Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

Domain: biology
Published: 2026-03-18
Editorial outcome: minor_revision

Abstract
The lysosome is a cell’s endogenous machinery responsible for degrading proteins. Here we describe two lysosome-targeting live attenuated vaccine approaches, LYTAR1.0 and LYTAR2.0, by harnessing the lysosome to conditionally degrade viral proteins of influenza virus. LYTAR1.0 incorporates a conditionally removable lysosome-targeting motif at the N- or C-terminus of viral proteins. LYTAR2.0 allows flexible placement of lysosome-targeting motifs at internal or terminal sites of viral proteins. The resulting lysosome-targeting vaccine strains are attenuated by lysosome-mediated viral protein degradation in conventional cells, while maintaining replication efficiencies comparable to the wild-type virus in producer cell lines. In mouse models, these vaccine candidates are attenuated, induce strong and broad adaptive immune responses, and provide cross-reactive protection against H1N1 and H3N2 influenza viral challenges. This study establishes a lysosome-targeting vaccine platform for developing safe and effective live attenuated vaccines.

Introduction
Vaccination is regarded as the most effective measure to protect populations from influenza infections and severe diseases1,2. However, despite the availability of existing vaccines and advancements in vaccine development technologies, one century after the 1918 influenza pandemic–the most severe in history, which claimed over 40 million lives worldwide-influenza is still a major global public health challenge and a large economic burden to all societies3–6. The World Health Organization (WHO) estimates that annual influenza epidemics cause approximately 1 billion infections, 3–5 million cases of severe illness, and 0.3–0.5 million deaths worldwide3. Therefore, new vaccine strategies are urgently needed to produce safer and more effective vaccine candidates.

Viral proteins are essential for the assembly of virions and play a crucial role in determining the viral lifecycle7. Thus, manipulating viral protein degradation through the host cell’s protein degradation machinery represents a rational strategy to control viral replication and attenuate viruses for use as live vaccines. The lysosome is one major protein degradation machinery present in all eukaryotic cells, and chaperone-mediated autophagy (CMA) is a cell-intrinsic lysosomal degradation pathway that targets protein substrates to the lysosome for degradation8–11. In this system, a cytosolic chaperone, the heat shock-cognate protein of 70 KDa (HSC70), recognizes a Lys-Phe-Glu-Arg-Gln (KFERQ)-like motif on protein substrates and then targets them to the lysosome-associated membrane protein type 2A (LAMP2A) receptor on the lysosomal membrane, which facilitates the translocation of protein substrates from the cytosol lumen to the lysosomal lumen where they are subsequently degraded8,10–13.

To explore the feasibility of leveraging the lysosomal degradation system to manipulate the degradation of viral proteins for vaccine development, we designed lysosome-targeting (LYTAR) influenza A viruses as live attenuated vaccines by incorporating lysosome-targeting motifs (LTMs) that are derived from KFERQ(-like) motifs into the genome of influenza A virus. Screening of potential sites to incorporate LTM led to the generation of two types of LYTAR influenza viruses, referred to as LYTAR1.0 and LYTAR2.0, that are highly attenuated through lysosome-mediated degradation of viral proteins in conventional cells but can replicate efficiently in engineered vaccine-manufacturing cell lines. In animal models, both LYTAR1.0 and LYTAR 2.0 vaccine candidates exhibited sufficient attenuation, and immunization by a single intranasal exposure to the LYTAR1.0 or LYTAR2.0 vaccine candidate induced robust humoral, mucosal, and cellular immune responses and conferred complete protection against lethal challenges of both homologous and heterologous influenza viruses.

Results
Generation of the first-generation LYTAR (LYTAR1.0) influenza virus

In our design, a conditionally removable LTM, VKKDQKFERQ, was fused to the N-terminus or the C-terminus of each influenza A/WSN/33 (H1N1) viral protein via a tobacco etch virus cleavage site (TEVcs) linker, ENLYFQG14 (Fig. 1a, b, Supplementary Table 1). The LTM was designed to contain two tandem KFERQ(-like) motifs, VKKDQ and KFERQ, to potentially increase the binding affinity of the LTM to HSC70 (Fig. 1b). Both motifs are recognized by HSC70, leading to the translocation of tagged proteins to the lysosome for degradation13,15,16. Therefore, the LTM was used to induce lysosomal degradation of LTM-tagged viral proteins and thus attenuation of LYTAR 1.0 viruses in conventional cells. The TEVcs linker can be selectively cleaved by TEV protease (TEVp) in engineered TEVp-expressing cells, resulting in the removal of the LTM from viral proteins and thus protecting viral proteins from lysosomal degradation (Fig. 1a), which enables efficient replication of LYTAR1.0 viruses for large-scale production.

Six viral proteins (PA, PB2, PB1, NP, NS1, and M1) were assessed for the incorporation of the conditionally removable LTM at either their N- or C-terminus (Supplementary Table 2). To generate LYTAR1.0 viruses, each of the LTM-modified viral constructs was co-transfected with the other viral constructs from the 12-plasmid influenza reverse genetics system into TEVp-expressing HEK293T cells and MDCK-TEVp cells. After 3–4 days post-transfection, the culture supernatants were harvested and used to infect MDCK-TEVp cells for viral amplification. One virus strain, NS1-NLTM, which contains the removable LTM at the N-terminus of viral NS1 protein, caused obvious cytopathic effect (CPE) (Supplementary Table 2), a cell phenotypic observation indicating the presence of replication-competent virus17,18. However, no CPE was observed for other virus strains, suggesting that other viral constructs did not generate viable virus (Supplementary Table 2). This is potentially due to the fact that the LTM incorporation could affect the structures or functions of these viral proteins. The successful generation of NS1-NLTM virus was further confirmed by the TCID50 assay (Fig. 1c). To compare the in vitro replication properties of NS1-NLTM and the wild-type (WT) virus, we measured their multi-cycle viral replication kinetics in MDCK-TEVp cells and conventional MDCK cells (Fig. 1d). The viral titers of NS1-NLTM at 24, 48, 72, and 96 h after infection in MDCK-TEVp cells were 103.8, 106.6, 107.9, 107.5 TCID50/mL, respectively, comparable to those of the WT virus (104.8, 107.7, 108.3, 107.9 TCID50/mL, respectively) (Fig. 1d, left), which suggests that NS1-NLTM can efficiently replicate in MDCK-TEVp cells, essential for vaccine production purposes. In contrast, viral titers of NS1-NLTM in conventional MDCK cells were approximately 104-fold lower than those of the WT virus (Fig. 1d, right), indicating a significant attenuation of NS1-NLTM relative to the WT virus in conventional MDCK cells. In addition, we generated a virus strain NS1-NLTM mutant that contains alanine mutations at key amino acids15,19–22 positions within the LTM of NS1-NLTM virus (Fig. 1b bottom, 1d right). Importantly, the mutation of LTM abolished the attenuation of NS1-NLTM virus in conventional MDCK cells (Fig. 1d right), indicating that the attenuation of NS1-NLTM virus is mediated by the LTM. These data suggest that the conditional control over lifecycle and thus virulence of influenza A virus can be achieved by incorporating a removable LTM at the N-terminus of its NS1 protein, enabling efficient replication in engineered TEVp-expressing cells for large-scale production while conferring sufficient attenuation in conventional cells.

Mechanistic characterization of NS1-NLTM attenuation

Next, we sought to determine whether the degradation of viral protein and the attenuation of the NS1-NLTM virus depends specifically on the lysosomal degradation system. We began with a mechanistic investigation of the degradation pathways of the viral NS1 protein and its mutants. HEK293T cells were transfected with a plasmid expressing N-terminal removable LTM-tagged NS1 protein (NS1-NLTM) or a mutant version (NS1-NLTM mutant). The mutant contains alanine mutations at key amino acids15,19–22 positions within the LTM (Fig. 1b, bottom). Western Blot showed that the alanine mutations of key amino acid residues in the LTM restored the levels of NS1 protein (Fig. 2a, left), indicating an LTM-dependent degradation of the NS1 protein. Comparable levels of NS1 mRNA were detected in the NS1-NLTM- and NS1-NLTM mutant-expressing cells (Fig. 2a, right), demonstrating that LTM-mediated NS1 degradation occurred at the protein level rather than at the transcription level.

We then tested whether incorporating the removable LTM at the N-terminus of the viral NS1 protein could direct the viral protein to the lysosome for degradation. HEK293T cells were transfected with a construct expressing the NS1-NLTM protein (Fig. 1b, top), cultured for 18 h, and treated with inhibitors of different protein degradation machineries, including the proteasome inhibitor MG-132, the macroautophagy inhibitor 3-methyladenine (3-MA), the lysosome inhibitor Bafilomycin A1 (BafA1), or the lysosome inhibitor Chloroquine (CQ) for 6 h. Inhibition of the lysosome by BafA1 and CQ increased levels of NS1-NLTM protein (Fig. 2b, left), whereas inhibition of the proteasome by MG-132 or inhibition of macroautophagy by 3-MA did not (Fig. 2b, left), indicating that NS1-NLTM degradation is lysosome-dependent rather than mediated by the ubiquitin-proteasome system (UPS) or macroautophagy. This conclusion was further validated by the finding that alanine substitution of key residues within the LTM (Fig. 1b, bottom) abolished the effect of lysosome inhibition on levels of NS1-NLTM mutant protein (Fig. 2b, right).

In this lysosomal degradation system, HSC70 is responsible for recognizing KFERQ(-like) motifs in protein substrates15,16. We examined the interaction between the NS1-NLTM protein and HSC70 using a co-immunoprecipitation (Co-IP) assay. NS1-NLTM protein showed Co-IP with HSC70, whereas this interaction was abolished by alanine mutation in the NS1-NLTM mutant protein (Fig. 2c), confirming the LTM-mediated interaction of NS1-NLTM protein with HSC70. Given that LAMP2A on the lysosome membrane is the receptor for binding KFERQ(-like) motif-containing protein substrates and is required for substrate translocation from the cytosol to the lysosomal lumen8,10,13, we confirmed the LAMP2A dependence of the lysosomal degradation of NS1-NLTM protein by measuring levels of NS1-NLTM protein in conventional HEK293T cells and LAMP2A-knockout (KO) HEK293T cells. The LAMP2A KO restored the level of NS1-NLTM protein (Fig. 2d, left) but had no effect on that of the NS1-NLTM mutant protein (Fig. 2d, right). Moreover, confocal microscopy analysis revealed that NS1-NLTM protein formed cytoplasmic puncta, whereas NS1-NLTM mutant protein exhibited diffused distribution in the cell nucleus (Fig. 2e, f; Supplementary Figs. 1 and 2), which is consistent with the interaction of NS1-NLTM but not NS1-NLTM mutant protein with HSC70 and LAMP2A in the cytoplasm. More importantly, the cytoplasmic puncta formed by NS1-NLTM protein exhibited colocalization with HSC70 and LAMP2A, whereas this effect was not observed for NS1-NLTM mutant protein (Fig. 2e, f; Supplementary Figs. 1 and 2), further verifying the LTM-mediated interaction of viral NS1 protein with the lysosomal degradation system.

