Recombinant Influenza Virus Research Articles

Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a.

Mycoplasma pneumoniae (MP) is a common respiratory pathogen affecting the longevity of the elderly and the health of children. However, the human vaccine against MP has not been successfully developed till now due to the poor immunogenicity and side effects of MP inactivated or attenuated vaccine. Therefore, it is necessary to develop a MP genetic engineering vaccine with influenza virus strain as vector. In this study, the major antigen genes P1a of MP adhesion factor P1(3862-4554 bases) and P30a of P30(49-822 bases) were inserted into the nonstructural protein (NS) gene of Influenza A virus strain A/Puerto Rio/8/34(H1N1), PR8 for short, to construct the recombinant vectors NS-P1a or NS-P30a. The recombinant pHW2000 plasmids containing NS-P1a or NS-P30a were cotransfected with the rest 7 fragments of PR8 into HEK293T cells. After inoculating chicken embryos, the recombinant influenza viruses rFLU-P1a and rFLU-P30a were rescued. RT-PCR and sequencing were used to identify the recombinant viruses. The hemagglutination titers of rFLU-P1a and rFLU-P30a were determined after five successive generations in chicken embryos so as to indicate the genetic stability of the recombinant viruses. The morphology of recombinant influenza viruses was observed under electron microscopy. P1a or P30a was designed to be inserted into the modified NS gene sequence separately and synthesized successfully. RT-PCR identification of the recombinant viruses rFLU-P1a and rFLU-P30a showed that P1a (693 bp), P30a (774 bp), NS-P1a (1992bp) and NS-P30a (2073 bp) bands were found, and the sequencing results were correct. After five successive generations, each virus generation has a certain hemagglutination titer (from 1:32 to 1:64), and the band of P1a or P30a can be seen in the corresponding positions. The virus particles under the electron microscope appeared as spheres or long strips connected by several particles, revealing a complete viral membrane structure composed of virus lipid bilayer, hemagglutinin, neuraminidase, and matrix proteins. The recombinant viruses rFLU-P1a and rFLU-P30a which carried the advantaged immune regions of the P1 and P30 genes in MP were successfully constructed and identified. And the genetic stability of rFLU-P1a or rFLU-P30a was relatively high. The typical and complete morphology of influenza virus was observed under the electron microscope. Our research provided a foundation for the further development of MP vaccines for human.

Development of a Recombinant Live Attenuated Influenza Vaccine Virus Expressing Pneumococcal Surface Antigen A as a Strategy for Combined Protection Against Influenza and Bacterial Coinfection Caused by Streptococcus pneumoniae

Introduction Bacterial superinfection with Streptococcus pneumoniae following the influenza virus infection complicates the course of the disease and is a major cause of mortality during influenza virus epidemics. The effectiveness of licensed polysaccharide vaccines is limited by the serotypes included in the vaccine and possible immune tolerance during revaccination. Pneumococcal surface protein A (PspA), which includes conserved regions and has at least two functions in pathogen virulence, is considered a promising target for the development of new-generation vaccines, including gene-engineering constructions. Therefore, the development of an influenza virus vector-based vaccine expressing conserved bacterial proteins seems to be a promising strategy for designing combined vaccines against influenza and bacterial pathogens. Methods A recombinant live attenuated influenza virus (LAIV) expressing PspA fragment in a modified hemagglutinin was rescued on the A/Leningrad/134/17/57 (H2N2) backbone. This recombinant virus was assessed for its growth characteristics in vitro, as well as for its immunogenicity and protective capacity, using a mouse model of influenza-bacterial coinfection. Results The rescued recombinant LAIV/HA+PspA virus was genetically stable after sequential passaging in embryonated chicken eggs and possessed an attenuated phenotype similar to the classical LAIV strain. The LAIV/HA+PspA bivalent vaccine-induced IgG antibodies specific to both influenza virus and S. pneumoniae and provided complete protection of vaccinated mice against lethal influenza infection, as well as a 40% survival rate for lethal homologous and heterologous influenza infection complicated by concomitant bacterial infection with S. pneumoniae. Conclusion The presented design of a recombinant influenza virus carrying immunogenic fragments of a bacterial pathogen can be considered a promising strategy for the combined protection of vaccinated individuals against influenza and its bacterial complications, and further in-depth studies of such recombinant viruses in preclinical studies are warranted.

