Recombinant Influenza Virus Research Articles

Tissue-Resident Memory T Cells in the Lungs Protect against Acute Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young children. The T cell response plays a critical role in facilitating clearance of an acute RSV infection, and memory T cell responses are vital for protection against secondary RSV exposures. Tissue-resident memory (TRM) T cells have been identified as a subset of memory T cells that reside in nonlymphoid tissues and are critical for providing long-term immunity. There is currently limited information regarding the establishment and longevity of TRM T cell responses elicited following an acute RSV infection as well as their role in protection against repeated RSV infections. In this study, we examined the magnitude, phenotype, and protective capacity of TRM CD4 and CD8 T cells in the lungs of BALB/c mice following an acute RSV infection. TRM CD4 and CD8 T cells were established within the lungs and waned by 149 d following RSV infection. To determine the protective capacity of TRMs, FTY720 administration was used to prevent trafficking of peripheral memory T cells into the lungs prior to challenge of RSV-immune mice, with a recombinant influenza virus expressing either an RSV-derived CD4 or CD8 T cell epitope. We observed enhanced viral clearance in RSV-immune mice, suggesting that TRM CD8 T cells can contribute to protection against a secondary RSV infection. Given the protective capacity of TRMs, future RSV vaccine candidates should focus on the generation of these cell populations within the lung to induce effective immunity against RSV infection.

Generation of recombinant influenza virus bearing strep tagged PB2 and effective identification of interactional host factors

The genome of influenza A virus is negative-sense and segmented RNA, which is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp) during the virus life cycle. The viral RdRp is thought to be an important host range and virulence determinant factor, and the 627 site of PB2 subunit is a highly acceptable key site of RdRp function. Besides, the function of RdRp is modulated by several host factors. Identification of the host factors interacting with RdRp is of great interest. Here, we tried to explore an effective method to study virus-host interaction by rescuing replication-competent recombinant influenza viruses carrying Strep tagged PB2. Subsequently, we tested several biological characteristics of recombinant viruses in cells and pathogenicity in mice. Then, we purified of protein complex of Strep tagged PB2 and host factors of interest from 293 T cells infected with recombinant viruses. After purification, we performed mass spectrometry to identify these proteins that interacting with PB2. We identified 57 host factors in total. Through Gene Ontology (GO) and Protein-Protein interaction (PPI) network analysis, we revealed the function and network of these proteins. In summary, we generated replication-competent recombinant influenza viruses by inserting a Strep-Tag into PB2 and purified host factors interacting with viral RdRp bearing a 627 K or 627E PB2. These proteins might function as host range and virulence determinants of influenza virus.

One step closer to influenza vaccine inclusiveness.

Flu virus infection is a common cause of acute respiratory illness, with the major incidence in pediatric age, high morbidity, and mortality. The flu vaccine is recommended for all people aged ≥6 months, unless specific contraindications are present. Younger and older age, pregnancy, chronic diseases like asthma, and immunodeficiency are risk factors for severe complications following flu infection. Thus, these categories represent the target for flu vaccine strategies in most countries. Inactivated influenza vaccine (IIV), recombinant influenza vaccine (RIV) or live‐attenuated influenza virus (LAIV) are currently available, with specific precautions and contraindications. We aim to resume the current indications for vaccines in the vulnerable populations to support flu vaccination inclusiveness, in anticipation of a “universal vaccine” strategy.

Immunogenicity and protective activity of recombinant influenza viruses expressing fragments of ScaAB lipoprotein of group B streptococci in a mouse model

Group B streptococci (GBS) cause a number of serious diseases in humans. The development of an effective vaccine against GBS requires special approaches. In the present study, three recombinant influenza viruses were constructed on the backbone of H7N9 live attenuated influenza vaccine (LAIV) strain expressing fragments of the ScaAB lipoprotein of Streptococcus agalactiae, fused to the surface protein of the virus, hemagglutinin, using a flexible linker. Recombinant viruses with ScaAB inserts of 85, 141, and 200 amino acids were successfully rescued by the means of reverse genetics. The recombinant strains were able to grow in developing chicken embryos and MDCK cells and retained the temperature-sensitive phenotype attributable to the LAIV viruses. Studies of immunogenicity and protective activity of the vaccine candidates in BALB/c mice revealed that the most promising strain was a strain with an insert of 141 amino acids: this variant had optimal immunogenicity against influenza and GBS and had a protective effect against both pathogens. These data indicate that further studies of the recombinant vectored vaccine H7-ScaAB-141 as a combined viral-bacterial vaccine capable of protection against both influenza virus and bacterial infections caused by group B streptococci are warranted.

