Establishing a Multicolor Flow Cytometry to Characterize Cellular Immune Response in Chickens Following H7N9 Avian Influenza Virus Infection

Avian influenza virus (AIV) emerged and has continued to re-emerge, continuously posing great threats to animal and human health. The detection of hemagglutination inhibition (HI) or virus neutralization antibodies (NA) is essential for assessing immune protection against AIV. However, the HI/NA-independent immune protection is constantly observed in vaccines’ development against H7N9 subtype AIV and other subtypes in chickens and mammals, necessitating the analysis of the cellular immune response. Here, we established a multi-parameter flow cytometry to examine the innate and adaptive cellular immune responses in chickens after intranasal infection with low pathogenicity H7N9 AIV. This assay allowed us to comprehensively define chicken macrophages, dendritic cells, and their MHC-II expression, NK cells, γδ T cells, B cells, and distinct T cell subsets in steady state and during infection. We found that NK cells and KUL01⁺ cells significantly increased after H7N9 infection, especially in the lung, and the KUL01⁺ cells upregulated MHC-II and CD11c expression. Additionally, the percentages and numbers of γδ T cells and CD8 T cells significantly increased and exhibited an activated phenotype with significant upregulation of CD25 expression in the lung but not in the spleen and blood. Furthermore, B cells showed increased in the lung but decreased in the blood and spleen in terms of the percentages or/and numbers, suggesting these cells may be recruited from the periphery after H7N9 infection. Our study firstly disclosed that H7N9 infection induced local and systemic cellular immune responses in chickens, the natural host of AIV, and that the flow cytometric assay developed in this study is useful for analyzing the cellular immune responses to AIVs and other avian infectious diseases and defining the correlates of immune protection.     

Influenza A Virus Entry Inhibitors Targeting the Hemagglutinin

Influenza A virus (IAV) has caused seasonal influenza epidemics and influenza pandemics, which resulted in serious threat to public health and socioeconomic impacts. Until now, only 5 drugs belong to two categories are used for prophylaxis and treatment of IAV infection. Hemagglutinin (HA), the envelope glycoprotein of IAV, plays a critical role in viral binding, fusion and entry. Therefore, HA is an attractive target for developing anti‑IAV drugs to block the entry step of IAV infection. Here we reviewed the recent progress in the study of conformational changes of HA during viral fusion process and the development of HA-based IAV entry inhibitors, which may provide a new choice for controlling future influenza pandemics.     

Influenza Virus RNA Synthesis and the Innate Immune Response

Infection with influenza A and B viruses results in a mild to severe respiratory tract infection. It is widely accepted that many factors affect the severity of influenza disease, including viral replication, host adaptation, innate immune signalling, pre-existing immunity, and secondary infections. In this review, we will focus on the interplay between influenza virus RNA synthesis and the detection of influenza virus RNA by our innate immune system. Specifically, we will discuss the generation of various RNA species, host pathogen receptors, and host shut-off. In addition, we will also address outstanding questions that currently limit our knowledge of influenza virus replication and host adaption. Understanding the molecular mechanisms underlying these factors is essential for assessing the pandemic potential of future influenza virus outbreaks.     

Application of the CRISPR/Cas9 System to Study Regulation Pathways of the Cellular Immune Response to Influenza Virus

Influenza A virus (IAV) causes a respiratory infection that affects millions of people of different age groups and can lead to acute respiratory distress syndrome. Currently, host genes, receptors, and other cellular components critical for IAV replication are actively studied. One of the most convenient and accessible genome-editing tools to facilitate these studies is the CRISPR/Cas9 system. This tool allows for regulating the expression of both viral and host cell genes to enhance or impair viral entry and replication. This review considers the effect of the genome editing system on specific target genes in cells (human and chicken) in terms of subsequent changes in the influenza virus life cycle and the efficiency of virus particle production.     

