Experimental Influenza Research Articles

Prophylactic and therapeutic mouse models for evaluating immunologic resilience to infection with influenza virus by Immulina® (Part 1)

BackgroundIllness resulting from influenza is a global health problem that has significant adverse socioeconomic impact. Although various strategies such as flu vaccination have beneficial effects, the risk of this illness has not been eliminated. The use of botanicals may provide a complementary approach by enhancement of the host antiviral immune response. PurposeGenerate preclinical data using rodent models to determine the most effective utility of a Limnospira (formerly Arthrospira)-derived oral supplement (Immulina®) for enhancing host immunity to improve antiviral resilience. Study designTwo non-lethal mouse models (prophylactic and therapeutic) were used to evaluate the impact of Immulina® on increasing host resilience against experimental influenza infection. MethodsMice were fed Immulina® only for the 2 weeks prior to viral infection (prophylactic regime) or starting 3 days post-viral infection (at the onset of symptoms, therapeutic design). Three doses of Immulina® were evaluated in each model using both female and male mice. ResultsSignificant protective effect of Immulina® against viral illness was observed in the prophylactic model (improved clinical scores, less body weight loss, decreased lung/body weight ratio, lower lung viral load, and increased lung IFN-γ and IL-6). Substantially less (minimal) protective effect was observed in the therapeutic model. ConclusionThis study demonstrates that Immulina® exerts a protective effect against influenza illness when administered using a prophylactic regime and may not be effective if given after the onset of symptoms. The results will help to optimally design future clinical trials.

Study of the Protective Activity of Thymogen® Dosed Nasal Spray on a Model of Experimental Influenza Pneumonia in Laboratory Animals

Introduction. Due to the ability of the influenza virus to mutate, it is necessary to constantly search for new drugs with preventive and direct antiviral effects.The aim of the study is to investigate the protective antiviral properties of the drug Thymogen®, a dosed nasal spray, on a model of lethal influenza pneumonia in laboratory animals.Material and methods. White mature mice were selected for the experiment on the antiviral activity of the tested drug samples against H1N1 influenza virus; the mice were divided into the following groups (30 animals each): two negative control groups; two groups receiving Thymogen®, a dosed nasal spray (before and after infection); one group of animals received Tamiflu®, and one received Hexoral®. The mortality of animals with influenza pneumonia was assessed; histological and morphometric analyses were also carried out.Results and discussion. The morphological picture of the lungs of animals correlated with their protective activity when analyzing the survival of animals. The normalization of tissue structure was pronounced the most in the group of animals infected with the virus, pre-incubated with Thymogen®, a dosed nasal spray, and in the group receiving Tamiflu®. Moreover, the viral load in the lungs was lower by 1.5 and 1.2 orders of magnitude in the group receiving Thymogen®, dosed nasal spray, compared to the negative control group (the 3rd and the 6th days of the experiment, respectively), which corresponds to a decrease in the intensity of viral reproduction by 31.6 and 15.8 times

Antiviral Activity of Double-Stranded Ribonucleic Acid and Interferon Alpha Composition in the Model of Experimental Influenza Infection of Mice

High variability of influenza viruses requires the development of agents for nonspecific resistance stimulation, along with the development of new drugs for prevention and treatment. Among the antiviral drugs, interferons and their inducers are known to exhibit a universally wide spectrum of action. The Institute of Medical Biotechnology, a branch of SRC VB «Vector», Rospotrebnadzor, has developed the technology and obtained pharmaceutical compositions containing an interferon inducer — double stranded ribonucleic acid (dsRNA) from the killer strain of Saccharomyces cerevisiae yeast and recombinant human interferon-alpha-2b (IFN- alpha-2b). The specific antiviral activity of the preparations and synergistic effect of the components within the compositions were shown in L-68 and L-929 cell cultures. The aim of this work was to study antiviral activity of intranasal forms of the pharmaceutical compositions containing yeast dsRNA and recombinant human interferon-alpha-2b in a model of lethal influenza infection in mice. The outbred ICR/CD1 mice were intranasally infected with influenza A/Aichi/2/68 (H3N2) virus. The study compositions were intranasally administered 3 hours before infection with influenza virus, as well as 1 and 3 days post infection. The doses of active components in the administered compositions were as follows: for dsRNA — 2.5 mg/kg, for IFN-alpha-2b — 500 IU/kg, 2500 IU/kg, or 5000 IU/kg. The antiviral activity of the drugs was assessed based on the mortality rate and the average life expectancy of mice. It was shown that a three-time intranasal administration of the composition of dsRNA (2.5 mg/kg) and IFN-alpha-2b (2500 IU) into the infected mice according to therapeutic-prophylactic regimen led to an increase in the rates of survival and average life expectancy of animals, which were comparable to the effect of Tamiflu. The comparison preparations — dsRNA and IFN-alpha-2b — administered intranasally at the same doses and regimen exerted no antiviral effect in this mouse model of viral infection. The data obtained confirm the prospects for further development of new dosage forms of dsRNA and interferons for intranasal application as agents for prevention and treatment of influenza.