Having confirmed that the degradation of the LTM-tagged viral NS1 protein was through the lysosomal degradation system, we further validated that the same mechanism is responsible for NS1 protein degradation in the virus and subsequent viral attenuation by culturing NS1-NLTM virus in conventional and LAMP2A-KO HEK293T or A549 cells. LAMP2A KO led to the restoration of the levels of viral NS1 protein (Fig. 2g, h; left) and an increase in viral replication competence of the NS1-NLTM virus (Fig. 2i, j). In contrast, LAMP2A KO had no effect on viral NS1 protein levels (Fig. 2g, h; right) or the replication competence of the NS1-NLTM mutant virus (Fig. 2i, j).

In addition to the NS1 protein, the influenza viral NS gene segment also expresses an alternative spliced protein, i.e., the nuclear export protein (NEP). Therefore, we tested whether incorporating the LTM at the N-terminus of the NS gene segment affects NEP expression. The expression level of LTM-tagged NEP (NEP-NLTM) was comparable to that of WT NEP (Supplementary Fig. 3a). In addition, treatment with MG-132, 3-MA, BafA1, or CQ had no detectable effect on NEP-NLTM protein levels (Supplementary Fig. 3b). These data suggest that LTM incorporation does not interfere with NEP expression.

Generation of the second-generation LYTAR influenza viruses

In the LYTAR1.0 vaccine study, the LTM must be incorporated at the N- or C-terminus of viral proteins, and only N-terminal incorporation of the LTM into the NS1 protein permitted successful generation of a LYTAR1.0 virus, designated NS1-NLTM. The limited number of terminal sites for LTM incorporation could hinder the flexibility and universality of the LYTAR1.0 approach in vaccine development. Given that viral proteins have thousands of amino acid sites within their internal regions, we sought to expand the incorporation sites of LTM from the terminal ends of viral proteins to the internal sites of viral proteins. To this aim, we developed the next-generation LYTAR vaccine strategy, termed LYTAR 2.0. The LYTAR 2.0 approach could allow the incorporation of LTM at any site of the viral proteins of interest as long as the LTM within viral proteins can be accessible and recognized by the lysosomal degradation system in conventional cells and has no or minimal effect on virus propagation in LAMP2A-knockout (KO) cells during vaccine production (Fig. 3a). The LYTAR 2.0 vaccines were designed to contain LTM at different sites within four influenza viral proteins (PA, NP, M1, and NS1). These viral proteins were chosen as representatives for LTM incorporation based on their distinct functions during viral lifecycle7,23: PA was chosen as a representative of influenza viral polymerases (PA, PB1, and PB2) that are responsible for viral genome replication and transcription; NP is involved in encapsulating influenza viral genome by forming ribonucleoprotein (RNP); M1 mediates virus assembly by forming an endoskeleton beneath the virus membrane; and NS1 represents nonstructural proteins and plays a central role in evading the host innate immune system to facilitate viral replication. A total of 29 sites were selected for LTM incorporation: eight from PA (Asp294, Asn350, Glu372, Leu425, His510, Ala553, Glu604, and Ser624), eight from NP (Gly126, Asn247, Arg317, Val353, Ala366, Gln409, Glu465, and Met481), eight from M1 (Ala33, Val68, Asp89, Arg105, Met135, Gln164, His222, and Ala239), and five from NS1 (Ala76, Ala82, His101, Ala122, and Leu163) (Supplementary Fig. 4). These sites were chosen because they distribute on the surface of viral proteins (Supplementary Fig. 4), providing a structural basis for accessibility and recognition by the lysosomal degradation system. KFERQ, the minimal peptide motif recognized by the lysosomal degradation system, was first used as the LTM to screen for LTM incorporation sites. Each LTM-containing viral construct was co-transfected together with the remaining plasmids of the 12-plasmid influenza virus reverse genetics system into co-cultured LAMP2A-KO HEK293T and LAMP2A-KO MDCK cells. Culture supernatants were harvested 3–4 days post-transfection and subsequently inoculated into LAMP2A-KO MDCK cells for viral amplification. Seven virus strains (7/29, 24%), PA-Asn350LTM (N350LTM), PA-Glu372LTM (E372LTM), NP-Gly126LTM (G126LTM), NP-Gln409LTM (Q409LTM), M1-Met135LTM (M135LTM), NS1-His101LTM (H101LTM), and NS1-Ala122LTM (A122LTM) caused obvious CPE, indicating successful generation of potential LYTAR 2.0 viruses, which was verified by the TCID50 assay (Supplementary Fig. 5 and Supplementary Table 3).

We then tested whether LTM incorporation in internal regions of viral proteins could induce lysosome-dependent viral protein degradation. HEK293T cells expressing LTM-containing or WT viral proteins were treated with the lysosome inhibitor BafA1 for 6 h. Inhibition of the lysosome by Baf A1 increased levels of all LTM-containing viral proteins (Fig. 3b) but not WT viral proteins (Supplementary Fig. 6a), suggesting that the degradation of these LTM-containing viral proteins is lysosome-dependent. We then confirmed the LAMP2A dependence of the lysosomal degradation of LTM-containing viral proteins by measuring their levels in conventional HEK293T cells and LAMP2A-KO HEK293T cells: the LAMP2A KO restored the levels of LTM-containing viral proteins (Fig. 3c) but had no effect on those of WT viral proteins (Supplementary Fig. 6b). We further validated the lysosome-dependent viral protein degradation by culturing LYTAR 2.0 viruses or WT virus in conventional and LAMP2A-KO HEK293T (Fig. 3d) or A549 cells (Fig. 3e). LAMP2A KO led to the restoration of the levels of LTM-containing viral proteins of LYTAR 2.0 viruses (Fig. 3d, e) but had no effect on the levels of WT viral proteins (Supplementary Fig. 6c). These data suggest that LTM incorporation at these sites can induce lysosome-dependent degradation of viral proteins and convert WT viruses into LYTAR 2.0 viruses.

To potentially enhance the strength of recognition of LYTAR 2.0 viral proteins by the lysosomal degradation system, we incorporated three tandem KFERQ(-like) motifs (triLTM), VKKDQ, KFERQ, and KILDQ, into LYTAR 2.0 viral proteins. PA-N350 and NP-G126 were chosen as representatives for incorporation of the triLTM because PA-N350LTM and NP-G126LTM viruses exhibited the highest replication efficiencies among the LYTAR 2.0 virus strains containing a single LTM in LAMP2A-KO MDCK cells (Supplementary Fig. 5), which is important for large-scale vaccine production. This led to the generation of PA-N350triLTM and NP-G126triLTM viruses (Supplementary Table 3). One advantage of the LYTAR 2.0 over the LYTAR 1.0 vaccine approach is its capacity to accommodate multiple LTMs. This feature not only enhances viral protein degradation and attenuation, thereby improving safety, but also increases the genetic barrier to LTM mutation, reducing the likelihood of viral escape from lysosomal degradation. On this basis, we combined two triLTMs into the genome of one virus by incorporating one triLTM at PA-N350 and the other at NP-G126. This led to the generation of LYTAR 2.0dual triLTMs virus (Supplementary Table 3). Growth kinetics curves showed that LYTAR 2.0dual triLTMs virus replicated efficiently in LAMP2A-KO MDCK cells but was attenuated by 103-fold in conventional MDCK cells (Fig. 4a). Importantly, the attenuation factor of LYTAR 2.0dual triLTMs virus in conventional MDCK cells was 101.8-fold higher than that of LYTAR 2.0dual LTMs virus (101.2-fold attenuation), a virus strain containing KFERQ motifs instead of triLTMs at PA-N350 and NP-G126 (Fig. 4a). These data suggest that increasing the number of LTMs within one LYTAR virus can enhance viral attenuation and increase viral safety. This is consistent with the observation that increasing the number of LTMs induces stronger degradation of viral proteins (Supplementary Fig. 7).

Next, we sought to confirm that the triLTM-mediated viral protein degradation and attenuation by the lysosomal degradation system. HEK293T cells were transfected with a plasmid expressing triLTM-tagged viral protein (PA-N350triLTM or NP-G126triLTM) or mutated triLTM-tagged viral protein (PA-N350triLTM mutant or NP-G126triLTM mutant). The triLTM mutant contains alanine mutations at key amino acids15,19–22 positions (Supplementary Table 1). Western Blot showed that the alanine mutations of key amino acid residues in the triLTM restored the levels of PA and NP proteins (Fig. 4b), indicating the triLTM dependence of the PA and NP protein degradation. In addition, the same levels of viral mRNAs were detected in the cells expressing triLTM-tagged or mutated triLTM-tagged viral proteins (Fig. 4b), confirming that the triLTM-mediated viral protein degradation occurred post-transcriptionally by reducing protein abundance without affecting mRNA levels. Furthermore, the mutation of triLTM abolished the attenuation of LYTAR 2.0dual triLTMs virus in conventional MDCK cells (Fig. 4a), suggesting that the triLTM mediates the attenuation of LYTAR 2.0dual triLTMs virus.

To test whether incorporating the triLTM at PA-N350 and NP-G126 could direct the viral proteins to the lysosome for degradation. HEK293T cells were transfected with a construct expressing PA-N350triLTM or NP-G126triLTM protein, cultured for 18 h, and treated with the lysosome inhibitor Bafilomycin A1 (BafA1) for 6 h (Fig. 4c). Inhibition of the lysosome by BafA1 increased levels of PA-N350triLTM and NP-G126triLTM proteins (Fig. 4c), suggesting that the degradation of PA-N350triLTM and NP-G126triLTM proteins is lysosome-dependent. In a parallel experiment, alanine mutation of key amino acids in the triLTM abolished the effect of lysosome inhibition on levels of PA-N350triLTM mutant or NP-G126triLTM mutant protein (Fig. 4c), again confirming that the lysosomal degradation of PA-N350triLTM and NP-G126triLTM proteins is mediated by the triLTM. Furthermore, we examined the interaction between PA-N350triLTM or NP-G126triLTM protein and HSC70 using the Co-IP assay. Both PA-N350triLTM and NP-G126triLTM proteins showed Co-IP with HSC70, whereas this interaction was abolished by alanine mutation in the PA-N350triLTM mutant or NP-G126triLTM mutant protein (Fig. 4d), confirming the triLTM-mediated interaction of PA-N350triLTM or NP-G126triLTM protein with HSC70.