Cross protection by inactivated recombinant influenza viruses containing chimeric hemagglutinin conjugates with a conserved neuraminidase or M2 ectodomain epitope

Influenza virus neuraminidase (NA) contains a universally conserved epitope (NAe, NA222-230). However, no studies have reported vaccines targeting this NA conserved epitope and inducing antibodies recognizing NAe. The extracellular domain of M2 (M2e) is considered as an attractive target for a universal influenza vaccine. We generated recombinant influenza H1N1 viruses expressing conserved epitopes in hemagglutinin (HA) molecules: NAe (NAe-HA) or M2e (M2e-HA) within the HA head domain. Inactivated recombinant NAe-HA and M2e-HA viruses were more effective in inducing IgG antibodies specific for an inserted conserved epitope than live recombinant virus. Recombinant inactivated M2e-HA virus vaccination induced cross protection against H3N2 virus with less weight loss compared to NAe-HA and was more effective in inducing humoral and cellular M2e immune responses. This study provides insight into developing recombinant influenza virus vaccines compatible with current platforms to induce antibody responses to conserved poorly immunogenic epitopes.

High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines.

Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.

Live Visualization of Hemagglutinin Dynamics During Infection by Using a Novel Reporter Influenza A Virus.

Live visualization of influenza A virus (IAV) structural proteins during viral infection in cells is highly sought objective to study different aspects of the viral replication cycle. To achieve this, we engineered an IAV to express a Tetra Cysteine tag (TC tag) from hemagglutinin (HA), which allows intracellular labeling of the engineered HA protein with biarsenic dyes and subsequent fluorescence detection. Using such constructs, we rescued a recombinant IAV with TC tag inserted in HA, in A/Puerto Rico/8/1934(H1N1) background (HA-TC). This recombinant HA-TC tag reporter IAV was replication-competent; however, as compared to wild type PR8 IAV, it was attenuated in multicycle replication. We confirmed expression of TC tag and biarsenical labeling of HA by immunofluorescence assay in cells infected with an HA-TC tag reporter IAV. Further, we used this reporter virus to visualize HA expression and translocation in IAV infected cells by live confocal imaging. We also tested the utility of the HA-TC IAV in testing chemical inhibitors of the HA translocation. Overall, HA-TC IAV is a versatile tool that will be useful for studying viral life cycle events, virus-host interactions, and anti-viral testing.

Assessing the efficacy of a recombinant H9N2 avian influenza virus–inactivated vaccine

The H9N2 avian influenza virus has been widely spread in poultry around the world. It is proved to the world that the avian influenza virus can directly infect human beings without any intermediate host adaptation in “1997 Hong Kong avian influenza case,” which shows that the avian influenza virus not only causes significant losses to the poultry industry but also affects human health. In this study, we aimed to address the problem of low protection of avian H9N2 subtype influenza virus vaccine against H9N2 wild-type virus. We have rescued the H9.4.2.5 branched avian influenza virus isolated in South China by reverse genetics technology. We have recombined these virus (rHA/NA-GD37 and rHA/NA-GD38) which contain hemagglutinin and neuraminidase genes from the H9N2 avian influenza virus (MN064850 or MN064851) and 6 internal genes from the avian influenza virus (KY785906). We compared the biological properties of the virus for example virus proliferation, virus elution, thermostability, and pH stability. Then, we evaluated the immune effects between rHA/NA-GD37 and GD37, which show that the recombinant avian influenza virus–inactivated vaccine can stimulate chickens to produce higher antibody titers and produce little inflammatory response after the challenge. It is noticeable that the recombinant virus-inactivated vaccine had better immune impact than the wild-type inactivated vaccine. Generally speaking, this study provides a new virus strain for the development of a H9N2 vaccine.

A Novel Synthetic Dual Agonistic Liposomal TLR4/7 Adjuvant Promotes Broad Immune Responses in an Influenza Vaccine With Minimal Reactogenicity.