An experimental universal swine influenza a virus (IAV) vaccine candidate based on the M2 ectodomain (M2e) peptide does not provide protection against H1N1 IAV challenge in pigs

Swine flu is a common disease problem in North American pig populations and swine influenza A viruses (IAV) are extremely diverse and the lack of cross protection between heterologous strains is impacting vaccine efficacy in the field. The objective of this study was to design and test a novel swine flu vaccine targeting the M2 ectodomain (M2e) of IAV, a highly conserved region within the IAV proteome. In brief, an M2e peptide was designed to match the predominant swine IAV M2 sequence based on global analysis of sequences from pigs and humans. The resulting sequence was used to synthesize the M2e peptide coupled to a carrier protein. The final vaccine concentration was 200 µg per dose, and a commercial, microemulsion-based aqueous adjuvant was added. Nine 3-week-old IAV negative piglets were randomly assigned to three groups and rooms including non-vaccinated pigs (NEG-CONTROLs) and vaccinated pigs using the intramuscular (M2e-IM) or the intranasal route (M2e-IN). Vaccinations were done at weaning and again at 2 weeks later. An in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated to study the M2e IgG antibody response and demonstrated M2e-IM pigs had a higher systemic antibody response compared to M2e-IN pigs. Subsequently, an IAV challenge study was conducted. The results indicated that M2e-IM vaccinated pigs were not protected from H1N1 (US pandemic clade, global clade 1A.3.3.2) challenge despite having a strong humoral anti-M2e immune response. In conclusion, while the experimental IAV vaccine was able to induce anti-M2e antibodies, when challenged with H1N1, the vaccinated pigs were not protected, perhaps indicating that reactivity to the M2e antigen alone is not sufficient to reduce clinical signs, lesions or shedding associated with experimental IAV challenge.

Influenza A(H1N1)pdm09 Virus Alters Expression of Endothelial Factors in Pulmonary Vascular Endothelium in Rats.

Influenza virus infection may cause endothelial activation and dysfunction. However, it is still not known to what extent the influenza virus can dysregulate the expression of various endothelial proteins. The aim of the study is to identify the level of expression of endothelial nitric oxide synthase (eNOS), plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (tPA) in the pulmonary vascular endothelium, as well as the concentration of PAI-1 and tPA in the blood plasma in Wistar rats. Animals were intranasally infected with rat-adapted influenza A(H1N1)pdm09 virus. The expression of eNOS, PAI-1 and tPA in the pulmonary vascular endothelium was determined by immunohistochemistry; the concentration of PAI-1 and tPA was analyzed by ELISA at 24 and 96 h post infection (hpi). Thus, the expression of eNOS in the pulmonary vascular endothelium decreased by 1.9-fold at 24 hpi and increased by 2-fold at 96 hpi. The expression of PAI-1 in the pulmonary vascular endothelium increased by 5.23-fold and 6.54-fold at 24 and 96 hpi, respectively. The concentration of PAI-1 in the blood plasma of the rats decreased by 3.84-fold at 96 hpi, but not at 24 hpi. The expression of tPA in the pulmonary vascular endothelium was increased 2.2-fold at 96 hpi. The obtained data indicate the development of endothelial dysfunction that is characterized by the dysregulation of endothelial protein expression in non-lethal and clinically non-severe experimental influenza virus infection.