We further confirmed the LAMP2A dependence of the lysosomal degradation of PA-N350triLTM and NP-G126triLTM proteins by culturing LYTAR 2.0dual triLTMs virus in conventional and LAMP2A-KO HEK293T (Fig. 4e) or A549 cells (Fig. 4f). LAMP2A KO led to the restoration of the levels of both PA-N350triLTM and NP-G126triLTM proteins (Fig. 4e, f; left) and an increase of viral replication competence of LYTAR 2.0dual triLTMs virus (Fig. 4g, h). In contrast, in parallel experiments with LYTAR 2.0dual triLTMs mutant virus wherein key amino acids in the triLTMs were mutated to alanine, LAMP2A KO had no effect on the levels of viral PA-N350triLTM mutant or NP-G126triLTM mutant protein (Fig. 4e, f; right) or the viral replication competence of LYTAR 2.0dual triLTMs mutant virus (Fig. 4g, h). Overall, these data above suggest that we successfully developed two versions of the LYTAR vaccine approaches by using the cell’s lysosomal degradation system to manipulate viral protein degradation. In addition, viral genome sequencing showed that no mutation was detected in NS1-NLTM and LYTAR dual triLTMs virus strains after multiple passages of culture, suggesting the genetic stability (Supplementary Fig. 8).

LYTAR1.0 and LYTAR2.0 viruses are highly attenuated in vivo

Next, we investigated the in vivo safety of the LYTAR1.0 and LYTAR2.0 viruses by intranasally infecting C57BL/6J mice with NS1-NLTM, LYTAR 2.0dual triLTMs, or the WT virus, followed by monitoring body weight changes, survival rates, and clinical symptoms over a 14-day period. The median lethal dose (LD50) of the WT virus in C57BL/6J mice was approximately 103 TCID50. A dose of 105 TCID50 (100×LD50) of WT virus caused death of all mice within 9 days, accompanied by rapid body weight loss, fur ruffling, and tremors before death (Fig. 5a, b). In contrast, infection with the same dose of NS1-NLTM or LYTAR 2.0dual triLTMs did not cause any death, body weight loss, or any other disease signs (Fig. 5a, b). We further measured viral titers in the mouse lungs on day 3 post-infection with 105 TCID50 of NS1-NLTM, LYTAR 2.0dual triLTMs, or WT virus. The viral titers in the lungs of C57BL/6J mice infected with WT virus were approximately 106.8 TCID50/mL, whereas those infected with NS1-NLTM or LYTAR 2.0dual triLTMs virus were approximately 102.5 TCID50/mL (a 104.3-fold decrease) and below the detection limit (>104.8-fold decrease), respectively (Fig. 5c, d). Remarkably, while low levels of viral titers in mouse lungs were detected for the NS1-NLTM virus, the titer of LYTAR 2.0dual triLTMs virus was undetectable (Fig. 5c, d). These data suggest that both NS1-NLTM and LYTAR 2.0dual triLTMs viruses are highly attenuated in vivo and that the LYTAR 2.0dual triLTMs virus has higher degrees of attenuation than the NS1-NLTM virus.

We further investigated the in vivo safety profiles of NS1-NLTM and LYTAR 2.0dual triLTMs viruses in immunodeficient mice. To this end, immunodeficient BALB/c nude mice were intranasally infected with 105 TCID50 of NS1-NLTM, LYTAR 2.0dual triLTMs, or WT virus and monitored for body weight, survival, and disease signs for 14 days. The WT virus caused 100% mortality within 8 days, accompanied by rapid body weight loss and tremors before death (Fig. 5e). In contrast, infection with the same dose of NS1-NLTM or LYTAR 2.0dual triLTMs did not cause any death, body weight loss, or other disease signs (Fig. 5e). In addition, detection of viral titers in the mouse lungs on day 3 post-infection revealed that the titers in the lungs of BALB/c nude mice infected with WT virus were approximately 106 TCID50/mL, whereas those infected with NS1-NLTM or LYTAR 2.0dual triLTMs virus were approximately 102 TCID50/mL or lower than 102 TCID50/mL (>104-fold decrease) (Fig. 5f). These data suggest that NS1-NLTM and LYTAR 2.0dual triLTMs viruses are highly attenuated in immunodeficient mice.

LYTAR1.0 and LYTAR2.0 vaccines induce robust humoral, mucosal, and cellular immune responses

We next assessed the capacity of the LYTAR 1.0 vaccine to elicit humoral, mucosal, and cellular immune responses by intranasal immunization of C57BL/6J mice with 105 TCID50 of the NS1-NLTM virus. Clinically approved IIV and CAIV were used as controls and administered by intramuscular injection and intranasal inoculation, respectively. Detection of antibody responses in mouse sera three weeks after vaccination revealed that a single dose of NS1-NLTM induced robust humoral immune responses, including neutralization (NT) antibody responses (Fig. 6a), hemagglutinin inhibition (HI) antibody responses (Fig. 6b), viral surface HA protein-specific immunoglobulin G (IgG) antibody responses (Fig. 6c), and viral internal NP protein-specific IgG antibody responses (Fig. 6d). The titers of antibodies induced by NS1-NLTM were 10- to 768-fold higher than those induced by IIV and were higher than or comparable to those induced by CAIV (Fig. 6a–d). In addition, detection of secretory IgA antibody responses in mouse lungs three weeks after vaccination showed that NS1-NLTM induced high levels of influenza virus-specific IgA antibodies (Fig. 6e), indicating the induction of robust respiratory mucosal immune responses. This is significantly advantageous over IIV, which induced only background levels of IgA antibodies (Fig. 6e). Furthermore, an interferon-γ-based enzyme-linked immunospot (IFN-γ ELISpot) assay revealed that NS1-NLTM induced high levels of viral antigen-specific T-cell immune responses in mouse lungs and spleens one week after vaccination, which contrasts with IIV, which did not induce any T-cell immune response (Fig. 6f).

Similarly, a single dose of LYTAR 2.0dual triLTMs vaccine also induced broad and robust immune responses. The titers of NT, HI, viral surface HA protein-specific IgG, and viral internal NP protein-specific IgG antibodies induced by LYTAR 2.0dual triLTMs were 18-fold, 12-fold, 20-fold, and 640-fold higher than those induced by IIV, respectively, and higher than or comparable to those induced by CAIV (Fig. 6g–j). In addition, LYTAR 2.0dual triLTMs also induced high levels of influenza virus-specific IgA antibodies in mouse lungs (Fig. 6k). Furthermore, IFN-γ ELISpot assay revealed that LYTAR 2.0dual triLTMs induced high levels of viral antigen-specific T-cell immune responses in mouse lungs and spleens one week after vaccination, which contrasts with IIV, which did not induce any T-cell immune response and was significantly higher than those induced by CAIV (Fig. 6l).

These data suggest that both the LYTAR 1.0 vaccine candidate NS1-NLTM and the LYTAR 2.0 vaccine candidate LYTAR 2.0dual triLTMs at a single dose can induce robust and broad immune responses against viral surface and internal antigens, which is attributed to their ability to present the full complement of antigens identical to those of the circulating virus strain.

LYTAR1.0 and LYTAR2.0 vaccines confer complete protection against homologous and heterologous viral challenges

To evaluate the protective efficacy of LYTAR 1.0 vaccine candidate NS1-NLTM and LYTAR 2.0 vaccine candidate LYTAR 2.0dual triLTMs against homologous influenza virus challenge, C57BL/6J mice were vaccinated with 105 TCID50 of NS1-NLTM, LYTAR 2.0dual triLTMs, IIV, or CAIV and then intranasally challenged with 105 TCID50 of WT WSN virus three weeks after vaccination. Analysis of lung viral titers on day 3 post-challenge revealed high titers of WT virus in the lungs of unvaccinated mice (106.6 TCID50/mL) and IIV-vaccinated mice (104.8 TCID50/mL) (Fig. 7a). In contrast, the viral titers in the lungs of NS1-NLTM-vaccinated or LYTAR 2.0dual triLTMs-vaccinated mice were below the detection limit (<102 TCID50/mL) (Fig. 7a), indicating a complete protection against homologous viral challenge achieved by a single dose of NS1-NLTM or LYTAR 2.0dual triLTMs. In addition, homologous viral challenge caused death of all unvaccinated mice and 70% of IIV-vaccinated mice within 8 days after challenge (Fig. 7b), accompanied by rapid body weight loss, fur ruffling, and tremors before death (Fig. 7b). In contrast, all NS1-NLTM-vaccinated or LYTAR 2.0dual triLTMs-vaccinated mice survived the challenge and did not exhibit loss of body weight or any other disease signs (Fig. 7b).

To evaluate the cross-reactive protective efficacy of LYTAR 1.0 vaccine candidate NS1-NLTM and LYTAR 2.0 vaccine candidate LYTAR 2.0dual triLTMs against heterologous influenza virus challenge, C57BL/6J mice were vaccinated with 105 TCID50 of NS1-NLTM, LYTAR 2.0dual triLTMs, IIV, or CAIV and then intranasally challenged with 103 TCID50 (~10×LD50) of influenza A/X-31 (H3N2) virus three weeks after vaccination. Detection of viral titers in mouse lungs by day 3 after challenge revealed high titers of H3N2 virus in the lungs of both unvaccinated mice (103.3 TCID50/mL) and IIV-vaccinated mice (102.8 TCID50/mL) (Fig. 7c). In contrast, viral titers in the lungs of NS1-NLTM-vaccinated or LYTAR 2.0dual triLTMs-vaccinated mice were below the detection limit (<102 TCID50/mL) (Fig. 7d), indicating a complete protection against heterologous viral challenge achieved by a single dose of NS1-NLTM or LYTAR 2.0dual triLTMs. In addition, the heterologous viral challenge caused death of all unvaccinated mice and IIV-vaccinated mice within 11 days after challenge (Fig. 7d), accompanied by rapid body weight loss, fur ruffling, and trembling before death (Fig. 7d). The failure of IIV to protect against heterologous viral challenge is consistent with those clinical observations that current IIVs are strain-specific and poorly effective against variant or mismatched viruses24. Remarkably, all NS1-NLTM-vaccinated or LYTAR 2.0dual triLTMs-vaccinated mice survived the same heterologous viral challenge and did not show loss of body weights or any other disease signs (Fig. 7d).

These data suggest that the LYTAR 1.0 vaccine candidate NS1-NLTM and LYTAR 2.0 vaccine candidate LYTAR 2.0dual triLTMs, when intranasally administered at a single dose, can provide complete and cross-reactive protection against both homologous and heterologous viral challenges. This is attributed to the live whole-virion vaccine properties of NS1-NLTM and LYTAR 2.0dual triLTMs, which can induce robust and broad immune responses against both variable and conserved antigens.