The limited efficacy of seasonal influenza vaccines is usually attributed to ongoing variation in the major antigenic targets for protective antibody responses including hemagglutinin (HA) and neuraminidase (NA). Hence, vaccine development has largely focused on broadening antigenic epitopes to generate cross-reactive protection. However, the vaccine adjuvant components which can accelerate, enhance and prolong antigenic immune responses, can also increase the breadth of these responses. We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus HA, inducing rapid, and sustained antibody responses that are protective against influenza viruses in homologous and heterologous murine challenge models. To further enhance adjuvant efficacy, we performed a structure-activity relationship study for the TLR4 ligand, N-cyclohexyl-2-((5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)thio)acetamide (C25H26N4O2S; 1Z105), and identified the 8-(furan-2-yl) substituted pyrimido[5,4-b]indole analog (C29H28N4O3S; 2B182C) as a derivative with higher potency in activating both human and mouse TLR4-NF-κB reporter cells and primary cells. In a prime-boost immunization model using inactivated influenza A virus [IIAV; A/California/04/2009 (H1N1)pdm09], 2B182C used as adjuvant induced higher serum anti-HA and anti-NA IgG1 levels compared to 1Z105, and also increased the anti-NA IgG2a responses. In combination with a TLR7 ligand, 1V270, 2B182C induced equivalent levels of anti-NA and anti-HA IgG1 to 1V270+1Z105. However, the combination of 1V270+2B182C induced 10-fold higher anti-HA and anti-NA IgG2a levels compared to 1V270+1Z105. A stable liposomal formulation of 1V270+2B182C was developed, which synergistically enhanced anti-HA and anti-NA IgG1 and IgG2a responses without demonstrable reactogenicity after intramuscular injection. Notably, vaccination with IIAV plus the liposomal formulation of 1V270+2B182C protected mice against lethal homologous influenza virus (H1N1)pdm09 challenge and reduced lung viral titers and cytokine levels. The combination adjuvant induced a greater diversity in B cell clonotypes of immunoglobulin heavy chain (IGH) genes in the draining lymph nodes and antibodies against a broad spectrum of HA epitopes encompassing HA head and stalk domains and with cross-reactivity against different subtypes of HA and NA. This novel combination liposomal adjuvant contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

PD-1hi CD8+ tissue-resident memory T cells balance immunity and fibrotic sequelae following influenza infection

Abstract CD8+ tissue-resident memory T (TRM) cells provide frontline immunity in mucosal tissues. The mechanisms regulating CD8+ TRM maintenance, heterogeneity, and protective and pathological functions are largely elusive. Here, we identify a population of CD8+ TRM cells that is maintained by major histocompatibility complex class I (MHC-I) signaling, and CD80 and CD86 co-stimulation after acute influenza infection. These TRM cells have both “exhausted-like” phenotypes and memory features, and provide heterologous immunity against secondary infection. Using recombinant influenza virus, we demonstrate that the development of these PD-1hi “exhausted-like” TRM cells is dependent on the doses of the antigen during primary infection. Furthermore, we showed that lung CD11c+ antigen presenting cells are required for sustaining these TRM cells at the memory stage. Our data suggest that continuous antigen presentation in situ is required for the development and maintenance of these “exhausted-like” TRM cells in the lung. Strikingly, PD-L1 blockade after the resolution of primary infection promotes the rejuvenation of these “exhausted-like” TRM cells, restoring protective immunity at the cost of promoting post-infection inflammatory and fibrotic sequelae. Thus, PD-1 serves to limit the pathogenic capacity of “exhausted-like” TRM cells at the memory phase under specific tissue environment with chronic low-levels of antigen presentation following viral clearance. Our data indicate that TRM cell “exhaustion” is the result of a tissue-specific cellular adaptation that balances fibrotic sequelae with protective immunity.