H1N1 G4 swine influenza T cell epitope analysis in swine and human vaccines and circulating strains uncovers potential risk to swine and humans.

Pandemic influenza viruses may emerge from animal reservoirs and spread among humans in the absence of cross-reactive antibodies in the human population. Immune response to highly conserved T cell epitopes in vaccines may still reduce morbidity and limit the spread of the new virus even when cross-protective antibody responses are lacking. We used an established epitope content prediction and comparison tool, Epitope Content Comparison (EpiCC), to assess the potential for emergent H1N1 G4 swine influenza A virus (G4) to impact swine and human populations. We identified and computed the total cross-conserved T cell epitope content in HA sequences of human seasonal and experimental influenza vaccines, swine influenza vaccines from Europe and the United States (US) against G4. The overall T cell epitope content of US commercial swine vaccines was poorly conserved with G4, with an average T cell epitope coverage of 35.7%. EpiCC scores for the comparison between current human influenza vaccines and circulating human influenza strains were also very low. In contrast, the T cell epitope coverage of a recent European swine influenza vaccine (HL03) was 65.8% against G4. Poor T cell epitope cross-conservation between emergent G4 and swine and human influenza vaccines in the US may enable G4 to spread in swine and spillover to human populations in the absence of protective antibody response. One European influenza vaccine, HL03, may protect against emergent G4. This study illustrates the use of the EpiCC tool for prospective assessment of existing vaccine strains against emergent viruses in swine and human populations.

Alternating 3 different influenza vaccines for swine in Europe for a broader antibody response and protection

Heterologous prime-boost vaccination with experimental or commercial influenza vaccines has been successful in various animal species. In this study, we have examined the efficacy of alternating 3 different European commercial swine influenza A virus (swIAV) vaccines: the trivalent Respiporc® FLU3 (TIV), the bivalent GRIPORK® (BIV) and the monovalent Respiporc® FLUpan H1N1 (MOV). Five groups of 6 pigs each received 3 vaccinations at 4–6 week intervals in a homologous or heterologous prime-boost regimen. A sixth group served as a mock-vaccinated challenge control. Four weeks after the last vaccination, pigs were challenged intranasally with a European avian-like H1N1 (1C.2.1) swIAV, which was antigenically distinct from the vaccine strains. One heterologous prime-boost group (TIV–BIV–MOV) had higher hemagglutination inhibition (HI) and neuraminidase inhibition antibody responses against a panel of antigenically distinct H1N1, H1N2 and H3N2 IAVs than the other heterologous prime-boost group (BIV–TIV–MOV) and the homologous prime-boost groups (3xTIV; 3xBIV; 3xMOV). Group TIV–BIV–MOV had seroprotective HI titers (≥ 40) against 56% of the tested viruses compared to 33% in group BIV–TIV–MOV and 22–39% in the homologous prime-boost groups. Post-challenge, group TIV–BIV–MOV was the single group with significantly reduced virus titers in all respiratory samples compared to the challenge control group. Our results suggest that the use of different commercial swIAV vaccines for successive vaccinations may result in broader antibody responses and protection than the traditional, homologous prime-boost vaccination regimens. In addition, the order in which the different vaccines are administered seems to affect the breadth of the antibody response and protection.

Influenza A Virus Causes Histopathological Changes and Impairment in Functional Activity of Blood Vessels in Different Vascular Beds.