Methods
Viruses and vaccines
Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2) viruses were used in this study. The influenza vaccine controls, IIV and CAIV, were generated from the WSN virus and validated25,38,44,45. Briefly, culture supernatant containing WSN virus was treated with 0.2% formalin (Solarbio, Cat# G2161). After 7 days of incubation at 4°C, viral inactivation was confirmed by the absence of detectable infectious virus in MDCK cells. The doses of IIV and LYTAR vaccines were normalized to the same amount of HA protein measured by ELISA44,45. To generate CAIV, five cold-adapted amino acid mutations from the clinically approved cold-adapted live attenuated influenza vaccine FluMist were introduced into the segmented genome of the WSN virus. The five amino acid mutations are 265S in viral PB2 protein, 391E, 581G, and 661T in viral PB1 protein, and 34G in viral NP protein25,38. CAIV was verified by the cold-adapted attenuation phenotypes25,38.

Plasmids
The 12-plasmid reverse genetics system for generating influenza A/WSN/33 (H1N1) virus was a gift from Dr. George F. Gao from the CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences. Engineered plasmids for generating LYTAR viruses were obtained by inserting the gene sequence of the indicated LTM at the indicated site of the WT viral genome via PCR-based site-directed mutagenesis (Supplementary Table 1). Plasmids expressing indicated viral proteins with a Flag tag at the C-terminus were constructed by cloning the corresponding genes into the pCMV-3×Flag-1b vector (Addgene). Plasmid expressing HSC70 protein with HA tag at N-terminus was constructed by cloning the corresponding gene into the pCMV-3×HA-1b vector. All plasmids were confirmed by gene sequencing (RUIBIOTECHNOLOGY, Guangzhou) and prepared using EndoFree Maxi Plasmid Kit (TianGen, Cat# DP117).

Cell lines
The MDCK (CRL-2936) and HEK293T (CRL-3216) cell lines were purchased from the American Type Culture Collection; A549 cells (ab255450) were provided by Abcam. Proteasome-deficient A549 cell line (ab267149) was obtained from Abcam. These cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Cat# c11995500BT) containing 10% fetal bovine serum (FBS; PAN, Cat# ST30-3302), supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin, and incubated at 37°C under 5% CO2. HEK293T-TEVp and MDCK-TEVp cell lines were established by stably integrating the gene expressing TEVp into the genome of HEK293T and MDCK cells23 and cultured in DMEM supplemented with 10% FBS, 2 µg/mL puromycin (InvivoGen, Cat# ant-pr-1), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in 5% CO2.

LAMP2A-KO A549, LAMP2A-KO HEK293T, and LAMP2A-KO MDCK cell lines were established by lentiviral transduction. 5×105 cells/well of HEK293T cells were seeded into a 6-well plate in DMEM medium (Gibco, without sodium pyruvate, Cat# C11995500BT) supplemented with 10% FBS (PAN, Cat# ST30-3302) 24 h before transfection. LAMP2A target sequence (ATAGCAGTGCAGTTCGGACC for A549 and HEK293T cell lines or CGAGGTCATGGACAGTCAAA for MDCK cell line) was cloned into Lenti-CRISPRv2 by cutting with BsmBI. A total of 400 ng of Lenti-CRISPRv2, 800 ng of pLP1-gag pol, 400 ng of pLP-2 Rev, and 200 ng of pLP-VSVG were co-transfected into the HEK293T cells. The lentivirus-containing supernatants were collected 48 h after transfection, filtered through a 0.45-µm filter, and then used for transduction. A549, HEK293T, or MDCK cells (5×105 cells/well) were seeded into 6-well plates and transduced 24 h later with the lentiviral filtrates, supplemented with 8 µg/mL of polybrene. Selection was carried out by applying 2 µg/mL puromycin until the parental cells were completely eliminated. The stably transduced cells were subjected to monoclonal selection by limiting dilution in 96-well plates. Successful LAMP2A knockout in A549, HEK293T, and MDCK cell lines was verified by Western blot analysis and genomic sequencing.

Generation of WT, LYTAR1.0, and LYTAR2.0 influenza viruses
For generation of WT and LYTAR1.0 influenza viruses, 2×105 cells/well of HEK293T-TEVp cells and 5×104 cells/well of MDCK-TEVp cells were seeded in 6-well plates in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin and co-cultured for 24 h at 37°C in 5% CO2. To generate the WT influenza virus, the 12 WT viral plasmids (0.2 µg/plasmid/well) were co-transfected into the co-cultured HEK293T-TEVp and MDCK-TEVp cells. To generate LYTAR 1.0 influenza virus, the engineered LYTAR viral plasmid and the remaining plasmids of the 12-plasmid reverse genetics system were co-transfected into the co-cultured HEK293T-TEVp and MDCK-TEVp cells. Six hours later, the transfection medium was replaced with infection medium (DMEM supplemented with 0.5% FBS, 2 µg/mL TPCK-treated trypsin, 100 U/mL penicillin, and 100 µg/mL streptomycin). After 3–4 days of culture, the supernatants were collected and added to fresh MDCK-TEVp cells for viral amplification. CPE was daily monitored for 4 days. When CPE reached ~80%, supernatants were collected and subjected to quantification of viral titers by TCID50 assay. The generated viruses were further confirmed by viral genome sequencing (RUIBIOTECHNOLOGY, Guangzhou).

To generate LYTAR 2.0 influenza viruses, an almost identical procedure was performed with the following modifications: (1) the HEK293T-TEVp cells and MDCK-TEVp cells were replaced by LAMP2A-KO HEK293T cells and LAMP2A-KO MDCK cells, respectively; (2) the viral plasmids for LYTAR1.0 influenza viruses were replaced by viral plasmids for LYTAR 2.0 influenza viruses.

TCID50 assay
Viral titers were quantified by TCID50 assay23. Briefly, MDCK-TEVp or LAMP2A-KO cells were plated in 96-well plates at a density of 5000 cells per well. Cells were then infected with serial 10-fold dilutions of viral supernatant prepared in infection medium, with eight replicate wells per dilution. Following incubation at 37°C under 5% CO2 for four days, wells were scored as positive or negative for cytopathic effect. The Reed-Muench method was used to calculate the TCID50/mL46.

Measurement of in vitro replication kinetics curves of influenza viruses
Conventional or engineered MDCK cells in 6-well plates were infected with MOI=0.001 of influenza viruses. The cells were maintained in infection medium. Supernatants were harvested at 24, 48, 72, and 96 h post-infection, and viral titers were quantified by the TCID50 assay.

Analysis of the LTM dependence of viral protein degradation
2.5×105 cells/well of HEK293T cells cultured in 12-well plates were transfected with 300 ng/well of plasmid expressing the indicated viral protein with a Flag tag at the C-terminus. 6 h after transfection, the transfection medium was replaced with fresh culture medium. After another 24 h of culture, cells were collected and subjected to Western blotting assay for the detection of target proteins. The primary antibodies were anti-Flag antibody (Beijing Ray Antibody Biotech, Cat# RM1002-T, 1:5000 dilution) and anti-β-actin (Proteintech Group, Cat# HRP-60008, 1:6000 dilution). The secondary antibody was Goat anti-Mouse IgG (H+L) HRP (Beijing Ray Antibody Biotech, Cat# RM-3001, 1:8000 dilution).

Quantitative reverse transcription PCR
Cells were collected, and RNA was extracted by use of total RNA isolation kit (Vazyme, Cat# RC112-01). For the reverse transcription quantitative PCR (RT-qPCR), cDNA was generated with the HiScript III RT SuperMix for qPCR kit (Vazyme, Cat# R323–01) and was amplified by qPCR using ChamQ Universal SYBR qPCR MasterMix (Vazyme, Cat# Q712–02) with specific PCR primers (the forward and reverse primers for NS1 were CCCAAGCAGAAAGTGG and GAGGGCAGTGGTGAAAT, the forward and reverse primers for PA were TGGCAAAGTCGGTATT and AGGTTGTCCCTAAGAGC, the forward and reverse primers for NP were AGACCCTTTCAGACTGCT and CCCTCTTGGGACCACT). The expression levels of the specified viral genes were normalized against the internal control β-actin and expressed as fold changes relative to the control group. This analysis of viral gene expression was conducted in a minimum of three independent experiments.

Analysis of the lysosome dependence of viral protein degradation
2.5×105 cells/well of HEK293T cells cultured in 12-well plates were transfected with 300 ng/well of plasmid expressing the indicated viral protein with a Flag tag at the C-terminus. 6 h after transfection, the transfection medium was replaced with fresh culture medium. After another 18 h of culture, the culture medium was further supplemented with the indicated inhibitor (10 µM MG-132, 10 mM 3-MA, 0.4 µM Baf A1, or 50 µM CQ). These inhibitors and their concentrations used in this study were selected based on previous reports47–49. We further confirmed that they did not show detectable cytotoxicity at the concentrations used in this study using Cell Counting Kit-8 (CCK-8) assay (Beyotime, Cat# C0037), according to the manufacturer’s instructions (Supplementary Fig. 11). 6 h after treatment, cells were collected and subjected to a Western blotting assay for the detection of target proteins. The primary antibodies were anti-Flag antibody (Beijing Ray Antibody Biotech, Cat# RM1002-T, 1:5000 dilution) and anti-β-actin (Proteintech Group, Cat# HRP-60008, 1:6000 dilution). The secondary antibody was Goat anti-Mouse IgG (H+L) HRP (Beijing Ray Antibody Biotech, Cat# RM-3001, 1:8000 dilution).

Analysis of LTM-mediated interaction between viral proteins and HSC70 by Co-IP assay
5×105 cells/well of HEK293 were co-transfected with 1 µg/well of plasmid expressing HSC70 with an HA tag at the N-terminus and 1 µg/well of plasmid expressing the indicated viral protein with a Flag tag at the C-terminus. 6 h after transfection, the transfection medium was replaced with fresh culture medium. After another 24 h of culture, cells were lysed in cell lysis buffer (Beyotime, Cat# P0013) supplemented with protease inhibitor cocktail (MCE, Cat# HY-K0010) on ice. The cell lysates were collected and incubated with anti-Flag (Proteintech Group, Cat# 20543-1-AP, 1:500 dilution) and protein A/G beads (7 Sea Biotech, Cat# P001-3) overnight at 4°C, followed by three washes with cell lysis buffer. The immunoprecipitates were eluted from the beads with 1× sodium dodecyl sulfate (SDS) loading buffer (ACE, Cat# AL014) and separated on 4–12% SDS-polyacrylamide gels (SDS-PAGE) (ACE, Cat# F15412 Gel). Then proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Cat# 1620177), followed by incubation with 5% skim milk (YEASEN, Cat# 36120ES76) in TBST (EpiZyme, Cat# PS103) for 1 h at room temperature. Then the membranes were subsequently incubated with primary antibodies and secondary antibodies. The protein bands were visualized by Immunobilon Western Chemiluminescent HRP Substrate (Proteintech Group, Cat# PK10001). The primary antibodies were anti-Flag (Proteintech Group, Cat# 20543-1-AP, 1:5000 dilution) and anti-HA (MBL, Cat# M180-3, 1:8000 dilution). The secondary antibodies were anti-rabbit IgG-HRP (Beijing Ray Antibody Biotech, Cat# RM-3002, 1:8000 dilution) and anti-mouse IgG-HRP (Beijing Ray Antibody Biotech, Cat# RM-3001, 1:8000 dilution).

