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Review
. 2022 Jul 22:12:954811.
doi: 10.3389/fcimb.2022.954811. eCollection 2022.

Live attenuated influenza A virus vaccines with modified NS1 proteins for veterinary use

Aitor Nogales  1 , Marta L DeDiego  2 , Luis Martínez-Sobrido  3
Affiliations
Review

Live attenuated influenza A virus vaccines with modified NS1 proteins for veterinary use

Aitor Nogales et al. Front Cell Infect Microbiol. .

Abstract

Influenza A viruses (IAV) spread rapidly and can infect a broad range of avian or mammalian species, having a tremendous impact in human and animal health and the global economy. IAV have evolved to develop efficient mechanisms to counteract innate immune responses, the first host mechanism that restricts IAV infection and replication. One key player in this fight against host-induced innate immune responses is the IAV non-structural 1 (NS1) protein that modulates antiviral responses and virus pathogenicity during infection. In the last decades, the implementation of reverse genetics approaches has allowed to modify the viral genome to design recombinant IAV, providing researchers a powerful platform to develop effective vaccine strategies. Among them, different levels of truncation or deletion of the NS1 protein of multiple IAV strains has resulted in attenuated viruses able to induce robust innate and adaptive immune responses, and high levels of protection against wild-type (WT) forms of IAV in multiple animal species and humans. Moreover, this strategy allows the development of novel assays to distinguish between vaccinated and/or infected animals, also known as Differentiating Infected from Vaccinated Animals (DIVA) strategy. In this review, we briefly discuss the potential of NS1 deficient or truncated IAV as safe, immunogenic and protective live-attenuated influenza vaccines (LAIV) to prevent disease caused by this important animal and human pathogen.

Keywords: differentiating infected from vaccinated animals (DIVA); influenza A virus; interferon; live-attenuated influenza vaccine (LAIV); non-structural 1 (NS1) protein.

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Conflict of interest statement