It has been established that blood vessels are a target for influenza virus; however, the mechanism by which virus affects the cardiovascular system remains unknown. The aim of the study is the identification of histological changes and changes in the functional activity of the pulmonary and mesenteric blood vessels of Wistar rats. Wistar rats were intranasally infected with the influenza A(H1N1)pdm09 virus. At 24 and 96 h post infection (hpi), histopathological changes were observed in lung tissues with the absence of histological changes in mesenteric tissues. The functional activity of pulmonary and mesenteric arteries was determined using wire myography. In pulmonary arteries, there was a tendency towards an increase in integral response to the vasodilator and a decrease in the integral response to the vasoconstrictor at 24 hpi (compared with control). At 96 hpi, a tendency towards a decrease in the integral response to the vasoconstrictor persisted, while the response to acetylcholine was slightly increased. The functional activity of the mesenteric blood vessels was inverted: a significant decrease in the integral response to the vasodilator and an increase in the response to the vasoconstrictor at 24 hpi were observed; at 96 hpi, the integral response to the vasoconstrictor persisted, while the response to the vasodilator remained significantly reduced. Obtained data indicate the development of endothelial dysfunction in non-lethal and clinically non-severe experimental influenza virus infection.

Growth characteristics of experimental live influenza vaccine strains with modified NP and NS genes

BACKGROUND: Vaccination is the most effective means of fighting influenza epidemics, but the immunogenicity of licensed influenza vaccines is not always satisfactory. One of the ways to increase the immunogenicity of an attenuated live influenza vaccine is to shorten the open reading frame of the NS1 protein, a modulator of innate antiviral immunity. In addition, the T-cell response to vaccination can be optimized by including the NP gene from the epidemic parental virus into the genome of vaccine strains.
 MATERIALS AND METHODS: The open reading frame of the NS1 protein of the master donor virus A/Leningrad/134/17/57 was truncated to 126 amino acids by site-directed mutagenesis. The HA, NA, and NP genes of the model virus A/Anhui/1/2013 (H7N9) were cloned into the pCIPolISapIT vector. The rescue of recombinant influenza viruses was performed by transfection of Vero cells with a desired set of plasmids. The growth properties of the recombinant viruses were determined in embryonated chicken eggs incubated at different temperatures, as well as in the tissues of the respiratory tract of mice (nasal turbinates, lungs).
 RESULTS: Experimental live influenza vaccine strains of subtype H7N9 with genome compositions 6:2 and 5:3 and carrying a full-length or truncated NS1 gene were actively replicated in eggs under optimal conditions, while maintaining the temperature-sensitive and cold-adapted phenotypes characteristic of classical live influenza vaccine strains. All viruses lacked the ability to grow in the lungs of C57BL/6J mice, which confirms the attenuated phenotype of the viruses. In the nasal passages of mice, only viruses with the full-length NS1 gene replicated, while viruses expressing the truncated NS1 protein were not detected in the respiratory tract of animals.
 CONCLUSIONS: The results indicate that modification of the NS1 gene of the vaccine virus and the inclusion of wild-type NP gene in its genome does not affect its growth characteristics in eggs. A decrease in the activity of viral replication in the upper respiratory tract of mice with a shortening of the NS1 open reading frame indicates an increase in the attenuating properties of modified vaccines, which opens up prospects for the use of new vaccines in children under three years of age.

Generation and in vitro characterization of engineered cold-adapted influenza A strains with modified NS gene

BACKGROUND: The high variability of influenza strains and the emergence of new variants of viruses lead to the need for constant updating of the composition of influenza vaccines. One of the options for solving this problem is the development of vaccines with enhanced cross-protection against a wide range of influenza strains. Genetically engineered preparations based on live influenza vaccine can be used for targeted stimulation of the cellular immune response. It has been experimentally established that CTL epitopes inserted into the NS gene of the live influenza vaccine strain cause the activation of lymphocytes and the formation of a pool of resident memory T-cells in the lungs of model animals. It is optimal to use experimentally confirmed immunogenic regions for insertion.
 AIM: The aim of this study was to rescue a panel of experimental cold-adapted live attenuated influenza vaccine strains with a modified NS gene using A/Leningrad/134/17/57 backbone and recent influenza strains of H1N1, H3N2 and H7N9 subtypes, and evaluate their properties in vitro.
 MATERIALS AND METHODS: A cassette encoding immunogenic, conserved among a wide range of influenza strains T-cell epitopes of the influenza virus PB1 protein restricted by common HLA-allotypes was inserted into the gene encoding the NS1 protein. The modified NS gene was cloned into the pCIPolISapIT influenza virus reverse genetics vector. Chimeric influenza viruses were rescued by transfection of Vero cells by electroporation using a standard 8-plasmid system. The growth characteristics of viruses were assessed in developing chicken embryos.
 Results: Three strains were successfully obtained based on the live influenza vaccine master donor virus A/Leningrad/ 134/17/57 with a modified NS gene and influenza viruses of the H1N1, H3N2, H7N9 subtypes. Thus, modification of NS gene by insertion of immunogenic PB1 epitopes did not affect the viability and replicative activity of the rescued chimeric live influenza vaccine strains, regardless of the composition of the surface proteins. The strains replicated well at an optimal temperature, had temperature-sensitive phenotype and were able to grow at low temperature.
 CONCLUSIONS: The strains will be further studied as candidates for influenza prophylaxis as an experimental universal influenza vaccine.