Analysis of the LAMP2A dependence of viral protein degradation
2×105 cells/well of conventional or LAMP2A-KO HEK293T cells cultured in 12-well plates were transfected with 300 ng/well of plasmid expressing the indicated viral protein with Falag tag at the C-terminus and plasmid expressing HSC70 protein. 6 h after transfection, the transfection medium was replaced with fresh culture medium. After another 24 h of culture at 37°C in 5% CO2, cells were collected and subjected to a Western blotting assay for the detection of the indicated proteins. The primary antibodies were anti-Flag antibody (Beijing Ray Antibody Biotech, Cat# RM1002-T, 1:5000 dilution), anti-LAMP2A (Santa Cruz, Cat# sc-18822, 1:1000 dilution), and anti-β-actin (Proteintech Group, Cat# HRP-60008, 1:6000 dilution). The secondary antibody was Goat anti-Mouse IgG (H+L) HRP (Beijing Ray Antibody Biotech, Cat# RM-3001, 1:8000 dilution).

1.2×105 cells/well of conventional or LAMP2A-KO HEK293T cells cultured in 12-well plates were infected with the indicated virus (MOI=0.01). After 48 h of culture at 37°C in 5% CO2, cells were collected and subjected to a Western blotting assay for the detection of the indicated proteins. The primary antibodies were anti-NS1 antibody (GeneTex, Cat# GTX125990, 1:2000 dilution), anti-PA antibody (GeneTex, Cat# GTX118991, 1:2000 dilution), anti-NP antibody (GeneTex, Cat# GTX125989, 1:5000 dilution), anti-M1 antibody (Sino Biological, Cat# 40010-RP01; 1:1000 dilution), anti-LAMP2A (Santa Cruz, Cat# sc-18822, 1:1000 dilution), and anti-β-actin (Proteintech Group, Cat# HRP-60008, 1:6000 dilution). The secondary antibodies were Goat anti-Mouse IgG (H+L) HRP (Beijing Ray Antibody Biotech, Cat# RM-3001; 1:8000 dilution) and Goat anti-Rabbit IgG (H+L) HRP (Beijing Ray Antibody Biotech, Cat# RM-3002; 1:8000 dilution).

1.2×105 cells/well of conventional or LAMP2A-KO A549 cells cultured in 12-well plates were infected with the indicated virus (MOI=0.01). After 48 h of culture at 37°C in 5% CO2, cells were collected and subjected to a Western blotting assay for the detection of the indicated proteins. The primary antibodies were anti-NS1 antibody (GeneTex, Cat# GTX125990, 1:2000 dilution), anti-PA antibody (GeneTex, Cat# GTX118991, 1:2000 dilution), anti-NP antibody (GeneTex, Cat# GTX125989, 1:5000 dilution), anti-M1 antibody (Sino Biological, Cat# 40010-RP01, 1:1000 dilution), anti-LAMP2A (Santa Cruz, Cat# sc-18822, 1:1000 dilution), and anti-β-actin (Proteintech Group, Cat# HRP-60008, 1:6000 dilution). The secondary antibodies were Goat anti-Mouse IgG (H+L) HRP (Beijing Ray Antibody Biotech, Cat# RM-3001; 1:8000 dilution) and Goat anti-Rabbit IgG (H+L) HRP (Beijing Ray Antibody Biotech, RM-3002; 1:8000 dilution).

Immunofluorescence and FFU assay
2.5×105 cells/well of conventional or LAMP2A-KO A549 or HEK293T cells cultured in 12-well plates were infected with the indicated virus (MOI=0.01). After 48 h of culture at 37°C in 5% CO2, the culture supernatants were collected. 5×103 cells/well of MDCK-TEVp or LAMP2A-KO MDCK cells cultured in 96-well plates were infected with two-fold serially diluted supernatants for 48 h. Then the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA), washed with PBS, permeabilized with PBS containing 0.1% Triton X-100 (PBST) for 5 min, blocked with PBST containing 10% goat serum for 1 h at room temperature, and incubated with anti-M1 antibody (Sino Biological, Cat# 40010-RP01; 1:400 dilution) diluted in PBST containing 1% goat serum overnight at 4°C and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, Cat# A11034; 1:1000 dilution) for 1 h at room temperature. Cell nuclei were stained with DAPI (Invitrogen). Fluorescence images were captured using a fluorescence microscope (NIKON ECLIPSE Ti2-E, Japan), and image processing was done using NIS-Elements (NIKON, Japan). The quantitative analysis of images was done using ImageJ (v1.8.0).

Analysis of protein colocalization by fluorescence confocal microscopy
8×104 HeLa cells were seeded in a confocal dish (Cellvis, Cat# D35-20-1.5-N) and transfected with 500 ng of plasmid expressing NS1-NLTM or NS1-NLTM mutant protein with a Flag tag at the C-terminus. 6 h after transfection, the transfection medium was replaced with fresh culture medium. After 18 h of culture at 37°C in 5% CO2, 0.2 µM BafA1 was added to the culture medium. After another 6 h of culture, the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, washed with PBS, permeabilized with PBS containing 0.1% Triton X-100 for 5 min, blocked with PBS containing with 0.1% Triton X-100 and 10% goat serum for 1 h at room temperature, and incubated with anti-Flag antibody (MBL, Cat# PM020; 1:1000 dilution) and anti-HSC70 antibody (Santa Cruz Biotechnology, Cat# sc-7298; 1:300 dilution) or anti-LAMP2A antibody (Santa Cruz Biotechnology, Cat# sc-18822; 1:300 dilution) diluted in PBS containing 0.1% Triton X-100 and 1% goat serum overnight at 4°C, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, Cat# A11034; 1:1000 dilution) and Alexa Fluor 555 goat anti-mouse IgG (Life Technologies, Cat# A32727; 1:1000 dilution) for 1 h at room temperature. Cell nuclei were counterstained with DAPI (Invitrogen, Cat# 2086723) after the secondary antibody staining. Intracellular fluorescence was captured by a confocal laser-scanning microscope (Nikon AX, Japan) under a 100× oil-immersion objective. The images were processed for gamma adjustments using NIS-Elements (NIKON, Japan).

Evaluation of in vivo safety in mice
Animal experiments followed the institutional guidelines of the Shenzhen Institute of Advanced Technology (Chinese Academy of Sciences). We employed female C57BL/6J mice and immunodeficient BALB/c nude mice (6–8 weeks old; Vital River Laboratory). Housing conditions included a 12-h light/dark cycle, a constant temperature of 24°C, and 50% humidity. Each group of ten mice was intranasally inoculated with 105 TCID50 of WT virus, LYTAR1.0 virus, LYTAR 2.0 virus, or DMEM (vehicle) control, under anesthesia with tribromoethanol. Three days post-inoculation, five mice from each group were euthanized, and their lungs were harvested and homogenized in 1 mL PBS. Viral titers in the lung samples were assessed via the TCID50 assay. The remaining five mice in each group were monitored daily for signs of death, weight loss, or any other symptoms for 14 days.

Evaluation of immunogenicity in mice
Ten mice per group were intranasally inoculated with 105 TCID50 of LYTAR1.0 vaccine, LYTAR 2.0 vaccine, CAIV, or DMEM control (vehicle), or intramuscularly inoculated with an equivalent dose of IIV, under tribromoethanol anesthesia. One week post-inoculation, five mice from each group were euthanized, and their lungs and spleens were collected for IFN-γ ELISpot assay to assess viral antigen-specific T-cell responses. Three weeks after inoculation, bronchoalveolar lavage (BAL) fluid and serum samples were collected from the remaining five mice in each group for hemagglutination inhibition (HI) assay, neutralization (NT) assay, and ELISA to measure HI antibody titers, NT antibody titers, viral antigen-specific IgG, and viral antigen-specific IgA antibodies23,25.

Evaluation of cross-reactive protection efficacy in mice
To evaluate the protective efficacy against a homologous viral challenge, ten mice per group were intranasally inoculated with 105 TCID50 of LYTAR 1.0 vaccine, LYTAR 2.0 vaccine, CAIV, or DMEM (vehicle) control, or intramuscularly with the same dosage of IIV, under anesthesia with tribromoethanol. Three weeks post-inoculation, mice were intranasally challenged with 105 TCID50 of homologous WT WSN virus. Three days after the infection, five mice from each group were euthanized, and their lungs were collected and homogenized in 1 mL PBS. Viral titers in the lung homogenates were measured using a TCID50 assay. The remaining five mice from each group were observed daily for mortality, body weight changes, and other clinical signs for 14 days.

To assess the protective efficacy against a heterologous viral challenge, ten mice per group received an intranasal inoculation with 105 TCID50 of LYTAR 1.0 vaccine, LYTAR 2.0 vaccine, CAIV, or DMEM (vehicle) control, or an intramuscular injection of the same dosage of IIV, under tribromoethanol anesthesia. After three weeks, mice were intranasally challenged with 103 TCID50 of heterologous influenza A/X-31 (H3N2) virus. Three days post-infection, five mice from each group were euthanized, and their lungs were harvested and homogenized in 1 mL PBS. Viral titers in the lung samples were determined using the TCID50 assay. The remaining five mice from each group were monitored daily for signs of mortality, weight loss, and other clinical manifestations for 14 days.

IFN-γ ELISpot assay
The viral antigen-specific T-cell responses were assessed using the IFN-γ ELISpot assay, according to the manufacturer’s instructions for the IFN-γ ELISpot kit (Mabtech, Cat# 3321-4AST-2). The viral antigen peptide (NP366-374: ASNENMETME), synthesized by GenScript, was used as the stimulant in this study.

