Viral Subpopulation Screening Guides in Designing a High Interferon-Inducing Live Attenuated Influenza Vaccine by Targeting Rare Mutations in NS1 and PB2 Proteins

Abstract

Influenza A viruses continue to circulate among wild birds and poultry worldwide, posing constant pandemic threats to humans. Effective control of emerging influenza viruses requires new broadly protective vaccines. Live attenuated influenza vaccines with truncations in non-structural protein 1 (NS1) have shown broad protective efficacies in birds and mammals, which correlate with the ability to induce elevated interferon responses in the vaccinated hosts. Given the extreme diversity of influenza virus populations, we asked if we could improve an NS1-truncated live attenuated influenza vaccine developed for poultry (PC4) by selecting viral subpopulations with enhanced interferon-inducing capacities. Here, we deconstructed a de novo population of PC4 through plaque isolation, created a large library of clones, and assessed their interferon-inducing phenotypes. While most of the clones displayed the parental interferon-inducing phenotype in cell culture, few clones showed enhanced interferon-inducing phenotypes in cell culture and chickens. The enhanced interferon-inducing phenotypes were linked to either a deletion in NS1 (NS1Δ76-86) or a substitution in polymerase basic 2 protein (PB2-D309N). The NS1Δ76-86 deletion disrupted the putative eukaryotic translation initiation factor 4GI-binding domain and promoted the synthesis of biologically active interferons. The PB2-D309N substitution enhanced the early transcription of interferon mRNA, revealing a novel role for the 309D residue in suppression of interferon responses. We combined these mutations to engineer a novel vaccine candidate that induced additive amounts of interferons and stimulated protective immunity in chickens. Therefore, viral subpopulation screening approaches can guide the design of live vaccines with strong immunostimulatory properties. IMPORTANCE Effectiveness of NS1-truncated live attenuated influenza vaccines relies heavily on their ability to induce elevated interferon responses in vaccinated hosts. Influenza viruses contain diverse particle subpopulations with distinct phenotypes. We show that live influenza vaccines can contain underappreciated subpopulations with enhanced interferon-inducing phenotypes. The genomic traits of such virus subpopulations can be used to further improve the efficacy of the current live vaccines.

Influenza A virus infection in turkeys induces respiratory and enteric bacterial dysbiosis correlating with cytokine gene expression

“…For examples, we did not collect data to assess how the extent of mucosal barrier injury ( Li et al, 2018 ) in gut and respiratory sites affected the observed associations among microbiota, influenza virus titers, and innate gene expression profiles. Furthermore, the expression of IFN-α/β/λ and IL-6 genes at high levels may not indicate the production of high amounts of functional proteins, as demonstrated in our recent work where a subpopulation of avian influenza virus blocked the synthesis of IFN-β protein without inhibiting the transcription of IFN-β mRNA ( Ghorbani et al, 2020 ). Moreover, without bacterial metabolite or transcriptome data, it remains unknown if the alteration of bacterial diversity, and abundance and following virus infection in this study indicated functional changes of microbiota.…”

“…The treatment group was the experimental unit. Sixteen birds per group were sufficient for statistical comparisons of viral shedding, gene expression, and microbiota data based on our published studies ( Ghorbani et al, 2020 ; Jang et al, 2018 ; Johnson et al, 2018 ; Ngunjiri et al, 2019b ; Taylor et al, 2020 ). Median egg infectious dose (EID 50 ) for each virus stock was calculated prior to normalization of virus concentration on the day of challenge.…”

“…Transcriptional analysis of several innate immune genes was performed with trachea samples collected during necropsy at 5 dpi, as described in our previous studies ( Ghorbani et al, 2020 ; Jang et al, 2018 ; Jang, Ngunjiri & Lee, 2016 ; KC et al, 2020 ). The uppermost 0.5 cm of tracheal tissue was excised using sterile scissors and forceps, placed into a tube with one mL TRIzol, snap-frozen in liquid nitrogen, and stored at −70 C until processing.…”

“…Differential gene expressions were normalized using GAPDH gene as the internal control. Relative mRNA expression levels were calculated as fold changes over the mock group using the 2−ΔΔCT method ( Ghorbani et al, 2020 ; Jang et al, 2018 ).…”

Salmonella Serovar Typhimurium Infection Modulates Expression of Immune-Related Genes in Avian Enriched Monocytes

“…The relative PCR amplicon concentration was determined by uorescence signals detected at the end of each qPCR cycle, and their logarithmic values were plotted against the cycle number as Ct values. Relative mRNA expression levels were calculated as fold changes over the Mock group using the 2 -ΔΔCT method [15].…”