Lung-resident memory CD8+ T cells prevent transmission of respiratory viruses

Abstract Lung tissue-resident memory CD8+ T cells (TRM) reduce viral replication and limit pathology following respiratory virus infections. In particular, TRM are crucial for protection against infections with heterosubtypic influenza viruses, where pre-existing antibody does not provide sterilizing immunity. In addition to providing individual host immunity, vaccination programs are designed to limit pathogen spread at population level. Despite the interest in lung TRM for intra-host protective immunity, their effect on the transmission respiratory viruses has been understudied due to the lack of animal models where influenza infection can be tracked longitudinally, and where the immunological parameters are easily identified and manipulated. To circumvent these issues, we used a luciferase-expressing Sendai virus, a natural mouse parainfluenza virus that readily transmits via the aerosol and contact routes, to evaluate the efficacy of lung TRM against transmission of respiratory viruses. Using a recombinant influenza virus expressing the immunodominant SenNP324–332Kb epitope to generate SenNP+ memory CD8+ T cells and IVIS imaging to non-invasively measure Sendai virus infection over time, we found that mice with pre-existing SenNP+ lung TRM don’t transmit Sendai virus to naïve mice when co-housed. In contrast, immunized mice that had pre-existing circulating effector memory CD8+ T cells (TEM), but no lung TRM, failed to prevent transmission of Sendai virus to naïve mice when co-housed. These results show the ability of lung TRM to contribute to herd immunity by preventing respiratory virus transmission and underscore the potential of a TRM-inducing vaccine for protection against respiratory viruses.

Protection against human metapneumovirus (hMPV) conveyed by influenza virus vectors carrying multiple epitope antigens of hMPV

Objective To evaluate the immune responses and protection against human metapneumovirus (hMPV) conveyed by influenza virus vectors carrying multiple epitope antigens of hMPV. Methods Two recombinant influenza viruses (rFLU/hMPV/B and rFLU/hMPV/CTL+ Th) carrying hMPV multi-epitope gene segments in NS gene were generated by reverse genetic techniques of eight-plasmid system. BALB/c mice were immunized intranasally with rFLU/hMPV/B and rFLU/hMPV/CTL+ Th twice at a two-week interval. Virus-specific antibody titers and splenocyte cytokines were detected two weeks after the boost immunization. Viral loads in lung tissues and turbinates were detected with digital PCR after the immunized mice were challenged with hMPV and influenza virus. Moreover, HE staining was used to observe lung injuries. Results Specific antibodies against both the influenza virus and hMPV were induced in mice immunized intranasally with rFLU/hMPV/B, while the influenza virus-specific antibody response and hMPV-specific cytotoxic lymphocyte response (significant IFN-γ secretion) were detected in mice immunized with rFLU/hMPV/CTL+ Th. Additionally, balanced Th1/Th2 responses were elicited by rFLU/hMPV/B and rFLU/hMPV/CTL+ Th. Both rFLU/hMPV/B and rFLU/hMPV/CTL+ Th conveyed effective protection against subsequent influenza virus and hMPV challenges with significantly alleviated histopathological damages and reduced viral loads. Conclusions Both rFLU/hMPV/B and rFLU/hMPV/CTL+ Th can induce specific humoral immune response against hMPV and/or the influenza virus. Moreover, rFLU/hMPV/CTL+ Th can also elicit hMPV-specific CTL immune response. These two recombinant strains can also protect BALB/c mice from the challenges with hMPV and influenza virus, suggesting that they are promising vaccine candidates. Key words: Human metapneumovirus; Influenza virus vector; Antigen epitope; Immune protection

A single dose of a vesicular stomatitis virus-based influenza vaccine confers rapid protection against H5 viruses from different clades

The avian influenza virus outbreak in 1997 highlighted the potential of the highly pathogenic H5N1 virus to cause severe disease in humans. Therefore, effective vaccines against H5N1 viruses are needed to counter the potential threat of a global pandemic. We have previously developed a fast-acting and efficacious vaccine against Ebola virus (EBOV) using the vesicular stomatitis virus (VSV) platform. In this study, we generated recombinant VSV-based H5N1 influenza virus vectors to demonstrate the feasibility of this platform for a fast-acting pan-H5 influenza virus vaccine. We chose multiple approaches regarding antigen design and genome location to define a more optimized vaccine approach. After the VSV-based H5N1 influenza virus constructs were recovered and characterized in vitro, mice were vaccinated by a single dose or prime/boost regimen followed by challenge with a lethal dose of the homologous H5 clade 1 virus. We found that a single dose of VSV vectors expressing full-length hemagglutinin (HAfl) were sufficient to provide 100% protection. The vaccine vectors were fast-acting as demonstrated by uniform protection when administered 3 days prior to lethal challenge. Moreover, single vaccination induced cross-protective H5-specific antibodies and protected mice against lethal challenge with various H5 clade 2 viruses, highlighting the potential of the VSV-based HAfl as a pan-H5 influenza virus emergency vaccine.