Adjuvanted nanoliposomes displaying six hemagglutinins and neuraminidases as an influenza virus vaccine

Inclusion of defined quantities of the two major surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA), could benefit seasonal influenza vaccines. Recombinant HA and NA multimeric proteins derived from three influenza serotypes, H1N1, H3N2, and type B, are surface displayed on nanoliposomes co-loaded with immunostimulatory adjuvants, generating "hexaplex" particles that are used to immunize mice. Protective immune responses to hexaplex liposomes involve functional antibody elicitation against each included antigen, comparable to vaccination with monovalent antigen particles. When compared to contemporary recombinant or adjuvanted influenza virus vaccines, hexaplex liposomes perform favorably in many areas, including antibody production, Tcell activation, protection from lethal virus challenge, and protection following passive sera transfer. Based on these results, hexaplex liposomes warrant further investigation as an adjuvanted recombinant influenza vaccine formulation.

An intranasal influenza virus vector vaccine protects against Helicobacter pylori in mice.

Helicobacter pylori is a human pathogen that infects almost half of the population. Antibiotic resistance in H. pylori threatens health and increases the demand for prophylactic and therapeutic vaccines. Traditional oral vaccine research faces considerable challenges because of the epithelial barrier, potential enterotoxicity of adjuvants, and the challenging conditions of the gastric environment. We developed an intranasal influenza A virus (IAV) vector vaccine based on two live attenuated influenza viruses with modified acidic polymerase protein (PA) genes encoding the A subunit of H. pylori neutrophil-activating protein (NapA), named IAV-NapA, including influenza virus A/WSN/33 (WSN)-NapA and A/Puerto Rico/8/34 (PR8)-NapA. These recombinant influenza viruses were highly attenuated and exhibited strong immunogenicity in mice. Vaccination with IAV-NapA induced antigen-specific humoral and mucosal immune responses while stimulating robust Th1 and Th17 cell immune responses in mice. Our findings suggest that prophylactic and therapeutic vaccination with influenza virus vector vaccines significantly reduces colonization of H. pylori and inflammation in the stomach of mice.IMPORTANCEHelicobacter pylori is the most common cause of chronic gastritis and leads to severe gastroduodenal pathology in some patients. Many studies have shown that Th1 and Th17 cellular and gastric mucosal immune responses are critical in reducing H. pylori load. IAV vector vaccines can stimulate these immune responses while overcoming potential adjuvant toxicity and antigen dosing issues. To date, no studies have demonstrated the role of live attenuated IAV vector vaccines in preventing and treating H. pylori infection. Our work indicates that vaccination with IAV-NapA induces antigen-specific humoral, cellular, and mucosal immunity, producing a protective and therapeutic effect against H. pylori infection in BALB/c mice. This undescribed H. pylori vaccination approach may provide valuable information for developing vaccines against H. pylori infection.

A chimeric influenza virus vaccine expressing fusion protein epitopes induces protection from human metapneumovirus challenge in mice.

Human metapneumovirus (HMPV) is a common virus associated with acute respiratory distress syndrome in pediatric patients. There are no HMPV vaccines or therapeutics that have been approved for prevention or treatment. In this study, we constructed a novel recombinant influenza virus carrying partial HMPV fusion protein (HMPV-F), termed rFLU-HMPV/F-NS, utilizing reverse genetics, which contained (HMPV-F) in the background of NS segments of influenza virus A/PuertoRico/8/34(PR8). The morphological characteristics of rFLU-HMPV/F-NS were consistent with the wild-type flu virus. Additionally, immunofluorescence results showed that fusion proteins in the chimeric rFLU-HMPV/F-NS could work well, and the virus could be stably passaged in SPF chicken embryos. Furthermore, intranasal immunization with rFLU-HMPV/F-NS in BALB/c mice induced robust humoral, mucosal and Th1-type dominant cellular immune responses in vivo. More importantly, we discovered that rFLU-HMPV/F-NS afforded significant protective efficacy against the wild-type HMPV and influenza virus challenge, with significantly attenuated pathological changes and reduced viral titers in the lung tissues of immunized mice. Collectively, these findings demonstrated that chimeric recombinant rFLU-HMPV/F-NS as a promising HMPV candidate vaccine has potentials for the development of HMPV vaccine.