Influenza Neuraminidase Characteristics and Potential as a Vaccine Target.

Neuraminidase of influenza A and B viruses plays a critical role in the virus life cycle and is an important target of the host immune system. Here, we highlight the current understanding of influenza neuraminidase structure, function, antigenicity, immunogenicity, and immune protective potential. Neuraminidase inhibiting antibodies have been recognized as correlates of protection against disease caused by natural or experimental influenza A virus infection in humans. In the past years, we have witnessed an increasing interest in the use of influenza neuraminidase to improve the protective potential of currently used influenza vaccines. A number of well-characterized influenza neuraminidase-specific monoclonal antibodies have been described recently, most of which can protect in experimental challenge models by inhibiting the neuraminidase activity or by Fc receptor-dependent mechanisms. The relative instability of the neuraminidase poses a challenge for protein-based antigen design. We critically review the different solutions that have been proposed to solve this problem, ranging from the inclusion of stabilizing heterologous tetramerizing zippers to the introduction of inter-protomer stabilizing mutations. Computationally engineered neuraminidase antigens have been generated that offer broad, within subtype protection in animal challenge models. We also provide an overview of modern vaccine technology platforms that are compatible with the induction of robust neuraminidase-specific immune responses. In the near future, we will likely see the implementation of influenza vaccines that confront the influenza virus with a double punch: targeting both the hemagglutinin and the neuraminidase.

Effect of Tilorone on the Dynamics of Viral Load and the Levels of Interferons and Interleukin-1β in the Lung Tissue and Blood Serum of Mice with Experimental Influenza.

We studied the effect of tilorone on the dynamics of IFNα, IFNγ, and IL-1β levels in the lung tissue and blood serum in relation to viral load in the lungs of BALB/c mice with pneumonia caused by influenza virus A/Aichi/2/68 (H3N2). Tilorone was administered per os in doses of 40, 150, and 540 μg per mouse 6, 30, and 78 h postinfection, which simulated the drug regimen used in the clinic for the treatment of influenza and acute respiratory viral infections in Russia and post-Soviet countries. Tilorone reduced viral load with the maximum amplitude (2-3 lg) after 1-2 administrations. The results of studying the dynamics of the cytokine levels in the infected animals in general support the previous hypothesis that, in repeated dosing, tilorone enhances the IFN response (compensates for its deficiency) at the early stages of acute respiratory viral infections and suppresses (damps) excessive production of IFN and proinflammatory cytokines at the later stages.

The potency of newly development H5N8 and H9N2 avian influenza vaccines against the isolated strains in laying hens from Egypt during 2019.

Avian influenza (AI) is a respiratory disease complex syndrome recently recorded in vaccinated flocks causing high economic losses. This study aimed to prepare inactivated vaccine from recently isolated field strains [highly pathogenic avian influenza (HPAI) (H5N8) and low pathogenic avian influenza (LPAI) (H9N2)] and compare the efficiency of the two experimental avian influenza vaccines and some commercial avian influenza H5 and H9N2 vaccines in laying hens. The obtained results indicated that the identified experimental vaccines (H5N8 and H9N2) were protected the flocks from AI as compared to commercial H5N1, H5N3, and H9N2 vaccines, which showed a protection level of 80, 70, and 90%, respectively, indicating a high efficacy for the developed vaccines. In addition, it significantly improved the virus shedding, especially when used in booster dose. The experimental vaccines were given high antibody titer higher than commercial vaccine which was reached to 9.3 log2, 9.7log2 for experimental H5N8 vaccine which was significantly higher than and groups 3 and 4 especially at 2nd WPV, while at the 3rd WPV, the significant difference was with group 4 only. The HI titer was 9.3 log2 at 2nd WPV for the experimental H9N2 vaccine that was significantly higher than group 9. In conclusion, the booster dose of the experimental vaccines could elicit strong immunity than single-dose and commercial vaccines.

