iGATE analysis improves the interpretability of single-cell immune landscape of influenza infection.

Influenza poses a persistent health burden worldwide. To design equitable vaccines effective across all demographics, it is essential to better understand how host factors such as genetic background and aging affect the single-cell immune landscape of influenza infection. Cytometry by time-of-flight (CyTOF) represents a promising technique in this pursuit, but interpreting its large, high-dimensional data remains difficult. We have developed a new analytical approach, in silico gating annotating training elucidating (iGATE), based on probabilistic support vector machine classification. By rapidly and accurately "gating" tens of millions of cells in silico into user-defined types, iGATE enabled us to track 25 canonical immune cell types in mouse lung over the course of influenza infection. Applying iGATE to study effects of host genetic background, we show that the lower survival of C57BL/6 mice compared with BALB/c was associated with a more rapid accumulation of inflammatory cell types and decreased IL-10 expression. Furthermore, we demonstrate that the most prominent effect of aging is a defective T cell response, reducing survival of aged mice. Finally, iGATE reveals that the 25 canonical immune cell types exhibited differential influenza infection susceptibility and replication permissiveness in vivo, but neither property varied with host genotype or aging. The software is available at https://github.com/UmichWenLab/iGATE.

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04-0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72-144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.

Enhancing the Yield and Quality of Influenza Virus-like Particles (VLPs) Produced in Insect Cells by Inhibiting Cytopathic Effects of Matrix Protein M2.

To provide broader protection and eliminate the need for annual update of influenza vaccines, biomolecular engineering of influenza virus-like particles (VLPs) to display more conserved influenza proteins such as the matrix protein M2 has been explored. However, achieving high surface density of full-length M2 in influenza VLPs has been left unrealized. In this study, we show that the ion channel activity of M2 induces significant cytopathic effects in Spodoptera frugiperda (Sf9) insect cells when expressed using M2-encoding baculovirus. These effects include altered Sf9 cell morphology and reduced baculovirus replication, resulting in impaired influenza protein expression and thus VLP production. On the basis of the function of M2, we hypothesized that blocking its ion channel activity could potentially relieve these cytopathic effects, and thus restore influenza protein expression to improve VLP production. The use of the M2 inhibitor amantadine indeed improves Sf9 cellular expression not only of M2 (∼3-fold), but also of hemagglutinin (HA) (∼7-fold) and of matrix protein M1 (∼3-fold) when coexpressed to produce influenza VLPs. This increased cellular expression of all three influenza proteins further leads to ∼2-fold greater VLP yield. More importantly, the quality of the resulting influenza VLPs is significantly improved, as demonstrated by the ∼2-fold, ∼50-fold, and ∼2-fold increase in the antigen density to approximately 53 HA, 48 M1, and 156 M2 per influenza VLP, respectively. Taken together, this study represents a novel approach to enable the efficient incorporation of full-length M2 while enhancing both the yield and quality of influenza VLPs produced by Sf9 cells.

The Coming Age of Insect Cells for Manufacturing and Development of Protein Therapeutics.

Protein therapeutics is a rapidly growing segment of the pharmaceutical market. Currently, the majority of protein therapeutics are manufactured in mammalian cells for their ability to generate safe and efficacious human-like glycoproteins. The high cost of using mammalian cells for manufacturing has motivated a constant search for alternative host platforms. Insect cells have begun to emerge as a promising candidate, largely due to the development of the baculovirus expression vector system. While there are continuing efforts to improve insect-baculovirus expression for producing protein therapeutics, key limitations including cell lysis and the lack of homogeneous humanized glycosylation still remain. The field has started to see a movement toward virus-less gene expression approaches, notably the use of clustered regularly interspaced short palindromic repeats to address these shortcomings. This review highlights recent technological advances that are realizing the transformative potential of insect cells for the manufacturing and development of protein therapeutics.