Protective efficacy of a novel recombinant influenza virus carrying partial human metapneumovirus F protein epitopes

ABSTRACT Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infections in infants and children. Currently, no approved HMPV vaccine is available. We developed a novel recombinant influenza virus, which carried partial HMPV F protein (HMPV-F) epitopes, utilizing reverse genetics. The novel single-stranded RNA virus, termed rFLU-HMPV/F-NA, was synthesized in the neuraminidase (NA) fragment of influenza virus A/PuertoRico/8/34 (PR8). The morphological characteristics of rFLU-HMPV/F-NA were consistent with the wild-type flu virus. The virus could passage in specific pathogen-free (SPF) chicken embryos for at least five consecutive generations with haemagglutinin (HA) titres of 28–9 or 8–9LogTCID50/mL. BALB/c mice were intranasally immunized at 21-day intervals with 104 TCID50 (low-dose group) or 106 TCID50 (high-dose group) rFLU-HMPV/F-NA, and PBS or PR8 vaccine was used for the control group. rFLU-HMPV/F-NA induced robust humoral, mucosal, and cellular immune responses in vivo in a dose-dependent manner. More importantly, wt clinical HMPV isolate challenge studies showed that rFLU-HMPV/F-NA provided significant immune protection against HMPV infection compared to the PBS or PR8 vaccine control group, as shown by improved histopathological changes and reduced viral titres in the lungs of immunized mice post-challenge. These findings demonstrate that rFLU-HMPV/F-NA has potential as a promising HMPV candidate vaccine and warrants further investigation into its control of HMPV infection.

A Recombinant Oncolytic Influenza Virus Carrying GV1001 Triggers an Antitumor Immune Response.

Oncolytic viruses are able to lyse tumor cells selectively in the liver without killing normal hepatocytes, in addition to activating the immune response. Oncolytic virus therapy is expected to revolutionize the treatment of liver cancer, including hepatocellular carcinoma (HCC), one of the most frequent and fatal malignancies. In this study, reverse genetics techniques were exploited to load NA fragments of the A/PuertoRico/8/34 virus (PR8) with GV1001 peptides derived from human telomerase reverse transcriptase. An in vitro assessment of the therapeutic effect of the recombinant oncolytic virus was followed by an in vivo study in mice with HCC. The recombinant virus was verified by sequencing of the recombinant viral gene sequence, and viral virulence was detected by hemagglutination assays and based on the 50% tissue culture infectious dose (TCID50). The morphological structure of the virus was observed by electron microscopy, and GV1001 peptide was localized by cellular immunofluorescence. The selective cytotoxicity of the recombinant oncolytic virus in vitro was demonstrated in cultured HCC cells and normal hepatocytes, as only the tumor cells were killed; the normal cells were not significantly altered. Consistent with the in vitro results, the recombinant oncolytic influenza virus significantly inhibited liver tumor growth in mice in vivo, in addition to inducing an antitumor immune response, including an increase in the number of CD4+ and CD8+ T lymphocytes and, in turn, improving survival. Our results suggest that oncolytic influenza virus carrying GV1001 is a promising immunotherapy in patients with HCC.

Immunity induced by vaccination with recombinant influenza B virus neuraminidase protein breaks viral transmission chains in guinea pigs in an exposure intensity-dependent manner.

Vaccines that can slow respiratory virus transmission in the population are urgently needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Here, we describe how a recombinant neuraminidase-based influenza virus vaccine reduces transmission in vaccinated guinea pigs in an exposure intensity-based manner.