HI assay
HI assay was performed to quantify HI antibody responses23,25,27. Briefly, mouse sera were treated with receptor-destroying enzyme (RED) solution (Denka Seiken, Cat# 340122) and two-fold serially diluted in 50 µL of PBS in V-shaped 96-well plates, followed by the addition of 4 hemagglutinating (HA) units of WT virus to each well. After 30 min of incubation at room temperature, 50 µL of 1% (v/v) isolated chicken red blood cells in PBS was added to each well. After another 30 min of incubation at room temperature, the highest dilutions of sera that inhibited hemagglutination were read as the HI antibody titers.

NT assay
NT assay was performed to quantify NT antibody responses23,25,27. Briefly, a mixture of two-fold serially diluted RDE-treated sera and MOI=0.01 of influenza virus in DMEM supplemented 1% FBS and 2 µg/mL TPCK-treated trypsin was added to confluent MDCK cells in 96-well plates and incubated for 4 days at 37°C in 5% CO2. The highest dilutions of sera that completely inhibited CPE were read as the NT antibody titers.

Detection of IgG and IgA antibodies by ELISA
ELISA was performed to quantify IgG and IgA antibody responses23,25. Influenza viral HA protein (Sino Biological, Cat# 11692-V08H), NP protein (Sino Biological, Cat# 11675-V08B), and WSN virus were used as antigens. 96-well ELISA plates (Corning, Cat# 3590) were coated overnight at 4°C with 50 ng/well of the protein antigen or 106 TCID50/well of the WSN virus, diluted in 50 µL of ELISA coating buffer (Solarbio, Cat# C1055). Following removal of the coating buffer, the plates were incubated for 1 h with 100 µL/well of blocking buffer (PBS containing 0.1% Tween 20 and 5% non-fat milk), then exposed to two-fold serial dilutions of mouse sera or BAL samples. After 2 h at 37°C, the plates were washed three times with PBS containing 0.1% Tween 20 (PBST) and incubated with 100 µL/well of HRP-conjugated anti-mouse IgG antibody (Proteintech, Cat# SA00001-1; 1:2000 dilution) or HRP-conjugated anti-mouse IgA antibody (Abcam, Cat# ab97235; 1:2000 dilution) diluted in blocking buffer. After another 1-h incubation at 37°C, the plates were washed five times with PBST, and 100 µL/well of TMB substrate solution (Beyotime, Cat# P0209) was added. After 15 min at room temperature, 100 µL of stop solution (Solarbio, Cat# C1058) was added to halt the reaction. Absorbance was measured at 450 nm using an ELISA microplate reader (BioTek Synergy H1, USA).