LM-S and AN have patented LAIV for the prevention of canine and equine IAV. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
IAV virion structure and genome organization. (A) Virion structure. IAV particles have a lipid envelope where the two major viral glycoproteins HA and NA and the ion channel M2 are located. Below the viral lipid membrane is a layer composed of M1 protein and the NEP. Inside the viral particle are the vRNP particles formed by the vRNA coated by the viral NP and linked to the heterotrimeric polymerase complex (PB2, PB1 and PA). (B) Genome organization. IAV contains eight vRNA segments (PB2, PB1, PA, HA, NP, NA, M, and NS) made of a coding region (gray boxes) flanked at the 3′ and 5′ terminal ends by untranslated non-coding regions (white boxes). At the end of the 3′ and 5′ coding regions are the specific viral segment packaging signals (Ψ) required for efficient encapsidation of the vRNP particles into new viruses.
Figure 2
Figure 2
Plasmid-based reverse genetics approach for the recovery of recombinant IAV. Schematic representation of the ambisense plasmid used to generate recombinant IAV is shown in the top. IAV cDNAs are cloned into the plasmid flanked by the human polymerase I promoter (hPol-I, gray arrow) and the mouse Pol-I terminator (TI, gray box) sequences to drive the synthesis of the vRNAs. In opposite orientation to the polymerase I cassette is the polymerase II (Pol-II) cassette made of a Pol-II dependent cytomegalovirus promoter (pCMV, white arrow) and the polyadenylation sequence of the bovine growth hormone (BGH, white box) to allow the expression of viral proteins from the same viral cDNAs. Co-cultures of human 293T (gray) and canine MDCK (black) cells are co-transfected with the eight (PB2, PB1, PA, HA, NP, NA, M, and NS) ambisense plasmids. Recovered virus is amplified in fresh cells or embryonated chicken eggs for vaccine production. Since NS1 truncated or deficient IAV have a ts phenotype, virus rescue and amplification is carried out at 33°C.
Figure 3
Figure 3
Schematic representation of NS segment and NS1 domains. (A) An IAV NS vRNA segment is shown by a gray box and non-coding regions (NCR) are indicated with white boxes. Packaging signals (Ψ) at the end of the 3′ and 5′ coding regions are also indicated. IAV NS1 and NEP transcripts are indicated with gray and black boxes, respectively. IAV NS1 and NEP ORFs share the first 10 amino acids in the N-terminus. (B) The NS1 protein is divided into four regions: The N-terminal RNA-binding domain (RBD; amino acids 1–73), the linker sequence (L; amino acids 74–88), the effector domain (ED; amino acids 89–202), and the C-terminal tail (CTT; amino acids 203-219/230/237). Note that both the L and the CTT can vary in length among different IAV strains, and, although a 237 amino-acids-length NS1 has been represented, IAV NS1 can be 219, 230, or 237 amino acids long. Nuclear localization and export signals (NLS and NES, respectively) are indicated with black boxes at the bottom, including their amino acid locations in the NS1.
Figure 4
Figure 4
Role of NS1 in counteracting IFN responses. 1) IAV NS1 decreases RIG-I activation, and therefore, IFN responses, through the sequestration of dsRNA (represented with two parallel lines), or by interaction with RIG-I, TRIM25 or Riplet, resulting in the suppressed ubiquitination and activation of RIG-I. 2) IAV NS1 inhibits the activation of IRF3, NF-κβ, and AP-1 transcription factors, impairing type I IFN production, and, therefore, the induction of ISGs. (3) IAV NS1 directly inhibits the antiviral activities of the ISGs PKR and OAS-RNaseL. NS1 protein binds dsRNA and PKR, leading to decreased PKR activity and impaired host translation inhibition mediated by PKR. IAV NS1 protein also inhibits OAS activation via the dsRNA-binding activity of its RBD, therefore, reducing RNA degradation mediated by RNAseL. 4) IAV NS1 impairs NLRP3 inflammasome activation as well as decreases the cleavage of pro-interleukin (IL)-1β and pro-IL-18 into their mature forms. Upon infection, these cytokines are released from the cell to stimulate inflammatory processes. 5) Depending on the IAV strain, NS1 proteins can bind to CPSF30. In addition, IAV NS1 binds to PABPII. These interactions of IAV NS1 with CPSF30 and PABPII block the cleavage of immature mRNAs (pre-mRNAs) and the recruitment of the poly(A) polymerase to add the poly(A) tail and function of PABPII to stimulate the synthesis of long poly(A) tails, respectively, leading to host protein shutoff. IFN: interferon; dsRNA: double-stranded RNA; RIG-I: retinoic acid-inducible gene I; TRIM25: tripartite motif containing 25; IRF3: interferon regulatory factor 3; NF-κβ: nuclear factor kappa beta; AP-1: activator protein 1; ISGs: IFN-stimulated genes; PKR: protein kinase R; OAS: 2’,5’-oligoadenylate synthetase; NLRP3: NLR family pyrin domain containing 3; CPSF30: cleavage and polyadenylation specificity factor 30; PABPII: poly(A)-binding protein II.
Figure 5
Figure 5
IFN responses in LAIV based on NS1 truncations or complete deletion. Representation of WT (A), truncated (B), or deficient (C) NS1 recombinant IAV. WT NS vRNA is represented in gray boxes and NCR located at the 3´and 5´ ends of the NS vRNA are indicated with white boxes. WT NS1 and NEP ORFs are represented as gray and black boxes, respectively. Black lines in panel B represent stop codons. Expression of WT NS1 protein (A) results in strong inhibition of IFN induction and, therefore, efficient viral replication. NS1 1–73 (top), 1–99 (middle), or 1–126 (bottom) truncations in the NS1 ORF (B) or deletion of the entire NS1 ORF (C) result in less efficient inhibition of IFN induction and, thus, reduced viral replication.
Figure 6
Figure 6
NS1 truncated or deficient IAV as LAIV for SIV (A and B), EIV (C), CIV (D), and AIV (E–I). Schematic representation of the WT and modified NS segments for SIV (A, B), EIV (C), CIV (D), and AIV (E–I). NS vRNA is represented in gray boxes and the NCR located at the 3´and 5´ ends of the NS vRNA are indicated with white boxes. NS1 and NEP ORFs are represented as gray and black boxes, respectively. Black lines represent stop codons. In (B), truncated NS1 protein is expressed as a single polyprotein together with 2A autoproteolytic cleavage site and NEP. NEP protein is released from NS1 protein during translation. IL-18 was incorporated between NS1 and NEP proteins via GSGG and GSG linkers (striped rectangles), and the 2A autoproteolytic cleavage site. Splice acceptor site was mutated to inhibit splicing. In (F), the NS segments encoded unmodified NEP and truncated NS1 protein products created by adding three serial stop codons comprising amino acids 1-73, 1-86, 1-101, and 1-122 without any nucleotide deletions. In (G), the internal deletion comprising nucleotides 370-426 (NS1-115-125) or 301-492 (NS1-91-93) in NS1 are indicated. The CCT of these NS1 proteins contains the same 115 and 91, respectively, amino acid residues than the WT NS1 but additional different residues (116 to 125 for NS1-115-125; and 92 to 93 in NS1-91/93, respectively) due to a frame shift in the ORF.
Figure 7
Figure 7
Principle of vaccination with NS1 truncated or deficient viruses as DIVA LAIV. (A) Specific antibodies (Abs) against IAV NS1 protein will be produced in infected animals, but not in animals vaccinated with NS1 truncated or deficient IAV (DIVA vaccine). However, specific antibodies against viral HA from circulating strains or the vaccine will be induced in infected and/or vaccinated animals, respectively. (B) Development of a DIVA serological test. Abs against IAV NS1 induced by natural infection can be identified by proper serological tests. In the figure an ELISA is used to differentiate sera from infected (containing antibodies against both NS1 and HA proteins) and not infected animals, before vaccination. Wells are coated using recombinant NS1 or HA IAV proteins. Then, the same serological test is carried out after vaccination to differentiate animals that has been infected from animals that have been not infected. Samples from infected animals will be positive for HA and NS1 in the ELISA tests, while samples from vaccinated and not infected animals will be positive only for HA in the ELISA tests.
Figure 8
Figure 8
Codon deoptimization of NS segment for the generation of LAIV. Schematic representation of WT (A) and codon deoptimized, cd (B) viral NS segments. NS vRNA is represented in gray boxes and the NCR located at the 3´and 5´ ends of the NS vRNA are indicated with white boxes. WT NS1 and NEP ORFs are represented as gray and black boxes, respectively. The cd region is represented with light gray boxes for viruses encoding cd NS1 (NS1cd, top), NEP (NEPcd, middle) or both NS1 and NEP (NScd, bottom) proteins. After infection with an IAV encoding a WT NS segment, expression of full-length NS1 results in inhibition of IFN induction, allowing efficient viral replication (A). Infection with IAV NS1cd (top, (B) results in reduced NS1 protein expression and inefficient inhibition of IFN responses, resulting in reduced viral replication. Infection with IAV NEPcd (middle, (B) results in lower expression of NEP, affecting viral replication. Infection with IAV NScd (bottom, (B) results in the virus showing higher attenuation, correlating with the amount of codon changes introduced in both NS1 and NEP in the modified NS viral segment.
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