Universally Immune: How Infection Permissive Next Generation Influenza Vaccines May Affect Population Immunity and Viral Spread

Next generation influenza vaccines that target conserved epitopes are becoming a clinical reality but still have challenges to overcome. Universal next generation vaccines are considered a vital tool to combat future pandemic viruses and have the potential to vastly improve long-term protection against seasonal influenza viruses. Key vaccine strategies include HA-stem and T cell activating vaccines; however, they could have unintended effects for virus adaptation as they recognise the virus after cell entry and do not directly block infection. This may lead to immune pressure on residual viruses. The potential for immune escape is already evident, for both the HA stem and T cell epitopes, and mosaic approaches for pre-emptive immune priming may be needed to circumvent key variants. Live attenuated influenza vaccines have not been immunogenic enough to boost T cells in adults with established prior immunity. Therefore, viral vectors or peptide approaches are key to harnessing T cell responses. A plethora of viral vector vaccines and routes of administration may be needed for next generation vaccine strategies that require repeated long-term administration to overcome vector immunity and increase our arsenal against diverse influenza viruses.     

Dendritic Cells in Innate and Adaptive Immune Responses against Influenza Virus

Dendritic cells (DC) are major players in both innate and adaptive immune responses against influenza virus. These immune responses, as well as the important interface between the innate and adaptive systems, are orchestrated by specialized subsets of DC, including conventional steady-state DC, migratory DC and plasmacytoid DC. The characteristics and efficacy of the responses are dependent on the relative activity of these DC subsets, rendering DC crucial for the development of both naïve and memory immune responses. However, due to their critical role, DC also contribute to the immunopathological processes observed during acute influenza, such as that caused by the pathogenic H5N1 viruses. Therein, the role of different DC subsets in the induction of interferon type I, pro-inflammatory cytokine and chemokine responses is important for the outcome of interaction between the virus and host immune defences. The present review will present current knowledge on this area, relating to the importance of DC activity for the induction of efficacious humoral and cell-mediated immune responses. This will include the main viral elements associated with the triggering or inhibition of DC activation. Finally, the current knowledge on understanding how differences in various vaccines influence the manner of immune defence induction will be presented.     

Cross-Reactive Fc-Fused Single-Domain Antibodies to Hemagglutinin Stem Region Protect Mice from Group 1 Influenza a Virus Infection

The continued evolution of influenza viruses reduces the effectiveness of vaccination and antiviral drugs. The identification of novel and universal agents for influenza prophylaxis and treatment is an urgent need. We have previously described two potent single-domain antibodies (VHH), G2.3 and H1.2, which bind to the stem domain of hemagglutinin and efficiently neutralize H1N1 and H5N2 influenza viruses in vivo. In this study, we modified these VHHs with Fc-fragment to enhance their antiviral activity. Reformatting of G2.3 into bivalent Fc-fusion molecule increased its in vitro neutralizing activity against H1N1 and H2N3 viruses up to 80-fold and, moreover, resulted in obtaining the ability to neutralize H5N2 and H9N2 subtypes. We demonstrated that a dose as low as 0.6 mg/kg of G2.3-Fc or H1.2-Fc administered systemically or locally before infection could protect mice from lethal challenges with both H1N1 and H5N2 viruses. Furthermore, G2.3-Fc reduced the lung viral load to an undetectable level. Both VHH-Fc antibodies showed in vivo therapeutic efficacy when delivered via systemic or local route. The findings support G2.3-Fc as a potential therapeutic agent for both prophylaxis and therapy of Group 1 influenza A infection.     

In the Shadow of Hemagglutinin: A Growing Interest in Influenza Viral Neuraminidase and Its Role as a Vaccine Antigen

Despite the availability of vaccine prophylaxis and antiviral therapeutics, the influenza virus continues to have a significant, annual impact on the morbidity and mortality of human beings, highlighting the continued need for research in the field. Current vaccine strategies predominantly focus on raising a humoral response against hemagglutinin (HA)—the more abundant, immunodominant glycoprotein on the surface of the influenza virus. In fact, anti-HA antibodies are often neutralizing, and are used routinely to assess vaccine immunogenicity. Neuraminidase (NA), the other major glycoprotein on the surface of the influenza virus, has historically served as the target for antiviral drug therapy and is much less studied in the context of humoral immunity. Yet, the quest to discern the exact importance of NA-based protection is decades old. Also, while antibodies against the NA glycoprotein fail to prevent infection of the influenza virus, anti-NA immunity has been shown to lessen the severity of disease, decrease viral lung titers in animal models, and reduce viral shedding. Growing evidence is intimating the possible gains of including the NA antigen in vaccine design, such as expanded strain coverage and increased overall immunogenicity of the vaccine. After giving a tour of general influenza virology, this review aims to discuss the influenza A virus neuraminidase while focusing on both the historical and present literature on the use of NA as a possible vaccine antigen.     