A recombinant chimeric influenza virus vaccine expressing the consensus H3 hemagglutinin elicits broad hemagglutination inhibition antibodies against divergent swine H3N2 influenza viruses

The global distribution and ongoing evolution of type A swine influenza virus (IAV-S) continue to pose significant challenges against developing broadly protective vaccines to control swine influenza. This study focuses on the hemagglutinin (HA) consensus-based approach towards developing a more broadly protective swine influenza vaccine against various H3 strains circulating in domestic pig populations. By computationally analyzing >1000 swine H3 full-length HA sequences, we generated a consensus H3 and expressed it in the context of influenza A WSN/33 reverse genetics system. The derived recombinant chimeric swine influenza virus with the consensus H3 was inactivated and further evaluated as a potential universal vaccine in pigs. The consensus H3 vaccine elicited broadly active hemagglutination inhibition (HI) antibodies against divergent swine H3N2 influenza viruses including human H3N2 variant of concern, and strains belong to genetic clusters IV, IV-A, IV-B, IV-C, IV-D and IV-F. Importantly, vaccinated pigs were completely protected against challenge with a clinical swine H3N2 isolate in that neither viral shedding nor replication in lungs of vaccinated pigs were observed. These findings warrant further study of the consensus H3 vaccine platform for broad protection against diverse swine influenza viruses.

Research Note: A recombinant duck-derived H6N2 subtype avian influenza virus can replicate and shed in young chickens and cause disease

The H6N2 subtype avian influenza virus (AIV) is commonly detected in the migratory waterfowl reservoirs. Previously, H6N2 AIV was believed to be nonpathogenic to young chickens and could not infect or shed in their respiratory tract under experimental conditions. However, in present study, a highly recombinant strain of duck-derived H6N2 AIV was discovered and isolated for pathogenicity tests. The results revealed that H6N2 could induce seroconversion in chickens and high morbidity of over 86.7%, along with evident upper respiratory tract hemorrhage. Moreover, 5 substitutions were detected in the upper respiratory tract shedding reisolated virus, with a high viral load in the target organs of infected chickens. In contrast, ducks failed to exhibit any symptoms, pathological lesions, or viral shedding, while demonstrated seroconversion and high viral load in the livers. These findings indicate that H6N2 AIV could also show pathogenicity to chickens under experimental conditions, thereby effectively replicating and shedding in chickens. Therefore, the study provides further elucidations on the pathogenicity of H6N2 AIV.

Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice.

Tick-borne encephalitis virus (TBEV) is one of the most relevant tick-transmitted neurotropic arboviruses in Europe and Asia and the causative agent of tick-borne encephalitis (TBE). Annually more than 10,000 TBE cases are reported despite having vaccines available. In Europe, the vaccines FSME-IMMUN® and Encepur® based on formaldehyde-inactivated whole viruses are licensed. However, demanding vaccination schedules contribute to sub-optimal vaccination uptake and breakthrough infections have been reported repeatedly. Due to its immunogenic properties as well as its role in viral replication and disease pathogenesis, the non-structural protein 1 (NS1) of flaviviruses has become of interest for non-virion based flavivirus vaccine candidates in recent years. Therefore, immunogenicity and protective efficacy of TBEV NS1 expressed by neuraminidase (NA)-deficient Influenza A virus (IAV) or Modified Vaccinia virus Ankara (MVA) vectors were investigated in this study. With these recombinant viral vectors TBEV NS1-specific antibody and T cell responses were induced. Upon heterologous prime/boost regimens partial protection against lethal TBEV challenge infection was afforded in mice. This supports the inclusion of NS1 as a vaccine component in next generation TBEV vaccines.

A recombinant oncolytic influenza virus expressing a PD-L1 antibody induces CD8+ T-cell activation via the cGas-STING pathway in mice with hepatocellular carcinoma

ObjectiveTo evaluate targeted killing of hepatocellular carcinoma (HCC) cells by a recombinant oncolytic influenza virus expressing a PD-L1 antibody (rgFlu/PD-L1) and to develop a novel immunotherapy for HCC. MethodsUsing influenza virus reverse genetics, a recombinant oncolytic virus was generated in the background of the A/Puerto Rico/8/34 (PR8) virus, then identified via screening and passage in specific pathogen-free chicken embryos. Hepatocellular carcinoma cell killing by rgFlu/PD-L1 was confirmed in vitro and in vivo. Transcriptome analyses were used to explore PD-L1 expression and function. Western blotting revealed that PD-L1 activated the cGas-STING pathway. ResultsrgFlu/PD-L1 expressed the PD-L1 heavy and light chain in PB1 and PA, respectively; PR8 served as the backbone. The hemagglutinin titer of rgFlu/PD-L1 was 29, and the virus titer was 9–10 logTCID50/mL. Electron microscopy revealed that the rgFlu/PD-L1 morphology and size were consistent with wild-type influenza virus. The MTS assay showed that rgFlu/PD-L1 induced significant killing of HCC cells but not normal cells. rgFlu/PD-L1 inhibited PD-L1 expression and induced apoptosis in HepG2 cells. Notably, rgFlu/PD-L1 controlled the viability and function of CD8+ T cells by activating the cGas-STING pathway. ConclusionrgFlu/PD-L1 activated the cGas-STING pathway in CD8+ T cells, causing them to kill HCC cells. This approach represents a novel immunotherapy for liver cancer.