Statistics and reproducibility
All experiments were performed independently with at least three repetitions. Data are presented as the mean ± standard deviation (s.d.). Graphical analysis and statistical tests were conducted using GraphPad Prism 8.0 and Microsoft Excel. A two-tailed Student’s t-test was applied for comparisons between two groups. For comparisons involving three or more groups, one-way ANOVA followed by Tukey’s or Dunnett’s multiple comparison test was used. P-values less than 0.05 were considered statistically significant; *P<0.05; **P<0.01; ***P<0.001; n.s. not significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Hao et al developed very interesting lysosome-targeting (LYTAR) live attenuated influenza vaccine approaches by\nleveraging the lysosomal degradation pathway, i.e. the chaperone-mediated autophagy, to manipulate the degradation of\nviral proteins of influenza virus. The authors created two types of LYTAR live attenuated vaccine approached, referred to as\nLYTAR 1.0 and LYTAR 2.0: LYTAR 1.0 allows the incorporation of a conditionally removable lysosome-targeting motif\n(LTM) at the N or C end of viral proteins, LYTAR 2.0 supports the flexible incorporation of LTM at internal regions and\nterminal ends of viral proteins. Both types of LYTAR vaccines were attenuated by lysosomal degradation of LTM-tagged\nviral proteins in normal cells but can replicate efficiently for large-scale production in engineered cells. In vivo study in\nanimal models revealed that both types of LYTAR vaccine strains were sufficiently attenuated, a single-dose intranasal\nimmunization induced strong and broad immunity and provided complete cross-reactive protection against homologous and\nheterologous viral challenges.\nThis study is well-executed, with solid data that support the conclusions drawn, making this study a valuable contribution to\nthe field of vaccine development. More importantly, in this manuscript, the authors perfectly solve major systemic\ndeficiencies in the proteasome-targeting (PROTAR) vaccine they previously developed: impairment or dysfunction of the\nproteasome in certain populations can compromise the vaccine's attenuation mechanism and safety.\nOverall, this present LYTAR live attenuated vaccine method represents a significant advancement in vaccine development,\noffering a promising avenue for attenuating viruses in a controlled and highly specific manner. This approach could be\ntransformative not only for influenza vaccines but also for the development of vaccines against other viral pathogens.\nDespite significant progress in vaccine development, emerging and re-emerging viruses still present major threats to global\npublic health. Therefore, there is an urgent need for new vaccine strategies that are safer, more effective, and easier to\ndesign and produce, which is a key priority for public health. This innovative LYTAR vaccine approach significantly\nadvances our understanding of virus attenuation and offers valuable insights into the development of next-generation\nvaccines.\nMajor Comments：\n1. The authors should perform a multi-step growth curve of the LTM-mutant virus in MDCK cells, as shown in Figure. 1d and\nFigure. 4a. If the virus is not attenuated and replicates to WT levels, it would support the conclusion that attenuation of the\nLTM virus is due to the KFERQ residues.\n2. In Figure. 2e and 2f, please calculate the Pearson correlation coefficient for the colocalization analysis of NS1 and\nHSC70, as well as NS1 and LAMP2A.\n3. In the LYTAR1.0 vaccine, the authors should indicate whether the introduction of the LTM sequence affects the\nexpression of the alternatively spliced protein NEP.\n4. In the LYTAR2.0 vaccine, the authors combined one triLTM at PA-N350 and one triLTM at NP-G126 in one virus. Why did\nthe author choose the two sites for triLTM incorporation? The rationale should be provided.\nMinor comments:\n1. Figure 1c and 1d, correct \"NS1-NCTM\" to \"NS1-NLTM\"\n2. Figure. 2b, the quantification of NS1 expression could provide a clearer assessment of the NS1 levels after treatment with\ndifferent inhibitors.\n3. Figure 2i and 2j, Figure 4g and 4h: Add statistical analysis to the bar graphs.\n4. Line 48，the abbreviation for chaperone-mediated autophagy (CMA) are not used elsewhere in the manuscript and are\nnot needed.\n5. In the LYTAR vaccine design concept, the chaperone-mediated autophagy pathway is crucial for generating attenuated\nviruses. Please use bioinformatics tools to present data on the expression of HSC70 and LAMP2A in tissues and cells.\n6. The source of the proteasome-deficient A549 cells used in this study is not provided. Please include the origin of the cell\nline used in your experiments.", "reviewer_2": "In this article “Lysosome-targeting degradation of viral proteins generates live attenuated influenza vaccines” by Jiawei\nHaoet al. live attenuated influenza vaccines (LAIV) are developed and tested in a mouse model of viral infection, for\nhomologous and heterologous protection against a lethal challenge. A single intranasal administration of the LAIV tested in\nmice was safe, showing immunogenicity and protection efficacy. Because influenza virus has an important impact in human\nhealth every year, and the zoonotic risk associated to influenza virus, the area of study is relevant. The paper is well written\nand authors and used a new and original strategy to develop LAIV (LYTAR 1.0 and LYTAR 2.0), using host cell’s lysosome\nto degrade viral proteins. However, the manuscript needs, in opinion of this reviewer, improvements before be considered for\npublication.\nSpecific comments:\n1. LYTAR 1.0. It is unclear and not well justified why authors have selected only NS1-NLTM to be tested. This reviewer is\nwondering if the other viruses were attenuated because the foreign sequence inserted or the region used for the insertion.\n2. What is the cytotoxicity of MG-132, 3-MA, Baf A1, and CQ in the cell lines used?\n3. Figures 2 e and f: authors should include additional images (no just one cell). This can be done as sup figure.\n4. The reason to include LTM seq to generate LYTAR 2.0 inside the sequence of viral genes is not well justified or the\nspecific viral genes selected.\n5. Why these PA-N350 and NP-G126 were selected for further studies?\n6. Authors have used CAIV and IIV for comparison with the generated LAIV. However, it is unclear if these caiv or IIV have\nthe same HA and NA in their formulation. If the ha and NA genes are not the same, these controls should be removed from\nthe paper, because the different vaccines cannot be directly compared. Instead, authors could test other vaccine doses.\n7. Are the sequences inserted in LYTAR vaccines conserved in mammalians? Please, discuss.", "reviewer_3": "In their current manuscript, Hao et al. present data on a novel approach towards an attenuated influenza virus vaccine. The\ndata is very interesting, but some points should be addressed:\nThe safety of the attenuated virus should also be evaluated for replication capacity in immunodeficient mice.\nThere is no side-by-side comparison of the protein levels of the different degradation-targeted proteins (single, triLTM, dual)\nand wild-type expression levels on the same blot. To allow for a direct comparison and to draw the conclusion that the\nprotein reduction is stronger with the advanced constructs, side-by-side blots should be provided.\nThe authors should provide proof for the safety of the system and show that there is no generation of revertants after\npassaging of the attenuated virus, e.g. by sequencing.\nThe images of LYTAR2.0 in LAMP2A-KO 293T and A549 in figure 4 g and h are showing identical cells, this has to be\ncorrected."}, "author_response": "RESPONSE TO REVIEWERS\n(NCOMMS-25-41531-T)\nReviewer #1:\nHao et al developed very interesting lysosome-targeting (LYTAR) live attenuated influenza\nvaccine approaches by leveraging the lysosomal degradation pathway, i.e. the chaperone-\nmediated autophagy, to manipulate the degradation of viral proteins of influenza virus. The\nauthors created two types of LYTAR live attenuated vaccine approached, referred to as LYTAR\n1.0 and LYTAR 2.0: LYTAR 1.0 allows the incorporation of a conditionally removable\nlysosome-targeting motif (LTM) at the N or C end of viral proteins, LYTAR 2.0 supports the\nflexible incorporation of LTM at internal regions and terminal ends of viral proteins. Both types\nof LYTAR vaccines were attenuated by lysosomal degradation of LTM-tagged viral proteins in\nnormal cells but can replicate efficiently for large-scale production in engineered cells. In vivo\nstudy in animal models revealed that both types of LYTAR vaccine strains were sufficiently\nattenuated, a single-dose intranasal immunization induced strong and broad immunity and\nprovided complete cross-reactive protection against homologous and heterologous viral\nchallenges.\nThis study is well-executed, with solid data that support the conclusions drawn, making this\nstudy a valuable contribution to the field of vaccine development. More importantly, in this\nmanuscript, the authors perfectly solve major systemic deficiencies in the proteasome-targeting\n(PROTAR) vaccine they previously developed: impairment or dysfunction of the proteasome in\ncertain populations can compromise the vaccine's attenuation mechanism and safety.\nOverall, this present LYTAR live attenuated vaccine method represents a significant\nadvancement in vaccine development, offering a promising avenue for attenuating viruses in a\ncontrolled and highly specific manner. This approach could be transformative not only for\ninfluenza vaccines but also for the development of vaccines against other viral pathogens.\nDespite significant progress in vaccine development, emerging and re-emerging viruses still\npresent major threats to global public health. Therefore, there is an urgent need for new vaccine\nstrategies that are safer, more effective, and easier to design and produce, which is a key priority\nfor public health. This innovative LYTAR vaccine approach significantly advances our\nunderstanding of virus attenuation and offers valuable insights into the development of next-\ngeneration vaccines.\nResponse: We thank the reviewer for recognizing the novelty, significance, and overall quality\nof our manuscript and for providing helpful comments. Please see our replies below.\nMajor Comments：\n1. The authors should perform a multi-step growth curve of the LTM-mutant virus in MDCK\ncells, as shown in Figure. 1d and Figure. 4a. If the virus is not attenuated and replicates to WT\nlevels, it would support the conclusion that attenuation of the LTM virus is due to the KFERQ\nresidues.\nResponse: We thank the reviewer for the suggestion to improve the manuscript. Accordingly,\nwe have tested the multi-step growth curves of the LTM-mutant viruses in MDCK cells. The mutation\nof LTMs abolished the attenuation of LYTAR virus strains in MDCK cells (revised Figs. 1d and 4a),\nsupporting that the attenuation of LYTAR virus strains is mediated by the LTMs. (Please see Lines\n105-109, 255-257)\n2. In Figure. 2e and 2f, please calculate the Pearson correlation coefficient for the colocalization\nanalysis of NS1 and HSC70, as well as NS1 and LAMP2A.\nResponse: Thank you for your suggestion to improve the manuscript. Accordingly, we have\nanalyzed the colocalization of NS1 with HSC70 and LAMP2A using the Pearson correlation\ncoefficient (new Supplementary Figs. 1b and 2b).\n3. In the LYTAR 1.0 vaccine, the authors should indicate whether the introduction of the LTM\nsequence affects the expression of the alternatively spliced protein NEP.\nResponse: Thank you for your suggestion to improve the manuscript. Accordingly, we have\ntested the levels of LTM-tagged NEP and untagged NEP proteins by western blotting and found that\nthe incorporation of LTM to NEP has no effect on the expression of NEP (new Supplementary Fig.\n3a). In addition, treatment with the proteasome inhibitor MG-132, the macroautophagy inhibitor 3-\nmethyladenine (3-MA), the lysosome inhibitor Bafilomycin A1 (Baf A1), or the lysosome inhibitor\nChloroquine (CQ) has no effect on the levels of LTM-tagged NEP (new Supplementary Fig. 3b),\nindicating that the LTM does not mediate NEP degradation.\nPlease refer to the revised manuscript for more details. (Please see Lines 164-170)\n4. In the LYTAR2.0 vaccine, the authors combined one triLTM at PA-N350 and one triLTM at\nNP-G126 in one virus. Why did the author choose the two sites for triLTM incorporation?\nThe rationale should be provided.\nResponse: We appreciate the opportunity to clarify this point. High viral replication efficiency\nin engineered cells is essential for vaccine production. The two sites, PA-N350 and NP-G126,\nwere chosen to generate PA-N350triLTM, NP-G126triLTM, and LYTAR 2.0dual triLTMs because PA-N350LTM and\nNP-G126LTM exhibited higher replication efficiencies compared to other LYTAR 2.0 strains\ncontaining a single LTM in LAMP2A-knockout MDCK cells, as measured by the viral growth curves\n(new Supplementary Fig. 5). We have included these data and relevant discussion in the revised\nmanuscript. (Please see Lines 226-229)\nMinor comments:\n1. Figure 1c and 1d, correct \"NS1-NCTM\" to \"NS1-NLTM\".\nResponse: We have changed ‘NS1-NCTM’ to ‘NS1-NLTM’.\n2. Figure. 2b, the quantification of NS1 expression could provide a clearer assessment of the\nNS1 levels after treatment with different inhibitors.\nResponse: We have included quantitative data of NS1 expression in the revised Fig. 2b.\n3. Figure 2i and 2j, Figure 4g and 4h: Add statistical analysis to the bar graphs.\nResponse: We have added statistical analysis in the revised Figs. 2i, 2j, 4g, and 4h.\n4. Line 48, the abbreviation for chaperone-mediated autophagy (CMA) are not used elsewhere\nin the manuscript and are not needed.\nResponse: The abbreviation ‘CMA’ has been retained because it is used multiple times in the\nrevised manuscript.\n5. In the LYTAR vaccine design concept, the chaperone-mediated autophagy pathway is crucial\nfor generating attenuated viruses. Please use bioinformatics tools to present data on the\nexpression of HSC70 and LAMP2A in tissues and cells.\nResponse: Thank you for your suggestion to improve this manuscript. Accordingly, we have\nincluded new data showing the expression and distribution of HSC70 and LAMP2A in tissues and\ncells (new Supplementary Fig. 10). (Please see Lines 434-445)\n6. The source of the proteasome-deficient A549 cells used in this study is not provided. Please\ninclude the origin of the cell line used in your experiments.\nResponse: Thank you for your kind reminder. Accordingly, we have included information\nabout the proteasome-deficient A549 cell line in the revised Materials and Methods section. (Please\nsee Lines 477-478)\nReviewer #2 (Remarks to the Author):\nIn this article “Lysosome-targeting degradation of viral proteins generates live attenuated\ninfluenza vaccines” by Jiawei Hao et al. live attenuated influenza vaccines (LAIV) are\ndeveloped and tested in a mouse model of viral infection, for homologous and heterologous\nprotection against a lethal challenge. A single intranasal administration of the LAIV tested in\nmice was safe, showing immunogenicity and protection efficacy. Because influenza virus has an\nimportant impact in human health every year, and the zoonotic risk associated to influenza\nvirus, the area of study is relevant. The paper is well written and authors and used a new and\noriginal strategy to develop LAIV (LYTAR 1.0 and LYTAR 2.0), using host cell’s lysosome to\ndegrade viral proteins. However, the manuscript needs, in opinion of this reviewer,\nimprovements before be considered for publication.