Time-Dependent Proinflammatory Responses Shape Virus Interference during Coinfections of Influenza A Virus and Influenza D Virus

Both influenza A virus (IAV) and influenza D virus (IDV) are enzootic in pigs. IAV causes approximately 100% morbidity with low mortality, whereas IDV leads to only mild respiratory diseases in pigs. In this study, we performed a series of coinfection experiments in vitro and in vivo to understand how IAV and IDV interact and cause pathogenesis during coinfection. The results showed that IAV inhibited IDV replication when infecting swine tracheal epithelial cells (STECs) with IAV 24 or 48 h prior to IDV inoculation and that IDV suppressed IAV replication when IDV preceded IAV inoculation by 48 h. Virus interference was not identified during simultaneous IAV/IDV infections or with 6 h between the two viral infections, regardless of their order. The interference pattern at 24 and 48 h correlated with proinflammatory responses induced by the first infection, which, for IDV, was slower than for IAV by about 24 h. The viruses did not interfere with each other if both infected the cells before proinflammatory responses were induced. Coinfection in pigs further demonstrated that IAV interfered with both viral shedding and virus replication of IDV, especially in the upper respiratory tract. Clinically, coinfection of IDV and IAV did not show significant enhancement of disease pathogenesis, compared with the pigs infected with IAV alone. In summary, this study suggests that interference during coinfection of IAV and IDV is primarily due to the proinflammatory response; therefore, it is dependent on the time between infections and the order of infection. This study facilitates our understanding of virus epidemiology and pathogenesis associated with IAV and IDV coinfection.     

Evasion of Influenza A Viruses from Innate and Adaptive Immune Responses

The influenza A virus is one of the leading causes of respiratory tract infections in humans. Upon infection with an influenza A virus, both innate and adaptive immune responses are induced. Here we discuss various strategies used by influenza A viruses to evade innate immune responses and recognition by components of the humoral and cellular immune response, which consequently may result in reduced clearing of the virus and virus-infected cells. Finally, we discuss how the current knowledge about immune evasion can be used to improve influenza A vaccination strategies.     

Adaptive Immune Response to Vaccinia Virus LIVP Infection of BALB/c Mice and Protection against Lethal Reinfection with Cowpox Virus

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 10⁵ PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD₅₀. The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection.     

Multiple Neuraminidase Containing Influenza Virus-like Particle Vaccines Protect Mice from Avian and Human Influenza Virus Infection

Avian influenza virus remains a threat for humans, and vaccines preventing both avian and human influenza virus infections are needed. Since virus-like particles (VLPs) expressing single neuraminidase (NA) subtype elicited limited heterosubtypic protection, VLPs expressing multiple NA subtypes would enhance the extent of heterosubtypic immunity. Here, we generated avian influenza VLP vaccines displaying H5 hemagglutinin (HA) antigen with or without avian NA subtypes (N1, N6, N8) in different combinations. BALB/c mice were intramuscularly immunized with the VLPs to evaluate the resulting homologous and heterosubtypic immunity upon challenge infections with the avian and human influenza viruses (A/H5N1, A/H3N2, A/H1N1). VLPs expressing H5 alone conferred homologous protection but not heterosubtypic protection, whereas VLPs co-expressing H5 and NA subtypes elicited both homologous and heterosubtypic protection against human influenza viruses in mice. We observed that VLP induced neuraminidase inhibitory activities (NAI), virus-neutralizing activity, and virus-specific antibody (IgG, IgA) responses were strongly correlated with the number of different NA subtype expressions on the VLPs. VLPs expressing all 3 NA subtypes resulted in the highest protection, indicated by the lowest lung titer, negligible body weight changes, and survival in immunized mice. These results suggest that expressing multiple neuraminidases in avian HA VLPs is a promising approach for developing a universal influenza A vaccine against avian and human influenza virus infections.     