Host-targeted self-attenuated influenza virus as an effective nasal spray vaccine in aged population

Abstract The elderly are at high risk for influenza infection and its complication. However, current inactivated influenza vaccines are often modestly immunogenic and show low protective immunity in the elderly, due to immunosenescence. New approaches are urgently needed for developing effective vaccines against influenza for the elderly. In current study, we have constructed new recombinant influenza viruses, named host-targeted self-attenuated influenza viruses (SAIVs), which can express functional mammalian species-specific artificial microRNAs (amiRNAs). The expression of these amiRNAs can inhibit expression of some host factors critical for influenza replication, and therefore the resultant recombinant influenza viruses are replication restricted and attenuated in the host cells. One of these SAIVs, which can express an amiRNA that inhibits expression of the host cellular Cdc2-like kinase 1 (CLK1), was produced in embryonic chicken eggs and evaluated in an aged mouse model of influenza infection. It elicited robust antibody and T cell responses against influenza virus and demonstrated significantly protective efficacy against lethal infection with wild type influenza virus H1N1 PR8 after single dose of intranasal vaccination. Our research finding provided a proof of concept that the new host targeted self-attenuated influenza virus can be further used as an effective vaccine against influenza in aged population. Supported by a research grant from National Institute of Allergy and Infectious Diseases (AI133207).

A rapid and flexible microneutralization assay for serological assessment of influenza viruses.

Serological responses from influenza vaccination or infection are typically measured by hemagglutinin inhibition (HAI) or microneutralization (MN). Both methods are limited in feasibility, standardization, and generalizability to recent strains. We developed a luciferase MN (LMN) assay that combines the advantages of the conventional MN assay with the ease of the HAI assay. Sera were obtained from the HIVE study, a Michigan household cohort. Reverse genetics was used to generate recombinant influenza viruses expressing the hemagglutinin and neuraminidase of test strains, all other viral proteins from an A/WSN/1933 backbone, and a NanoLuc reporter. Serum neutralization of luciferase-expressing targets was quantified as a reduction in light emission from infected cells. Neutralization titers were measured for cell- and egg-adapted versions of A/Hong Kong/4801/2014 and A/Singapore/INFIMH-16-0019/2016 and compared to HAI titers against egg-grown antigens. Three hundred thirty-three sera were collected from 259 participants between May 2016 and July 2018. Sampled participants were 7-68 years of age, and >80% were vaccinated against influenza. HAI and LMN titers were correlated for A/Hong Kong/4801/2014 (ρ= 0.52, p≤ 0.01) and A/Singapore/INFIMH-16-0019/2016 (ρ= 0.79, p≤ 0.01). LMN titers were lower for cell strains compared to egg strains (A/Hong Kong/4801/2014 mean log2 fold change = -2.66, p≤ 0.01 and A/Singapore/INFIMH-16-0019/2016 mean log2 fold change = -3.15, p≤ 0.01). The LMN assay was feasible using limited sample volumes and able to differentiate small antigenic differences between egg-adapted and cell-derived strains. The correspondence of these results with the commonly used HAI confirms the utility of this assay for high-throughput studies of correlates of protection and vaccine response.