Pathological and immunological protection induced by inactivated reverse genetics-based H3N8 equine influenza vaccine candidate in murine model.

Equine influenza (EI) is an important viral respiratory disease of equines caused by influenza A virus (IAV). The antigenic drift in IAVs necessitates regular updating and harmonization of vaccine strain with the circulating virus. The reverse genetics-based recombinant viruses could be easy instrument in generating vaccine against circulating virus in a quick and effective manner. Present study has been envisaged to evaluate the immunogenicity and protective efficacy of inactivated recombinant equine influenza virus (rgEIV) vaccine candidate having six segments from H1N1 virus (A/WSN/33/H1N1) and HA (hemaglutinin) and NA (neuraminidase) segments from H3N8 equine influenza virus [(A/eq/Jammu-Katra/06/08) of clade 2 of Florida sublineage] generated through reverse genetic engineering. BALB/c mice were immunized with inactivated rgEIV adjuvanted with aluminium hydroxide gel and challenged with H3N8 virus (A/eq/Jammu-Katra/06/08). The protective efficacy was evaluated through serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry and residual virus quantification. Immunizations induced robust humoral immune response as estimated through hemagglutination inhibition assay (HAI). The antibodies were isotyped and the predominant subclass was IgG1. The vaccine candidate produced mixed Th1 and Th2 responses through stimulation of IFN-γ, IL-2, IL-4 and IL-6 expression. Immunization protected mice against challenge as reflected through reduction in clinical signs and body weight loss, early recovery, mild pathological changes (gross and histopathological lesions) as evident through scoring of lesions, low residual virus in nasopharynx and lungs quantified through egg titration and quantitative reverse transcriptase PCR (qRT-PCR). The study demonstrates that inactivated recombinant EIV generated through reverse genetic approach provides equivalent protection to that observed with inactivated whole H3N8 EIV vaccine. Keywords: equine influenza; reverse genetics; vaccine; pathology; murine model.

HA-Dependent Tropism of H5N1 and H7N9 Influenza Viruses to Human Endothelial Cells Is Determined by Reduced Stability of the HA, Which Allows the Virus To Cope with Inefficient Endosomal Acidification and Constitutively Expressed IFITM3.

Previous studies revealed that certain avian influenza A viruses (IAVs), including zoonotic H5N1 and H7N9 IAVs, infect cultured human lung microvascular endothelial cells (HULEC) more efficiently than other IAVs and that tropism to HULEC is determined by viral hemagglutinin (HA). To characterize mechanisms of HA-mediated endotheliotropism, we used 2:6 recombinant IAVs harboring HAs from distinctive avian and human viruses and found that efficient infection of HULEC correlated with low conformational stability of the HA. We next studied effects on viral infectivity of single-point amino acid substitutions in the HA of 2:6 recombinant virus A/Vietnam/1203/2004-PR8 (H5N1). Substitutions H8Q, H103Y, T315I, and K582I (K58I in the HA2 subunit), which increased stability of the HA, markedly reduced viral infectivity for HULEC, whereas substitutions K189N and K218Q, which altered typical H5N1 virus-like receptor specificity and reduced binding avidity of the HA, led to only marginal reduction of infectivity. None of these substitutions affected virus infection in MDCK cells. We confirmed the previous observation of elevated basal expression of IFITM3 protein in HULEC and found that endosomal acidification is less efficient in HULEC than in MDCK cells. In accord with these findings, counteraction of IFITM3-mediated restriction by amphotericin B and reduction of endosomal pH by moderate acidification of the extracellular medium enhanced infectivity of viruses with stable HA for HULEC without significant effect on infectivity for MDCK cells. Collectively, our results indicate that relatively high pH optimum of fusion of the HA of zoonotic H5N1 and H7N9 IAVs allows them to overcome antiviral effects of inefficient endosomal acidification and IFITM3 in human endothelial cells.IMPORTANCE Receptor specificity of the HA of IAVs is known to be a critical determinant of viral cell tropism. Here, we show that fusion properties of the HA may also play a key role in the tropism. Thus, we demonstrate that IAVs having a relatively low pH optimum of fusion cannot efficiently infect human endothelial cells owing to their relatively high endosomal pH and increased expression of fusion-inhibiting IFITM3 protein. These restrictions can be overcome by IAVs with elevated pH of fusion, such as zoonotic H5N1 and H7N9. Our results illustrate that the infectivity of IAVs depends on an interplay between HA conformational stability, endosomal acidification and IFITM3 expression in target cells, and the extracellular pH. Given significant variation of levels of HA stability among animal, human, and zoonotic IAVs, our findings prompt further studies on the fusion-dependent tropism of IAVs to different cell types in humans and its role in viral host range and pathogenicity.

