Clinical validation of an RSV neutralization assay and analysis of cross-sectional sera associated with 2021-2023 RSV outbreaks to investigate the immunity debt hypothesis

Abstract

Respiratory syncytial virus (RSV) is a leading cause of acute respiratory infections and hospitalization in infants and the elderly. Newly approved vaccines and the prophylactic antibody nirsevimab have heightened interest in RSV immunologic surveillance, necessitating the development of high-throughput assays assessing anti-RSV neutralizing activity. Quantitative viral neutralization remains the best correlate of protection for RSV infection and the gold standard for RSV immunological testing. Here, we developed a high-throughput RSV strain A2 focus-reduction neutralization test validated to Clinical Laboratory Improvement Amendments (CLIA)/ Good Clinical Laboratory Practices (GCLP) standards using both clinical specimens and commercially available reference sera. The assay is highly accurate, generating reference serum neutralizing titers within twofold of established assays, with an analytical measurement range between 8 and 1,798 international units per mL (IU/mL). Neutralizing activity measured by the assay strongly correlated with antibody titer determined via indirect enzyme-linked immunosorbent assay (ELISA) (ρ = 1.0, P = 0.0014). Individuals recently having tested positive via quantitative reverse transcription polymerase chain reaction (RT-qPCR) for RSV had a 9.1-fold higher geometric mean neutralizing titer relative to RSV PCR negatives (P-value = 0.09). The validated assay was then used to investigate the immunity debt hypothesis for resurgent RSV outbreaks in the 2022-2023 season, using adult clinical remnant sera sent for herpes simplex virus (HSV)-1/2 antibody testing. There was no difference in geometric mean anti-RSV neutralizing titers between sera sampled before and after the 2022-2023 RSV outbreak (P = 0.68). These data are consistent with limited changes in RSV-neutralizing antibody levels in adults across the 2022-23 RSV outbreak.

Importance: Population surveillance studies of serum-neutralizing activity against RSV are crucial for evaluating RSV vaccine efficacy and vulnerabilities to new strains. Here, we designed and validated a high-throughput assay for assessing anti-RSV neutralizing activity, standardized its measurements for comparison with other methodologies, and demonstrated its applicability to real-world samples. Our assay is precise, linear, and yields measurements consistent with other standardized assays, offering a methodology useful for large-scale studies of RSV immunity. We also find no significant difference in neutralizing titers among adults between those taken before and after large RSV outbreaks associated with the latter stages of the coronavirus disease of 2019 (COVID-19) public health emergency, underlining the need for a greater understanding of the dynamics of serological responses to RSV infection.

Keywords: IU/mL; RSV; correlate of protection; epidemiology; neutralization; outbreaks; seroepidemiology; serology; titer; validation.

Fig 1

Accuracy, linearity, and precision of a respiratory syncytial virus (RSV) focus-reduction neutralization test (FRNT) assay. (A) Results of RSV-binding antibody measurement by indirect ELISA of the linearity panel specimens (black open circles; ) and the rubella serology remnant specimens (blue open circles) are shown. The dashed black line is the 4-parameter logistic curve fit of the ELISA results from the linearity panel. Sera was measured in duplicate, normalized relative to the assay cut-off control (relative absorbance of 10), and plotted as average values. The standard deviations of the measurements are plotted using a black line range, though these line ranges are small enough to be obscured by the larger diameter of the plotted points. The grey line depicts the approximate normalized ELISA cut-off value of approximately 10, with relative absorbance between 9 and 11 considered inconclusive. (B) Results of triplicate testing of the linearity panel, composed of a serial dilution series of reference serum (NR-21973), were plotted against the expected values for the series ranging from 6 to 5,543 IU/mL (international units per mL). The dashed line is the best fit from the linear regression analysis. The formula for this linear regression is displayed in the upper-right corner. (C) Precision testing results of select linearity panel specimens (2 through 5) that were within the expected analytical measurement range of the assay (expected values ranging from 22 to 1,386 IU/mL) were measured in duplicate over 3 days. Green boxes demarcate boundaries of expected variation for a geometric coefficient of variation (GCV) of 37%. Solid lines represent the mean IU/mL measurement for panel members. (D) Precision testing of five serum specimens from rubella remnant testing spanning the analytical measurement range (AMR) of the assay, tested in duplicate over two days. Green boxes demarcate boundaries of expected variation for a GCV of 37%. Solid lines represent mean IU/mL values for panel members.

Fig 2

The distribution of neutralizing titers among random population samples collected from late 2021 to early 2024. (A) Neutralizing titer results of HSV serology remnant serum specimens from individuals sampled around February 2022, August 2022, February 2023, and September 2023, with the date of serum collection on the x-axis. For each cross-sectional group, dashed and solid lines represent the geometric median and geometric mean, respectively. (B) Number of positive RSV antigen tests across time in Washington State taken from CDC Washington State trends (51), reported as a 5-week average. Log₂-transformed data were analyzed, using a two-sided student’s t-test to compare groups pairwise. IU/mL, international units per mL.

Fig 3

The distribution of neutralizing titers among respiratory syncytial virus (RSV) negative, positive, and future-infected individuals relative to a random population sample (50% neutralizing titer in international units per mL (IU/mL) for individuals sampled from October 2021 to February 2024). Solid lines represent the geometric mean. HSV serology remnant specimens: a random population sample of remnant sera sent for clinical HSV remnant testing, collected from February 2021 to September 2023, with no PCR data available for RSV. Future RSV-infected: HSV remnant samples (not part of the HSV serology remnant specimen group above) from individuals who tested PCR-positive for RSV, 25 or more days after the serum collection date. RSV PCR+, immunocompetent: emergency department (ED) patients PCR-positive for RSV, 98 days before to 7 days after serum collection. RSV PCR+, immunosuppressed: ED patients PCR-positive for RSV, 49 days before to 7 days after serum collection, with medical status of immunosuppression at the time of test/collection. Influenza+, RSV−: ED patients with no recorded RSV-positive or immunosuppression status who tested PCR-positive for influenza virus, 39 days before to 7 days after serum collection. Log₂-transformed data were analyzed using a two-sided student’s t-test to compare groups pairwise. *P < 0.05 and **P < 0.01.