Pathogenicity, transmissibility, and immunogenicity of recombinant H9N2 avian influenza viruses based on representative viruses of Southeast China

H9N2 is currently the main subtype of avian influenza in China. In order to use reverse genetics to rapid preparation of seed strains for vaccine production, and intend to prevent and control the H9N2 subtype epidemic strains of avian influenza virus (AIV). In this study, we successfully rescued 2 H9N2 recombinant viruses based on the representative viruses of Southeast China and confirmed by RT-PCR and sequencing. Genetic stability, pathogenicity, transmissibility, and antigenicity of 2 recombinant viruses were evaluated. Compared to the FZ1, the growth kinetics of H9N2(HA+NA)/PR8 showed no significant difference, H9N2(HA+NA+M+PB1)/PR8 was slightly lower. Our study also confirmed 2 recombinant viruses had good genetic stability after 10 passages but possessed lower pathogenicity than FZ1. Although both recombinant viruses led to seroconversion in all inoculated birds on 14 dpi, they complete loss of viral transmission of the virus to contact birds. In addition, birds were immunized via hypodermic route by inactivated vaccines of H9N2(HA+NA)/PR8, H9N2(HA+NA+M+PB1)/PR8 and wild-type virus with a single dose, and the results showed that the hemagglutination inhibition titers on 21 dpv were 10.5, 9.6, and 10.5 log2, respectively. And recombinant viruses both provided a certain protection against wild-type virus challenge. In conclusion, these data indicated that 2 recombinant viruses will be expected to be used as inactivated vaccines to controlling the spread of H9N2 subtype AIV even have potential application for attenuated viral vaccines, which provides a reference for the prevention and control of influenza virus pandemics.

Quantitative performance of digital ELISA for the highly sensitive quantification of viral proteins and influenza virus.

A single-molecule assay (SiMoA) using a digital enzyme-linked immunosorbent assay (ELISA) has been attracting attention as a promising method that can detect viruses with ultra-high sensitivity. However, the quantitative application of digital ELISA has not been adequately reported. Therefore, in this study, we first evaluated the linearity and sensitivity of digital ELISA using a Certified Reference Material of C-reactive protein (NMIJ CRM 6201-c) as a quality control material. Next, we originally screened those antibody pair that are suitable for detecting recombinant viral proteins of influenza A virus, nucleoprotein (NP), and hemagglutinin (HA), and established the measurement system. Under optimized conditions, the limit of detection (LOD) of NP and HA was 0.59 fM and 0.99 fM, and the coefficient of determination, R2, was 0.9998 and 0.9979, respectively. Two subtypes of influenza virus, A/Puerto Rico/8/1934 (H1N1) [PR8] and A/Panama/2007/99 (H3N2) [Pan99], were also quantified under established conditions, and the LOD of PR8 was 3.1 × 102 PFU/mL on targeting NP and 7.4 × 102 PFU/mL on targeting HA. The LOD of Pan99 was 5.3 × 102 PFU/mL on targeting NP. The specificity and robustness of the recombinant viral protein and influenza virus measurements using digital ELISA were also evaluated. Our measurement system showed enough specificity to discriminate the viral subtypes properly and showed sufficient inter- and intra-assay variations for both measurements of recombinant viral proteins and viruses, except for NP-targeting virus measurement.

Evaluation of the genetic stability of recombinant flu vectors encoding <i>Mycobacterium bovis</i> proteins using RT-PCR and optimization of their cultivation conditions

Prevention by immunizing cattle against tuberculosis with traditional vaccines and regular testing has long been the main method of controlling this infection. However, the non-specificity of the traditional method shows the need for alternative approaches in the creation of anti-infective vaccines. The development of recombinant vector vaccines based on influenza vectors shows great potential and advantages in providing a specific immune response.The purpose of the study is to evaluate the growth properties of the recombinant influenza virus strains expressing protective proteins of mycobacteria for further use in creating a vector vaccine against bovine tuberculosis.This article presents the results of work on the cultivation and reproduction of recombinant influenza virus strains. Using reverse genetics methods, recombinant strains of the influenza virus carrying the mycobacterial Mycobacterium bovis ESAT-6 and TB10.4 proteins in the NS gene sequence were constructed. Based on the results of the work carried out, the optimal conditions for cultivating recombinant influenza virus strains were determined. Both variants of the recombinant strain showed reproductive activity in the developing chick embryo system, under optimal cultivation conditions.The evaluation of the genetic stability of the insertion of mycobacterial proteins into the NS gene of the influenza virus was confirmed using the RT-PCR method. As a result, it was found that the NS gene segment contains an insertion of mycobacterial proteins TB10.4 and ESAT-6, which is retained throughout the studied 5 passages.