A Recombinant Influenza A/H1N1 Carrying A Short Immunogenic Peptide of MERS-CoV as Bivalent Vaccine in BALB/c Mice.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) became a global human health threat since its first documentation in humans in 2012. An efficient vaccine for the prophylaxis of humans in hotspots of the infection (e.g., Saudi Arabia) is necessary but no commercial vaccines are yet approved. In this study, a chimeric DNA construct was designed to encode an influenza A/H1N1 NA protein which is flanking immunogenic amino acids (aa) 736–761 of MERS-CoV spike protein. Using the generated chimeric construct, a novel recombinant vaccine strain against pandemic influenza A virus (H1N1pdm09) and MERS-CoV was generated (chimeric bivalent 5 + 3). The chimeric bivalent 5 + 3 vaccine strain comprises a recombinant PR8-based vaccine, expressing the PB1, HA, and chimeric NA of pandemic 2009 H1N1. Interestingly, an increase in replication efficiency of the generated vaccine strain was observed when compared to the PR8-based 5 + 3 H1N1pdm09 vaccine strain that lacks the MERS-CoV spike peptide insert. In BALB/c mice, the inactivated chimeric bivalent vaccine induced potent and specific neutralizing antibodies against MERS-CoV and H1N1pdm09. This novel approach succeeded in developing a recombinant influenza virus with potential use as a bivalent vaccine against H1N1pdm09 and MERS-CoV. This approach provides a basis for the future development of chimeric influenza-based vaccines against MERS-CoV and other viruses.

Influenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment.

We previously attempted to establish a reporter influenza virus by inserting the gene for the Venus fluorescent protein into the NS segment of influenza A/Puerto Rico/8/34 (PR8, H1N1) virus to yield WT-Venus-PR8. Although the inserted Venus gene was deleted during serial passages of WT-Venus-PR8, we discovered that the PB2-E712D mutation stabilizes the Venus gene. Here, we explored the mechanisms by which Venus gene deletion occurs and how the polymerase mutation stabilizes the Venus gene. Deep sequencing analysis revealed that PB2-E712D does not cause an appreciable change in the mutation rate, suggesting that the stability of the Venus gene is not affected by polymerase fidelity. We found by using quantitative real-time PCR that WT-Venus-PR8 induces high-level interferon beta (IFN-β) expression. The induction of IFN-β expression seemed to result from the reduced transcription/replication efficiency of the modified NS segment in WT-Venus-PR8. In contrast, the transcription/replication efficiency of the modified NS segment was enhanced by the PB2-E712D mutation. Loss of the Venus gene in WT-Venus-PR8 appeared to be caused by internal deletions in the NS segment. Moreover, to further our understanding of the Venus stabilization mechanisms, we identified additional amino acid mutations in the virus polymerase complex that stabilize the Venus gene. We found that some of these amino acids are located near the template exit or the product exit of the viral polymerase, suggesting that these amino acids contribute to the stability of the Venus gene by affecting the binding affinity between the polymerase complex and the RNA template and product.IMPORTANCE The reverse genetics method of influenza virus generation has enabled us to generate recombinant viruses bearing modified viral proteins. Recombinant influenza viruses expressing foreign genes have become useful tools in basic research, and such viruses can be utilized as efficient virus vectors or multivalent vaccines. However, the insertion of a foreign gene into the influenza virus genome often impairs virus replication, and the inserted genes are unstable. Elucidation of the mechanisms of foreign gene stabilization will help us to establish useful recombinant influenza viruses.