Aging imparts cell-autonomous dysfunction to regulatory T cells during recovery from influenza pneumonia.

Regulatory T (Treg) cells orchestrate resolution and repair of acute lung inflammation and injury after viral pneumonia. Compared with younger patients, older individuals experience impaired recovery and worse clinical outcomes after severe viral infections, including influenza and SARS coronavirus 2 (SARS-CoV-2). Whether age is a key determinant of Treg cell prorepair function after lung injury remains unknown. Here, we showed that aging results in a cell-autonomous impairment of reparative Treg cell function after experimental influenza pneumonia. Transcriptional and DNA methylation profiling of sorted Treg cells provided insight into the mechanisms underlying their age-related dysfunction, with Treg cells from aged mice demonstrating both loss of reparative programs and gain of maladaptive programs. Strategies to restore youthful Treg cell functional programs could be leveraged as therapies to improve outcomes among older individuals with severe viral pneumonia.

Safety, Immunogenicity, and Protective Efficacy of a Chimeric A/B Live Attenuated Influenza Vaccine in a Mouse Model.

Influenza A and B viruses cause significant morbidity and mortality worldwide. Current influenza vaccines are composed of three or four strains: A/H1N1, A/H3N2, and B (Victoria and Yamagata lineages). It is of great interest if immunization against both type A and B influenza viruses can be combined in a single vaccine strain, thus reducing the cost of vaccine production and the possibility of strain interference within the multicomponent vaccine. In the current study, we developed an experimental live cold-adapted influenza intertype reassortant (influenza A and B) vaccine on the live attenuated influenza vaccine (LAIV) A/Leningrad/134/17/57 backbone. Hemagglutinin (HA) and neuraminidase (NA) functional domains were inherited from the influenza B/Brisbane/60/2008 strain, whereas their packaging signals were substituted with appropriate fragments of influenza A virus genes. The recombinant A/B virus efficiently replicated in eggs and Madin–Darby Canine Kidney (MDCK) cells under optimal conditions, temperature-sensitive phenotype was maintained, and its antigenic properties matched the influenza B parental virus. The chimeric vaccine was attenuated in mice: after intranasal immunization, viral replication was seen only in nasal turbinates but not in the lungs. Immunological studies demonstrated the induction of IgG antibody responses against the influenza A and B virus, whereas hemagglutination inhibition (HAI) and neutralizing antibodies were detected only against the influenza B virus, resulting in significant protection of immunized animals against influenza B virus challenge. IFNγ-secreting CD8 effector memory T cells (CD44+CD62L−) were detected in mouse splenocytes after stimulation with the specific influenza A peptide (NP366); however, the T-cell response was not sufficient to protect animals against infection with a high-dose mouse-adapted A/California/07/2009 (H1N1pdm09) virus, most probably due to the mismatch of key T-cell epitopes of the H1N1 virus and the LAIV backbone. Overall, generation of the chimeric A/B LAIV virus on a licensed LAIV backbone demonstrated prospects for the development of safe and efficacious vaccine candidates that afford combined protection against both type A and type B influenza viruses; however, further optimization of the T-cell epitope content within the LAIV backbone may be required.