Recombinant single-cycle influenza virus with exchangeable pseudotypes allows repeated immunization to augment anti-tumour immunity with immune checkpoint inhibitors.

Virus-based tumour vaccines offer many advantages compared to other antigen-delivering systems. They generate concerted innate and adaptive immune response, and robust CD8+ T cell responses. We engineered a non-replicating pseudotyped influenza virus (S-FLU) to deliver the well-known cancer testis antigen, NY-ESO-1 (NY-ESO-1 S-FLU). Intranasal or intramuscular immunization of NY-ESO-1 S-FLU virus in mice elicited a strong NY-ESO-1-specific CD8+ T cell response in lungs and spleen that resulted in the regression of NY-ESO-1-expressing lung tumour and subcutaneous tumour, respectively. Combined administration with anti-PD-1 antibody, NY-ESO-1 S-FLU virus augmented the tumour protection by reducing the tumour metastasis. We propose that the antigen delivery through S-FLU is highly efficient in inducing antigen-specific CD8+ T cell response and protection against tumour development in combination with PD-1 blockade.

A replication-deficient H9N2 influenza virus carrying H5 hemagglutinin conferred protection against H9N2 and H5N1 influenza viruses in mice

H5N1 and H9N2 influenza viruses have been reported to cause human infections and are believed to have pandemic potential. The vaccine is an effective tool to prevent influenza virus infection. However, inactivated influenza vaccines sometimes result in low antigenicity as result leads to generating of incomplete immune protection in the form of low cellular and humoral immunity. While the low temperature adapted, traditional live attenuated influenza vaccine (LAIV) is associated with the potential risk to revert to a virulent phenotype, there appears an essential need for an alternative potent methodology to design and develop influenza vaccines with substantial safety and efficacy which may confer solid protection against H9N2 or H5N1 influenza virus infections. In the present study, a replication-deficient recombinant influenza virus, WM01ma-HA(H5), expressing hemagglutinin (HA) of both H9N2 and H5N1 subtypes was developed. The chimeric gene segment expressing HA(H5), was designed using the sequence of an open reading frame (ORF) of HA adopted from A/wild duck/Hunan/021/2005(H5N1)(HN021ma) which was flanked by the NA packaging signals of mouse-adapted strain A/Mink/Shandong/WM01/2014(H9N2)(WM01ma). Due to the absence of ORF of structural protein NA, the replication of this engineered H9N2 influenza viruses WM01ma-HA(H5) was hampered in vitro and in vivo but was well competent in MDCK cells stably expressing the NA protein of WM01ma. Intranasal vaccination of mice with WM01ma-HA(H5) stimulated robust immune response without any clinical signs and conferred complete protection from infection by H5N1 or H9N2 subtype influenza viruses.

Expanding the tolerance of segmented Influenza A Virus genome using a balance compensation strategy.

Reporter viruses provide powerful tools for both basic and applied virology studies, however, the creation and exploitation of reporter influenza A viruses (IAVs) have been hindered by the limited tolerance of the segmented genome to exogenous modifications. Interestingly, our previous study has demonstrated the underlying mechanism that foreign insertions reduce the replication/transcription capacity of the modified segment, impairing the delicate balance among the multiple segments during IAV infection. In the present study, we developed a "balance compensation" strategy by incorporating additional compensatory mutations during initial construction of recombinant IAVs to expand the tolerance of IAV genome. As a proof of concept, promoter-enhancing mutations were introduced within the modified segment to rectify the segments imbalance of a reporter influenza PR8-NS-Gluc virus, while directed optimization of the recombinant IAV was successfully achieved. Further, we generated recombinant IAVs expressing a much larger firefly luciferase (Fluc) by coupling with a much stronger compensatory enhancement, and established robust Fluc-based live-imaging mouse models of IAV infection. Our strategy feasibly expands the tolerance for foreign gene insertions in the segmented IAV genome, which opens up better opportunities to develop more versatile reporter IAVs as well as live attenuated influenza virus-based vaccines for other important human pathogens.