Extending the Stalk Enhances Immunogenicity of the Influenza Virus Neuraminidase.

Influenza viruses express two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). Anti-NA antibodies protect from lethal influenza virus challenge in the mouse model and correlate inversely with virus shedding and symptoms in humans. Consequently, the NA is a promising target for influenza virus vaccine design. Current seasonal vaccines, however, poorly induce anti-NA antibodies, partly because of the immunodominance of the HA over the NA when the two glycoproteins are closely associated. To address this issue, here we investigated whether extending the stalk domain of the NA could render it more immunogenic on virus particles. Two recombinant influenza viruses based on the H1N1 strain A/Puerto Rico/8/1934 (PR8) were rescued with NA stalk domains extended by 15 or 30 amino acids. Formalin-inactivated viruses expressing wild-type NA or the stalk-extended NA variants were used to vaccinate mice. The virus with the 30-amino-acid stalk extension induced significantly higher anti-NA IgG responses (characterized by increased in vitro antibody-dependent cellular cytotoxicity [ADCC] activity) than the wild-type PR8 virus, while anti-HA IgG levels were unaffected. Similarly, extending the stalk domain of the NA of a recent H3N2 virus enhanced the induction of anti-NA IgGs in mice. On the basis of these results, we hypothesize that the subdominance of the NA can be modulated if the protein is modified such that its height surpasses that of the HA on the viral membrane. Extending the stalk domain of NA may help to enhance its immunogenicity in influenza virus vaccines without compromising antibody responses to HA.IMPORTANCE The efficacy of influenza virus vaccines could be improved by enhancing the immunogenicity of the NA protein. One of the reasons for its poor immunogenicity is the immunodominance of the HA over the NA in many seasonal influenza virus vaccines. Here we demonstrate that, in the mouse model, extending the stalk domain of the NA protein can enhance its immunogenicity on virus particles and overcome the immunodominance of the HA without affecting antibody responses to the HA. The antibody repertoire is broadened by the extended NA and includes additional ADCC-active antibodies. Our findings may assist in the efforts toward more effective influenza virus vaccines.

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection.

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Highly pathogenic avian influenza H7N9 viruses with reduced susceptibility to neuraminidase inhibitors showed comparable replication capacity to their sensitive counterparts

BackgroundHuman infection with avian influenza H7N9 virus was first reported in 2013. Since the fifth epidemic, a highly pathogenic avian influenza (HPAI) H7N9 virus has emerged and caused 33 human infections. Several potential NAI resistance sites have been found in human cases. However, the drug susceptibility and replication ability of HPAI H7N9 virus with such substitutions have not yet been studied.MethodsThirty-three HPAI H7N9 virus strains were isolated from human cases in China, and then sequences were analyzed to identify potential NAI resistance sites. Recombinant influenza viruses were generated to evaluate the effect of NA amino acid substitutions on NAI (oseltamivir or zanamivir) susceptibility and viral replication efficiency in MDCK cells.ResultsFour potential NAI resistance sites, R292 K, E119V, A246T or H274Y, were screened. All four substitutions conferred either reduced or highly reduced susceptibility to oseltamivir or zanamivir. 292 K not only highly reduced the susceptibility of HPAI H7N9 to oseltamivir but also induced an increase in the IC50 of zanamivir. 119 V or 274Y conferred reduced susceptibility of HPAI H7N9 to oseltamivir. Additionally, 246 T conferred reduced susceptibility to zanamivir. All tested NAI-resistant viruses were capable of replication in MDCK cells. The virus yields of rg006-NA292K were lower than those of rg006-NA292R at 24, 48, 72 and 96 h postinfection (P<0.05). Rg006-NA119V, rg006-NA246T or rg006-NA274Y showed comparable replication capacity to wild-type virus (except for rg006-NA274Y at 96 h, P<0.05).ConclusionsAll 4 amino acid substitutions (R292 K, E119V, A246T or H274Y) in NA reduced the susceptibility of HPAI H7N9 to NAIs. The NAI-resistant mutations in HPAI H7N9, in most cases, did not reduce the replication ability of the virus in mammalian cells. Special attention needs to be paid to these mutations, and the development of new anti-H7N9 drugs is of great importance.