Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Dec 3.
Published in final edited form as: Vaccine. 2020 Nov 12;38(51):8185–8193. doi: 10.1016/j.vaccine.2020.10.076

Durability of Humoral Immune Responses to Rubella Following MMR Vaccination

Stephen N Crooke 1, Marguerite M Riggenbach 1, Inna G Ovsyannikova 1, Nathaniel D Warner 2, Min-Hsin Chen 3, Lijuan Hao 3, Joseph P Icenogle 3, Gregory A Poland 1, Richard B Kennedy 1
PMCID: PMC7716653  NIHMSID: NIHMS1646464  PMID: 33190948

Abstract

Background:

While administration of the measles-mumps-rubella (MMR-II®) vaccine has been effective at preventing rubella infection in the United States, the durability of humoral immunity to the rubella component of MMR vaccine has not been widely studied among older adolescents and adults.

Methods:

In this longitudinal study, we sought to assess the durability of rubella virus (RV)-specific humoral immunity in a healthy population (n = 98) of adolescents and young adults at two timepoints: ~7 and ~17 years after two doses of MMR-II® vaccination. Levels of circulating antibodies specific to RV were measured by ELISA and an immune-colorimetric neutralization assay. RV-specific memory B cell responses were also measured by ELISpot.

Results:

Rubella-specific IgG antibody titers, neutralizing antibody titers, and memory B cell responses declined with increasing time since vaccination; however, these decreases were relatively moderate. Memory B cell responses exhibited a greater decline in men compared to women.

Conclusions:

Collectively, rubella-specific humoral immunity declines following vaccination, although subjects’ antibody titers remain well above the currently recognized threshold for protective immunity. Clinical correlates of protection based on neutralizing antibody titer and memory B cell ELISpot response should be defined.

Keywords: Rubella, Antibodies, MMR-II Vaccine, Rubella Vaccine, Humoral Immunity, Waning Immunity

Introduction

Rubella was first formally defined as a human disease at the 1881 International Congress on Medicine [1]. Spread through respiratory secretions, rubella infection can easily go undetected. Symptoms can include generalized lymphadenopathy, mild fever, and rash [2]. Though rubella infection may be shortlived and lead to mild discomfort in children and adults, early trimester infection of the fetus can lead to congenital rubella syndrome (CRS). CRS includes several detrimental defects (e.g., intellectual delays, microcephaly, organ damage, sensory impairments) that can drastically diminish quality of life. In severe cases of congenital infection with rubella, miscarriage may result [3–5]. CRS develops in up to 90% of cases when maternal rubella infection occurs during the first trimester of pregnancy [6]. Although occurrences are now rare in the United States, nearly 100,000 cases of CRS are still estimated to occur globally each year [5]. Given its often asymptomatic presentation and the potential for damage to the fetus, maintaining durable immunity to rubella among the population is imperative.

The durability of protective immunity against rubella in vaccinated individuals remains poorly understood. Numerous surveillance studies have reported 90–100% seropositivity against rubella (i.e., antibody titer > 10 IU/mL) among vaccinated populations [7–13], and it is generally accepted that vaccination against rubella provides lifelong immunity after a single dose [5, 14]. Measurements of serostatus alone are not wholly representative of protective immunity, as standard serology tests do not account for functional antibody activity (e.g., neutralization) or memory B cell responses. Furthermore, a number of studies have reported waning immune responses to rubella. LeBaron et al. observed declines in rubella-specific IgG titers to pre-vaccination levels in a cohort of 307 U.S. schoolchildren 12 years post-vaccination with MMR-II® [15]. Davidkin et al. reported similar findings in a longitudinal study of Finnish children 15 years after vaccination [16]. A comparative study of schoolchildren in Ohio noted significant differences in rubella seropositivity (67% vs 90%) and neutralizing antibody titer (63% vs 100%) between older (11–13 years of age) and younger (4–6 years of age) subjects who received a single dose of MMR-II® at 15 months of age, suggesting that antibody titers significantly decline with time since vaccination [17]. Rates of antibody decline following MMR-II® vaccination have also been recently reported [18]. While the observed rate of decline was much slower for rubella titers (2.6% per year) compared to that of measles (9.7% per year) and mumps (9.2% per year) [18], other reports have noted the rate for rubella to be as high as 8.2% per year [8] and suggest that additional biological variables may influence the duration of protective immunity. Waning immunity against mumps virus and measles virus has been investigated as the cause of several disease outbreaks in recent years [19–25], but similar studies investigating waning immunity to rubella are lacking.

