Determining Immune and miRNA Biomarkers Related to Respiratory Syncytial Virus (RSV) Vaccine Types

Abstract

Respiratory Syncytial Virus (RSV) causes serious respiratory tract illness and substantial morbidity and some mortality in populations at the extremes of age, i.e., infants, young children, and the elderly. To date, RSV vaccine development has been unsuccessful, a feature linked to the lack of biomarkers available to assess the safety and efficacy of RSV vaccine candidates. We examined microRNAs (miR) as potential biomarkers for different types of RSV vaccine candidates. In this study, mice were vaccinated with a live attenuated RSV candidate that lacks the small hydrophobic (SH) and attachment (G) proteins (CP52), an RSV G protein microparticle (GA2-MP) vaccine, a formalin-inactivated RSV (FI-RSV) vaccine or were mock-treated. Several immunological endpoints and miR expression profiles were determined in mouse serum and bronchoalveolar lavage (BAL) following vaccine priming, boost, and RSV challenge. We identified miRs that were linked with immunological parameters of disease and protection. We show that miRs are potential biomarkers providing valuable insights for vaccine development.

Keywords: RSV; disease; immune; miR; microRNA; vaccines.

Figure 1

RSV vaccines types, serum IgG, and virus clearance. Sera at day 14 (A) and 5 (B) post-RSV A or B challenge of prime-boosted mice (C,D); IgG reactivity was determined against A2 (A,C) and B1 (B,D). Three weeks after the boost-vaccination mice were i.n. challenged with 10⁶ PFU of A2. (E) RSV neutralizing antibody levels were measured by microneutralization assay at day 5 post-RSV challenge. (F) Lung virus titers were determined 5 days post-challenge by plaque assay. PBS only-treated groups treated had no detectable effect and are not included. All samples were assayed in duplicate and n = 4 mice/group. Error bars represent the SEM and results were considered significant with a ^*p ≤ 0.05 and ^****p ≤ 0.0001 as determined by one-way ANOVA and Bonferroni's test.

Figure 2

Vaccine types and Th1/ Th2 memory responses. The number of M2₈₂₋₉₀, F₅₁₋₆₆, G_183−198, and eGFP_200−208-specific (irrelevant peptide control) IL4- and IFNγ- producing splenocytes were determined by ELISPOT harvested at 14 days post-boost vaccination. (A) IFNγ- producing splenocytes and (B) IL4-producing splenocytes. The data are presented as ELISPOTS/10⁶ splenocytes. Three weeks after the boost mice were i.n. challenged with 10⁶ PFU of A2. The level of (C) MCP1 and (D) RANTES were measured in sera and BAL supernatant by multiplex cytokine/chemokine assay and the data are presented as pg/mL of cytokine in BAL supernatant at day 3 post-challenge (n = 4–6 mice/group). The dashed line indicates the limit of detection (LOD) = 3.2 pg/ml. Error bars represent the SEM from n = 4 mice/group and results were considered significant with a ^*p ≤ 0.05, ^**p ≤ 0.01, and ^****p ≤ 0.0001 as determined by two-way ANOVA and Bonferroni's test using GraphPad Prism ver. 8.0.

Figure 3

The number of differentially expressed miRNAs during vaccination and post-RSV challenge. Sera miRNA profiles of vaccinated mice (n = 4/group) were evaluated at day 7 post-prime, day 14 post-prime, day 7 post-boost, day 14 post-boost, day 3 post-challenge, and day 5 post-challenge using a miRNA PCR array. The y-axis indicates the number of differentially expressed miRNAs. Significance was determined using a fold-change threshold of >2, the result was reported as a fold-upregulation. If the fold change was <0.5, the result was reported as a fold-downregulation.