Influenza Virus Infection during Pregnancy as a Trigger of Acute and Chronic Complications

Influenza A virus (IAV) infection during pregnancy disrupts maternal and fetal health through biological mechanisms, which are to date poorly characterised. During pregnancy, the viral clearance mechanisms from the lung are sub-optimal and involve hyperactive innate and adaptive immune responses that generate wide-spread inflammation. Pregnancy-related adaptations of the immune and the cardiovascular systems appear to result in delayed recovery post-viral infection, which in turn promotes a prolonged inflammatory phenotype, increasing disease severity, and causing maternal and fetal health problems. This has immediate and long-term consequences for the mother and fetus, with complications including acute cardiopulmonary distress syndrome in the mother that lead to perinatal complications such as intrauterine growth restriction (IUGR), and birth defects; cleft lip, cleft palate, neural tube defects and congenital heart defects. In addition, an increased risk of long-term neurological disorders including schizophrenia in the offspring is reported. In this review we discuss the pathophysiology of IAV infection during pregnancy and its striking similarity to other well-established complications of pregnancy such as preeclampsia. We discuss general features of vascular disease with a focus on vascular inflammation and define the “Vascular Storm” that is triggered by influenza infection during pregnancy, as a pivotal disease mechanism for short and long term cardiovascular complications.     

Modelling the Effect of MUC1 on Influenza Virus Infection Kinetics and Macrophage Dynamics

MUC1 belongs to the family of cell surface (cs-) mucins. Experimental evidence indicates that its presence reduces in vivo influenza viral infection severity. However, the mechanisms by which MUC1 influences viral dynamics and the host immune response are not yet well understood, limiting our ability to predict the efficacy of potential treatments that target MUC1. To address this limitation, we use available in vivo kinetic data for both virus and macrophage populations in wildtype and MUC1 knockout mice. We apply two mathematical models of within-host influenza dynamics to this data. The models differ in how they categorise the mechanisms of viral control. Both models provide evidence that MUC1 reduces the susceptibility of epithelial cells to influenza virus and regulates macrophage recruitment. Furthermore, we predict and compare some key infection-related quantities between the two mice groups. We find that MUC1 significantly reduces the basic reproduction number of viral replication as well as the number of cumulative macrophages but has little impact on the cumulative viral load. Our analyses suggest that the viral replication rate in the early stages of infection influences the kinetics of the host immune response, with consequences for infection outcomes, such as severity. We also show that MUC1 plays a strong anti-inflammatory role in the regulation of the host immune response. This study improves our understanding of the dynamic role of MUC1 against influenza infection and may support the development of novel antiviral treatments and immunomodulators that target MUC1.     

Dual R108K and G189D Mutations in the NS1 Protein of A/H1N1 Influenza Virus Counteract Host Innate Immune Responses

Influenza A viruses (IAV) modulate host antiviral responses to promote growth and pathogenicity. Here, we examined the multifunctional IAV nonstructural protein 1 (NS1) of influenza A virus to better understand factors that contribute to viral replication efficiency or pathogenicity. In 2009, a pandemic H1N1 IAV (A/California/07/2009 pH1N1) emerged in the human population from swine. Seasonal variants of this virus are still circulating in humans. Here, we compared the sequence of a seasonal variant of this H1N1 influenza virus (A/Urumqi/XJ49/2018(H1N1), first isolated in 2018) with the pandemic strain A/California/07/2009. The 2018 virus harbored amino acid mutations (I123V and N205S) in important functional sites; however, 108R and 189G were highly conserved between A/California/07/2009 and the 2018 variant. To better understand interactions between influenza viruses and the human innate immune system, we generated and rescued seasonal 2009 H1N1 IAV mutants expressing an NS1 protein harboring a dual mutation (R108K/G189D) at these conserved residues and then analyzed its biological characteristics. We found that the mutated NS1 protein exhibited systematic and selective inhibition of cytokine responses via a mechanism that may not involve binding to cleavage and polyadenylation specificity factor 30 (CPSF30). These results highlight the complexity underlying host–influenza NS1 protein interactions.     

H1N1 Influenza A Virus Protein NS2 Inhibits Innate Immune Response by Targeting IRF7

Influenza A virus (IAV) is a globally distributed zoonotic pathogen and causes a highly infectious respiratory disease with high morbidity and mortality in humans and animals. IAV has evolved various strategies to counteract the innate immune response, using different viral proteins. However, the mechanisms are not fully elucidated. In this study, we demonstrated that the nonstructural protein 2 (NS2) of H1N1 IAV negatively regulate the induction of type-I interferon. Co-immunoprecipitation experiments revealed that NS2 specifically interacts with interferon regulatory factor 7 (IRF7). NS2 blocks the nuclear translocation of IRF7 by inhibiting the formation of IRF7 dimers, thereby prevents the activation of IRF7 and inhibits the production of interferon-beta. Taken together, these findings revealed a novel mechanism by which the NS2 of H1N1 IAV inhibits IRF7-mediated type-I interferon production.     