\nResponse: We thank the reviewer for recognizing the originality and importance of our\nmanuscript and for providing constructive comments. Please see our replies below.\nSpecific comments:\n1. LYTAR 1.0. It is unclear and not well justified why authors have selected only NS1-NLTM to\nbe tested. This reviewer is wondering if the other viruses were attenuated because the foreign\nsequence inserted or the region used for the insertion.\nResponse: We appreciate the opportunity to clarify this point. To generate LYTAR 1.0 influenza\nvirus strains, we screened six viral proteins (PB2, PB1, PA, NP, M1, and NS1) to explore the\nfeasibility of incorporating a removable LTM at their N-terminus or C-terminus. We found that only\nwhen the removable LTM is fused to the N-terminus of NS1 protein, the LYTAR 1.0 virus, i.e. NS1-\nNLTM, can be successfully generated. Therefore, only NS1-NLTM was tested in the LYTAR 1.0 study.\nIncorporation of the LTM at the N-terminus or C-terminus of other viral proteins did not generate\nviable virus (Supplementary Table 2), potentially because LTM incorporation could disrupt the\nstructures or functions of these viral proteins. We have included the data for other viral proteins from\nour screening and the relevant discussion in the revised manuscript.\nAlthough the LYTAR 1.0 vaccine approach shows potential for virus attenuation and vaccine\ndesign, a major limitation is the restricted sites where the LTM can be added: the LTM must be fused\nto the N-terminus or C-terminus of viral proteins for removal from viral proteins to keep the viral\nintact in TEVp-expressing cells and can be efficiently recognized by the heat shock-cognate protein\nof 70 KDa (HSC70) for lysosomal degradation in conventional cells. Our screening revealed that only\nthe N-terminus of the influenza viral NS1 protein meets both requirements, thus only one single\nLYTAR 1.0 vaccine strain (i.e. NS1-NLTM) was successfully generated. To overcome this limitation,\nwe have developed the next-generation LYTAR vaccine approach (referred to as LYTAR 2.0) in the\nmanuscript. The LYTAR 2.0 vaccine approach removes the requirement of LTM incorporation at the\nN-terminus or C-terminus of viral proteins and supports the incorporation of LTM at any appropriate\nsite of the viral proteins of interest, including N-terminus, C-terminus, and internal regions,\nsignificantly broadening the diversity of feasible sites for LTM incorporation and thus enabling\ngeneration of a larger pool of vaccine candidates. We have also included the relevant discussion in\nthe revised manuscript. (Please see Lines 93-96)\n2. What is the cytotoxicity of MG-132, 3-MA, Baf A1, and CQ in the cell lines used?\nResponse: Thank you for your question to improve the manuscript. These inhibitors and their\nconcentrations used in our study were selected based on previous reports (Mol Cell 68, 308-322 e304\n(2017); PLoS Pathog 17, e1009300 (2021); Nat Commun 13, 5204 (2022)), wherein the inhibitors\ndid not show detectable cytotoxicity. In addition, we further evaluated the cytotoxicity of these\ninhibitors using Cell Counting Kit-8 (CCK-8) assay and confirmed that they did not show cytotoxicity\nat the concentrations used in this study (new Supplementary Fig. 11). We have included the data and\ncitations in the revised manuscript.\n3. Figures 2 e and f: authors should include additional images (no just one cell). This can be\ndone as sup figure.\nResponse: We appreciate your suggestion to improve the manuscript. Accordingly, we have\nincluded additional representative images in the revised manuscript (new Supplementary Figs. 1a\nand 2a). In addition, we have quantitatively analyzed the colocalization of NS1 with HSC70 and\nLAMP2A using the Pearson correlation coefficient (n = 10 independent biological replicates) (new\nSupplementary Figs. 1b and 2b). Please refer to the revised manuscript for more details.\n4. The reason to include LTM seq to generate LYTAR 2.0 inside the sequence of viral genes is\nnot well justified or the specific viral genes selected.\nResponse: Thank you for your comments to improve the manuscript. As also mentioned in our\nresponse to Comment #1, a major limitation of the LYTAR 1.0 vaccine approach is the restricted sites\nwhere the LTM can be added: the LTM must be fused to the N-terminus or C-terminus of viral proteins\nfor removal from viral proteins to keep the viral intact in TEVp-expressing cells and can be efficiently\nrecognized by the heat shock-cognate protein of 70 KDa (HSC70) for lysosomal degradation in\nconventional cells. Our screening revealed that only the N-terminus of the influenza viral NS1 protein\nmeets both requirements, thus only one single LYTAR 1.0 vaccine strain (i.e. NS1-NLTM) was\nsuccessfully generated. However, unlike influenza virus that has 8 gene segments and 10 viral\nproteins, most viruses have much smaller number of proteins, resulting in few sites potential suitable\nto LTM incorporation, which hinders an extensive application of the LYTAR 1.0 vaccine approach.\nTo overcome this limitation, we have developed the next-generation LYTAR vaccine approach\n(referred to as LYTAR 2.0). In contrast to the dependence of the TEVcs cleavage linker in LYTAR\n1.0, the LYTAR 2.0 relies on LAMP2A-knockout cells to achieve efficient viral propagation for\nproduction purpose. This LYTAR 2.0 vaccine design removes the requirement of LTM incorporation\nat a fixed locus (N-terminus or C-terminus) of viral proteins and supports the incorporation of LTM\nat any appropriate site of the viral proteins of interest, including N-terminus, C-terminus, and internal\nregions, significantly broadening the diversity of sites for incorporation of LTM and thus allowing\nthe generation of much larger pool of vaccine candidates for influenza virus and potentially for other\nviruses with even less number of viral proteins. Consequently, LYTAR 2.0 offers greater flexibility\nand universality than LYTAR 1.0.\nIn the LYTAR 2.0 study, four influenza viral proteins (PA, NP, M1, and NS1) were chosen as\nrepresentatives for LTM incorporation based on their distinct roles: PA is a representative of influenza\nviral polymerases (PA, PB1, and PB2) that are responsible for replication and transcription of viral\ngenome; NP is responsible for encapsulating influenza viral genome by forming ribonucleoprotein\n(RNP); M1 mediates virus assembly by forming an endoskeleton beneath the virus membrane; and\nNS1 is a representative of nonstructural proteins and plays an important role in evasion of the host\ninnate immune system for facilitating viral replication.\nWe have included these points in the revised manuscript. (Please see Lines 93-96, 172-176,\n184-191)\n5. Why these PA-N350 and NP-G126 were selected for further studies?\nResponse: We appreciate the opportunity to clarify this point. High viral replication efficiency\nin engineered cells is essential for vaccine production. The two sites, PA-N350 and NP-G126,\nwere chosen to generate PA-N350triLTM, NP-G126triLTM, and LYTAR 2.0dual triLTMs because PA-N350LTM and\nNP-G126LTM exhibited higher replication efficiencies compared to other LYTAR 2.0 strains\ncontaining one LTM in LAMP2A-knockout MDCK cells, as measured by the viral growth curves\n(new Supplementary Fig. 5). We have included these data and relevant discussion in the revised\nmanuscript. (Please see Lines 226-229)\n6. Authors have used CAIV and IIV for comparison with the generated LAIV. However, it is\nunclear if these CAIV or IIV have the same HA and NA in their formulation. If the HA and NA\ngenes are not the same, these controls should be removed from the paper, because the different\nvaccines cannot be directly compared. Instead, authors could test other vaccine doses.\nResponse: Thank you for your comments to improve the manuscript. The CAIV and IIV used\nin our study have the same HA and NA as the LYTAR vaccine strains. We have added more detailed\ninformation of the CAIV and IIV in the Materials and Methods section of the revised manuscript to\nclarify this point. (Please see Lines 454-462)\n7. Are the sequences inserted in LYTAR vaccines conserved in mammalians? Please, discuss.\nResponse: Thank you for your suggestion to improve the manuscript. The LTM sequences in\nLYTAR vaccines are canonical KFERQ-like motifs that were identified and well characterized in the\nchaperone-mediated autophagy (CMA) system (PLoS Biol 17 (5): e3000301 (2019); Nat Biotechnol\n28, 256-263 (2010); Nat Neurosci 17 (3): 471–480 (2014)). The CMA system is highly conserved in\nmammals and birds (Nat Rev Mol Cell Biol 19, 365-381 (2018); Autophagy 14, 1267-1270 (2018);\nPLoS Biol 17, e3000301 (2019)) and is ubiquitously detectable in almost all mammalian cells\nexperimentally tested so far (PLoS Biol 17, e3000301 (2019); Nat Commun 2: 386 (2011)). In addition,\nthe data from Human Protein Atlas indicates the ubiquitous expression of HSC70 and LAMP2A, the\ntwo key components of CMA, in human tissues and cells (new Supplementary Fig. 10). The high\nconservation and wide distribution of the CMA system provide critical basis for the safety of LYTAR\nvaccines.\nFurthermore, increasing number of studies are leading to identification of more KFERQ-like\nmotifs (PLoS Biol 17, e3000301 (2019)). While the present proof-of-concept study demonstrates the\nfeasibility of LYTAR vaccine strategy, a systematic investigation of the KFERQ-like motifs and their\nincorporation sites within viral proteins could support generation of diverse LYTAR vaccine\nstrains, which may lead to development of optimal LYTAR vaccine candidates in terms of safety and immune\nefficacy.\nWe have included the relevant discussion to the revised manuscript. (Please see Lines 434-455)\nReviewer #3 (Remarks to the Author):\nIn their current manuscript, Hao et al. present data on a novel approach towards an attenuated\ninfluenza virus vaccine. The data is very interesting, but some points should be addressed.\nResponse: We thank the reviewer for recognizing the novelty of our manuscript and for\nproviding constructive comments to improve the manuscript. Please see our replies below.\n1. The safety of the attenuated virus should also be evaluated for replication capacity in\nimmunodeficient mice.\nResponse: We appreciate your suggestion to strengthen the manuscript. Accordingly, we have\nevaluated the in vivo safety of LYTAR vaccine candidates (i.e., NS1-NLTM and LYTAR 2.0dual triLTMs)\nin immunodeficient mice in the revised manuscript (revised Fig. 5). Immunodeficient BALB/c nude\nmice were intranasally infected with 105 TCID of NS1-NLTM, LYTAR 2.0dual triLTMs, or wild-type\n50\n(WT) virus and monitored for body weight, survival, and disease signs for 14 days. The WT virus\ncaused death of all mice within 8 days after infection, accompanied by rapid body weight loss and\ntrembling before death (revised Fig. 5e). In contrast, infection with the same dose of NS1-NLTM or\nLYTAR 2.0dual triLTMs did not cause any death, body weight loss, or any other disease signs (revised\nFig. 5e). In addition, detection of viral titers in the mouse lungs on day 3 post-infection revealed that\nthe titers in the lungs of BALB/c nude mice infected with WT virus were approximately 106\nTCID /mL, whereas those infected with NS1-NLTM or LYTAR 2.0dual triLTMs virus were approximately\n50\n50 50\nsuggest that both NS1-NLTM and LYTAR 2.0dual triLTMs viruses are highly attenuated in\nimmunodeficient mice. We have included these data in the revised manuscript. (Please see Lines 304-\n315)\n2. There is no side-by-side comparison of the protein levels of the different degradation-targeted\nproteins (single, triLTM, dual) and wild-type expression levels on the same blot. To allow for a\ndirect comparison and to draw the conclusion that the protein reduction is stronger with the\nadvanced constructs, side-by-side blots should be provided.\nResponse: Thank you for your great suggestion to improve the manuscript. Accordingly, we\nhave performed side-by-side comparison of viral proteins containing single LTM, triLTM, dual\ntriLTMs, and wild-type viral proteins on the same blot. The data suggest that increasing the number\nof LTM can enhance viral protein degradation (new Supplementary Fig. 7). We have included these\ndata in the revised manuscript. (Please see Lines 242-244)\n3. The authors should provide proof for the safety of the system and show that there is no\ngeneration of revertants after passaging of the attenuated virus, e.g. by sequencing.\nResponse: Thank you for your suggestion to improve the manuscript. Accordingly, we have\nevaluated the genetic stability of LYTAR vaccine candidates (i.e., NS1-NLTM and LYTAR 2.0dual triLTMs)\nby passaging them over 20 passages and detecting potential revertants by viral genome sequencing.\nThe data revealed that no mutation occurred after over 20 passages of culture (Supplementary Fig.\n8), indicating the genetic stability of the LYTAR vaccine candidates.\nIn addition, the safety of the LYTAR vaccine system is also supported by the following points.\n(1) The chaperone-mediated autophagy (CMA) system is highly conserved in mammals and birds\n(Nat Rev Mol Cell Biol 19, 365-381 (2018); Autophagy 14, 1267-1270 (2018); PLoS Biol 17,\ne3000301 (2019)) and is ubiquitously detectable in almost all mammalian cells experimentally\ntested so far (PLoS Biol 17, e3000301 (2019); Nat Commun 2: 386 (2011)). The data from Human\nProtein Atlas confirms the ubiquitous expression of HSC70 and LAMP2A, the two key\ncomponents of CMA system, in human tissues and cells (new Supplementary Fig. 10). The high\nconservation and wide distribution of the CMA system provide a critical basis for the safety of\nLYTAR vaccines.\n(2) In cell models, the LYTAR vaccine candidates exhibit an attenuation factor of over 104 relative\nto the wild-type (WT) virus (Figs. 1d, 4a), which is significantly higher than the attenuation factor\nof ~100 for the clinically approved CAIV at 37 ℃.\n(3) The LYTAR vaccine candidates showed favorable in vivo safety profiles in immunocompetent\nand even immunodeficient mouse models (revised Fig. 5).\n(4) Increasing number of studies are leading to identification of more KFERQ-like motifs (PLoS Biol\n17, e3000301 (2019)). While the present proof-of-concept study demonstrates the feasibility of\nLYTAR vaccine strategy, a systematic investigation on the KFERQ-like motifs and their\nincorporation sites within viral proteins could support generation of diverse LYTAR vaccine\nstrains, which may lead to development of optimal LYTAR vaccine candidates in terms of safety\nand immune efficacy.\nWe have included these data and relevant discussion in the revised manuscript. (Please see Lines\n283-285)\n4. The images of LYTAR2.0 in LAMP2A-KO 293T and A549 in figure 4 g and h are showing\nidentical cells, this has to be corrected.\nResponse: We appreciate your kind reminder. We have corrected this in the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have address all the questions raised by reviewers.", "reviewer_2": "All comments raised in my previous review, as well as those from other reviewers, have been thoroughly addressed. The\nauthors have demonstrated considerable effort in responding to the feedback and have conducted additional experiments\nthat significantly strengthen the study, providing more robust evidence to support their conclusions. In light of these\nrevisions, I find the manuscript suitable for publication.", "reviewer_3": "My previous comments have been addressed in the revised version of the manuscript."}, "author_response": ""}]

Decision letter