Effect of infectious bronchitis and Newcastle disease vaccines on experimental avian influenza infection (H9N2) in broiler chickens

Despite the fact that H9N2 avian influenza virus (AIV) is considered a low-pathogenic agent, frequent outbreaks of this subtype have caused high mortality and economic losses in poultry farms around the world including Iran. Coinfection with a respiratory pathogen or environmental factors may explain the exacerbation of H9N2 AIV infection. In this study, the role of infectious bronchitis (IB) vaccines (H120 and 4/91) and Newcastle disease (ND) vaccines (B1 and LaSota) on experimental H9N2 AIV infection was investigated in 180 broiler chickens allotted into 6 groups (n=30). At the age of 18 days, groups 3 and 4 received H120 and 4/91 infectious bronchitis live vaccines (IBLVs) and groups 5 and 6 received B1 and LaSota Newcastle disease live vaccines (NDLVs), respectively. At the age of 20 days, all birds in the experimental groups except the negative control group (group 1), were inoculated intra-nasally with H9N2 AIV. After the inoculation, clinical signs, gross and microscopic lesions, and viral detection were examined. The results of this study revealed that clinical signs, gross and microscopic lesions were more severe in the AIV challenged groups which had been previously vaccinated with IB vaccines. In addition, AI viral RNA from tracheal and faecal samples in IB vaccinated birds were recovered at a higher rate. Moreover, in the 4/91 IB vaccinated group, the AI virus shedding period was longer than the other challenged groups. In conclusion, infectious bronchitis live vaccines (IBLVs) exacerbated the H9N2 AIV infection; also, 4/91 IBLV extended AI virus shedding period and increased the recovery rate of AI virus from feaces. However, the coinfection of Newcastle disease live vaccines (NDLVs) had no considerable adverse effects on AIV infection in broiler chickens.

GLP-1 Receptor Signaling Differentially Modifies the Outcomes of Sterile vs Viral Pulmonary Inflammation in Male Mice.

A number of disease states, including type 2 diabetes (T2D), are associated with an increased risk of pulmonary infection. Glucagon-like peptide-1 (GLP-1) receptor agonists are used to treat T2D and exert anti-inflammatory actions through a single, well-defined GLP-1 receptor (GLP-1R). Although highly expressed in the lung, little is known about the role of the GLP-1R in the context of pulmonary inflammation. Here we examined the consequences of gain or loss of GLP-1R activity in infectious and noninfectious lung inflammation. We studied wild-type mice treated with a GLP-1R agonist, and Glp1r-/- mice, in the setting of bleomycin-induced noninfectious lung injury and influenza virus infection. Loss of the GLP-1R attenuated the severity of bleomycin-induced lung injury, whereas activation of GLP-1R signaling increased pulmonary inflammation via the sympathetic nervous system. In contrast, GLP-1R agonism reduced the pathogen load in mice with experimental influenza virus infection in association with increased expression of intracellular interferon-inducible GTPases. Notably, the GLP-1 receptor agonist liraglutide improved the survival rate after influenza virus infection. Our results reveal context-dependent roles for the GLP-1 system in the response to lung injury. Notably, the therapeutic response of GLP-1R agonism in the setting of experimental influenza virus infection may have relevance for ongoing studies of GLP-1R agonism in people with T2D susceptible to viral lung injury.

T cell and chemokine receptors differentially control CD8 T cell motility behavior in the infected airways immediately before and after virus clearance in a primary infection.

In mice, experimental influenza virus infection stimulates CD8 T cell infiltration of the airways. Virus is cleared by day 9, and between days 8 and 9 there is an abrupt change in CD8 T cell motility behavior transitioning from low velocity and high confinement on day 8, to high velocity with continued high confinement on day 9. We hypothesized that loss of virus and/or antigen signals in the context of high chemokine levels drives the T cells into a rapid surveillance mode. Virus infection induces chemokine production, which may change when the virus is cleared. We therefore sought to examine this period of rapid changes to the T cell environment in the tissue and seek evidence on the roles of peptide-MHC and chemokine receptor interactions. Experiments were performed to block G protein coupled receptor (GPCR) signaling with Pertussis toxin (Ptx). Ptx treatment generally reduced cell velocities and mildly increased confinement suggesting chemokine mediated arrest (velocity <2 μm/min) (Friedman RS, 2005), except on day 8 when velocity increased and confinement was relieved. Blocking specific peptide-MHC with monoclonal antibody unexpectedly decreased velocities on days 7 through 9, suggesting TCR/peptide-MHC interactions promote cell mobility in the tissue. Together, these results suggest the T cells are engaged with antigen bearing and chemokine producing cells that affect motility in ways that vary with the day after infection. The increase in velocities on day 9 were reversed by addition of specific peptide, consistent with the idea that antigen signals become limiting on day 9 compared to earlier time points. Thus, antigen and chemokine signals act to alternately promote and restrict CD8 T cell motility until the point of virus clearance, suggesting the switch in motility behavior on day 9 may be due to a combination of limiting antigen in the presence of high chemokine signals as the virus is cleared.