Understanding the Role of HLA Class I Molecules in the Immune Response to Influenza Infection and Rational Design of a Peptide-Based Vaccine

Influenza A virus is a respiratory pathogen that is responsible for regular epidemics and occasional pandemics that result in substantial damage to life and the economy. The yearly reformulation of trivalent or quadrivalent flu vaccines encompassing surface glycoproteins derived from the current circulating strains of the virus does not provide sufficient cross-protection against mismatched strains. Unlike the current vaccines that elicit a predominant humoral response, vaccines that induce CD8⁺ T cells have demonstrated a capacity to provide cross-protection against different influenza strains, including novel influenza viruses. Immunopeptidomics, the mass spectrometric identification of human-leukocyte-antigen (HLA)-bound peptides isolated from infected cells, has recently provided key insights into viral peptides that can serve as potential T cell epitopes. The critical elements required for a strong and long-living CD8⁺ T cell response are related to both HLA restriction and the immunogenicity of the viral peptide. This review examines the importance of HLA and the viral immunopeptidome for the design of a universal influenza T-cell-based vaccine.     

Comprehensive Characterization of Serum MicroRNA Profile in Response to the Emerging Avian Influenza A (H7N9) Virus Infection in Humans

A novel avian-origin influenza A (H7N9) virus recently occurred in China and caused 137 human infection cases with a 32.8% mortality rate. Although various detection procedures have been developed, the pathogenesis of this emerging virus in humans remains largely unknown. In this study, we characterized serum microRNA (miRNA) profile in response to H7N9 virus infection using TaqMan Low Density Arrays. Upon infection, a total of 395 miRNAs were expressed in the serum pool of patients, far beyond the 221 in healthy controls. Among the 187 commonly expressed miRNAs, 146 were up-regulated and only 7 down-regulated in patients. Further analysis by quantitative RT-PCR revealed that the serum levels of miR-17, miR-20a, miR-106a and miR-376c were significantly elevated in patients compared with healthy individuals (p < 0.05). Receiver operating characteristic (ROC) curves were constructed to show that each miRNA could discriminate H7N9 patients from controls with area under the curve (AUC) values ranging from 0.622 to 0.898, whereas a combination of miR-17, miR-20a, miR-106a and miR-376c obtained a higher discriminating ability with an AUC value of 0.96. Our findings unravel the significant alterations in serum miRNA expression following virus infection and manifest great potential of circulating miRNAs for the diagnosis of viral diseases.     

Cloak and Dagger: Alternative Immune Evasion and Modulation Strategies of Poxviruses

As all viruses rely on cellular factors throughout their replication cycle, to be successful they must evolve strategies to evade and/or manipulate the defence mechanisms employed by the host cell. In addition to their expression of a wide array of host modulatory factors, several recent studies have suggested that poxviruses may have evolved unique mechanisms to shunt or evade host detection. These potential mechanisms include mimicry of apoptotic bodies by mature virions (MVs), the use of viral sub-structures termed lateral bodies for the packaging and delivery of host modulators, and the formation of a second, “cloaked” form of infectious extracellular virus (EVs). Here we discuss these various strategies and how they may facilitate poxvirus immune evasion. Finally we propose a model for the exploitation of the cellular exosome pathway for the formation of EVs.     

Henipavirus Immune Evasion and Pathogenesis Mechanisms: Lessons Learnt from Natural Infection and Animal Models

Nipah henipavirus (NiV) and Hendra henipavirus (HeV) are zoonotic emerging paramyxoviruses causing severe disease outbreaks in humans and livestock, mostly in Australia, India, Malaysia, Singapore and Bangladesh. Both are bat-borne viruses and in humans, their mortality rates can reach 60% in the case of HeV and 92% for NiV, thus being two of the deadliest viruses known for humans. Several factors, including a large cellular tropism and a wide zoonotic potential, con-tribute to their high pathogenicity. This review provides an overview of HeV and NiV pathogenicity mechanisms and provides a summary of their interactions with the immune systems of their different host species, including their natural hosts bats, spillover-hosts pigs, horses, and humans, as well as in experimental animal models. A better understanding of the interactions between henipaviruses and their hosts could facilitate the development of new therapeutic strategies and vaccine measures against these re-emerging viruses.