 7 (HAdV-7) and 55 (HAdV-55) are major pathogens of acute respiratory infections (ARI) in children and adults. More than one type of HAdV can infect patients simultaneously, and the infections are sometimes fatal. However, there is currently no vaccine approved for general use in children and adults. Thus, development of a multivalent HAdV vaccine to combat HAdV infection becomes imperative. In this study, we constructed a new recombinant trivalent human adenovirus vaccine (rAdMHE3-h55), which expresses the hexon protein of HAdV-55 in the E3 region of rAdMHE3, a previously prepared bivalent vaccine candidate against HAdV-3 and HAdV-7. The results of in vitro neutralization assays indicate that rAdMHE3-h55 can induce the production of neutralizing antibodies against HAdV-3, HAdV-7, and HAdV-55 in mice. Furthermore, immunization with the recombinant trivalent vaccine candidate completely protected the mice challenged with HAdV-3, HAdV-7, orHAdV-55, respectively, showing lower lung viral loads and less lung Pathological changes was compared with those in unvaccinated mice. The current findings contribute to the development of a new adenovirus vaccine candidate and also advance this construction method for the generation of recombinant adenovirus vaccines. In conclusion, our recombinant trivalent vaccine rAdMHE3-h55 can provides protection against challenge with HAdV-3, HAdV-7, or HAdV-55 in mice. Future work of optimizing this vaccine candidate may lead to a more effective way of preventing respiratory diseases caused by common human adenoviruses.

A Model to Detect Autochthonous Group 1 and 2 Brazilian Vaccinia virus Coinfections: Development of a qPCR Tool for Diagnosis and Pathogenesis Studies.

Vaccinia virus (VACV) is the etiological agent of bovine vaccinia (BV), an emerging zoonosis that has been associated with economic losses and social effects. Despite increasing reports of BV outbreaks in Brazil, little is known about the biological interactions of Brazilian VACV (VACV-BR) isolates during coinfections; furthermore, there are no tools for the diagnosis of these coinfections. In this study, a tool to co-detect two variants of VACV was developed to provide new information regarding the pathogenesis, virulence profile, and viral spread during coinfection with VACV-BR isolates. To test the quantitative polymerase chain reactions (qPCR) tool, groups of BALB/c mice were intranasally monoinfected with Pelotas virus 1—Group II (PV1-GII) and Pelotas virus 2—Group I (PV2-GI), or were coinfected with PV1-GII and PV2-GI. Clinical signs of the mice were evaluated and the viral load in lung and spleen were detected using simultaneous polymerase chain reactions (PCR) targeting the A56R (hemagglutinin) gene of VACV. The results showed that qPCR for the quantification of viral load in coinfection was efficient and highly sensitive. Coinfected mice presented more severe disease and a higher frequency of VACV detection in lung and spleen, when compared to monoinfected groups. This study is the first description of PV1 and PV2 pathogenicity during coinfection in mice, and provides a new method to detect VACV-BR coinfections.

Cross-Protective Efficacy of Influenza Virus M2e Containing Virus-Like Particles Is Superior to Hemagglutinin Vaccines and Variable Depending on the Genetic Backgrounds of Mice.

Influenza virus M2 extracellular domain (M2e) has been a target for developing cross-protective vaccines. However, the efficacy and immune correlates of M2e vaccination are poorly understood in the different host genetic backgrounds in comparison with influenza vaccines. We previously reported the cross-protective efficacy of virus-like particle (M2e5x VLP) vaccines containing heterologous tandem M2e repeats (M2e5x) derived from human, swine, and avian influenza viruses. In this study to gain better understanding of cross-protective influenza vaccines, we compared immunogenicity and efficacy of M2e5x VLP, H5 hemagglutinin VLP (HA VLP), and inactivated H3N2 virus (H3N2i) in wild-type strains of BALB/c and C57BL/6 mice, and CD4 and CD8 knockout (KO) mice. M2e5x VLP was superior to HA VLP in conferring cross-protection whereas H3N2i inactivated virus vaccine provided high efficacy of homologous protection. After M2e5x VLP vaccination and challenge, BALB/c mice induced higher IgG responses, lower lung viral loads, and less body weight loss when compared with those in C57BL/6 mice. M2e5x VLP but not H3N2i immune mice after primary challenges developed strong immunity against a secondary heterosubtypic virus as a model of future pandemics. M2e5x VLP and HA VLP vaccines were able to raise IgG isotypes in CD4 KO mice. T cells were found to contribute to cross-protection by playing a role in reducing lung viral loads. In conclusion, M2e5x VLP vaccination induced better cross-protection than HA VLP, and its efficacy varied depending on the genetic backgrounds of mice, supporting the important roles of T cells.

Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are three of the most economically important swine pathogens, causing immense economic losses to the global swine industry. Monovalent commercial vaccines against each of the three viruses are routinely used in pig farms worldwide. A trivalent vaccine against all three pathogens would greatly simplify the vaccination programme and reduce the financial burden to the swine industry. In this study, by using an attenuated strain of PRRSV (strain DS722) as a live virus vector, we generated a multi-component vaccine virus, DS722-SIV-PCV2, which expresses the protective antigens from SIV and PCV2. The DS722-SIV-PCV2 trivalent vaccine virus replicates well, and expresses PCV2 capsid and SIV HA proteins in vitro. A subsequent vaccination and challenge study in 48 pigs revealed that the DS722-SIV-PCV2-vaccinated pigs had significantly reduced lung lesions and viral RNA loads when challenged with PRRSV. Upon challenge with PCV2, the vaccinated pigs had partially reduced lymphoid lesions and viral DNA loads, and when challenged with SIV the vaccinated pigs had significantly reduced acute respiratory sign scores. The results from this study demonstrate the potential of DS722-SIV-PCV2 as a candidate trivalent vaccine, and also shed light on exploring PRRSV as a potential live virus vaccine vector.