Self-assembling influenza nanoparticle vaccines drive extended germinal center activity and memory B cell maturation.

Protein-based, self-assembling nanoparticles elicit superior immunity compared with soluble protein vaccines, but the immune mechanisms underpinning this effect remain poorly defined. Here, we investigated the immunogenicity of a prototypic ferritin-based nanoparticle displaying influenza hemagglutinin (HA) in mice and macaques. Vaccination of mice with HA-ferritin nanoparticles elicited higher serum antibody titers and greater protection against experimental influenza challenge compared with soluble HA protein. Germinal centers in the draining lymph nodes were expanded and persistent following HA-ferritin vaccination, with greater deposition of antigen that colocalized with follicular dendritic cells. Our findings suggest that a highly ordered and repetitive antigen array may directly drive germinal centers through a B cell-intrinsic mechanism that does not rely on ferritin-specific T follicular helper cells. In contrast to mice, enhanced immunogenicity of HA-ferritin was not observed in pigtail macaques, where antibody titers and lymph node immunity were comparable to soluble vaccination. An improved understanding of factors that drive nanoparticle vaccine immunogenicity in small and large animal models will facilitate the clinical development of nanoparticle vaccines for broad and durable protection against diverse pathogens.

Experimental PCEP-Adjuvanted Swine Influenza H1N1 Vaccine Induced Strong Immune Responses but Did Not Protect Piglets against Heterologous H3N2 Virus Challenge.

Vaccination is the most efficient method of protection against influenza infections. However, the rapidly mutating viruses and development of new strains make it necessary to develop new influenza vaccines annually. Hence, vaccines that stimulate cross-protection against multiple influenza subtypes are highly sought. Recent evidence suggests that adjuvants such as PCEP that promote Th1-type T cell and Th2-type T cell immune responses and broad-spectrum immune responses may confer cross-protection against heterologous influenza strains. In this study, we evaluated whether the immunogenic and protective potential of PCEP-adjuvanted inactivated swine influenza virus H1N1 vaccine can protect pigs immunized against live H3N2 virus. Piglets were vaccinated via the intradermal route with PCEP-adjuvanted inactivated swine influenza virus (SIV) H1N1 vaccine, boosted at day 21 with the same vaccines then challenged with infectious SIV H3N2 virus at day 35 via the tracheobronchial route. The pigs showed significant anti-H1N1 SIV specific antibody titres and H1N1 SIV neutralizing antibody titres, and these serum titres remained after the challenge with the H3N2 virus. In contrast, vaccination with anti-H1N1 SIV did not trigger anti-H3N2 SIV antibody titres or neutralizing antibody titres and these titres remained low until pigs were challenged with H3N2 SIV. At necropsy (six days after challenge), we collected prescapular lymph nodes and tracheobronchial draining the vaccination sites and challenge site, respectively. ELISPOTs from lymph node cells restimulated ex vivo with inactivated SIV H1N1 showed significant production of IFN-γ in the tracheobronchial cells, but not the prescapular lymph nodes. In contrast, lymph node cells restimulated ex vivo with inactivated SIV H1N1 showed significantly higher IL-13 and IL-17A in the prescapular lymph nodes draining the vaccination sites relative to unchallenged animals. Lung lesion scores show that intradermal vaccination with H1N1 SIV plus PCEP did not prevent lesions when the animals were challenged with H3N2. These results confirm previous findings that PCEP is effective as a vaccine adjuvant in that it induces strong immune responses and protects against homologous swine influenza H1N1 virus, but the experimental H1N1 vaccine failed to cross-protect against heterologous H3N2 virus.