The primary objective of our study was to assess the durability of rubella-specific humoral immune responses in a cohort of 98 subjects previously vaccinated with MMR-II®. Rubella-specific immune responses (total IgG titer, neutralizing antibody titer, and memory B cell ELISpot response) were measured in samples collected 7- and 17-years post-vaccination. To our knowledge, ours is the first study to investigate the durability of rubella-specific humoral immunity in such a comprehensive manner at such an extended timepoint from the last vaccination in order to evaluate the potential for waning immunity.

Methods

The methods described here are the same or similar to those in our previously published studies [26–32].

Human Subjects

Study participants (n=98) were selected from a cohort of 1,025 children and young adults (11–22 years of age) previously recruited from Olmsted County, MN, for a rubella vaccine study between 2001 and 2009 [21, 33]. All subjects had two documented doses of MMR-II® vaccine and completed a blood draw ~ 7 years post-vaccination as part of the original study (Draw 1). Subjects still living in the Olmsted County, MN, area were invited to complete an additional blood draw ~ 17 years post-vaccination as part of this study (Draw 2). Peripheral blood mononuclear cells (PBMCs) and serum samples were obtained from each subject and processed for cryopreservation using previously established protocols [34]. All study procedures were approved by the Mayo Clinic Institutional Review Board, and informed consent was provided by all study participants.

Rubella-specific IgG ELISA

Rubella-specific IgG antibody titers were measured using the Zeus Rubella IgG ELISA Test System (Zeus Scientific Inc.; Branchburg, NJ) according to the manufacturer’s instructions and as previously described [7, 35]. Briefly, serum samples were diluted (1:21) in the proprietary SAVe Diluent® and measured alongside the calibrator, positive control, and negative control provided in the kit. The optical density (OD) ratio was calculated for each sample by dividing the sample OD by the cutoff OD (i.e., the mean calibrator OD value multiplied by the correction factor provided with the kit). All measurements were converted into rubella-specific IgG antibody titer (in IU/mL) for final reporting according to the manufacturer’s instructions. The coefficient of variation (CV) for this assay in our laboratory was 2.6%.

Rubella Neutralizing Antibody Assay

Rubella-specific neutralizing antibody titer was measured using a high-throughput immune-colorimetric neutralization method (sICNA) as previously described [26–29, 35, 36]. The neutralization titer (NT50) was calculated as the reciprocal of the highest dilution of the serum at which the input virus signal was reduced by 50%. The reported intra-class correlation coefficient (ICC) for this assay (based on log-transformed estimates from repeated measurements) was 0.89, demonstrating a high degree of reproducibility [27].

Memory B Cell ELISpot Assay

Rubella-specific IgG memory B cells were quantified in PBMC samples using ELISpotPLUS human IgG kits (Mabtech Inc.; Cincinnati, OH) as previously described [32, 37, 38]. Briefly, PBMCs were thawed from cryogenic storage and incubated with the Toll-like receptor (TLR) agonist R848 in the presence of recombinant human IL-2 at 37°C for 72 h to provide non-specific B cell stimulation. Assay plate wells were coated with rubella virus antigen (20 μg/mL strain HPV77, Meridian Life Science Inc.; Memphis, TN), monoclonal antibody MT91/145 (1:30), or saline solution at 4°C overnight. Plates were rinsed with PBS and blocked with 2% milk for 2 h, and stimulated B cells were added and incubated at 37°C for 16 h. Assay plates were subsequently developed according to the manufacturer’s protocol, and spot-forming units (SFUs) were measured in quadruplicate for each condition using an Immunospot® S6 VERSA Analyzer (Cellular Technology Limited; Cleveland, OH). Antigen-specific memory B cell frequencies were reported as the total number of rubella-specific SFUs as well as the percentage of rubella-specific SFUs relative to the total IgG SFUs for each subject.

Statistical Analysis

The Wilcoxon signed-rank test was used to compare assay results between the first and second blood draws. Linear mixed-effect models were used to test the relationship between the time from last vaccination and the immune response assays. Subject was included in the models as a random intercept accounting for multiple measurements from each subject, and the time since last vaccination was included as the fixed-effect. The Wilcoxon rank-sum test was used to test for differences in assays between male and female subjects. Alpha levels for all statistical tests were set at 0.05. All analyses described were performed using RStudio.

Results

Subject demographics and clinical variables

Demographic and clinical variables for the study cohort are summarized in Table 1. The study cohort was comprised of 98 total subjects, and 56.1% of the cohort was female. The majority of the study subjects were Caucasian/Non-Hispanic with a median age of 15.21 months (IQR 15.05, 15.89) at the time of initial MMR vaccination and 7.67 years (IQR 5.03, 11.96) at the time of their second vaccine dose. The median age at recruitment (first blood draw) was 14.79 years, and the median age at the second blood draw was 24.23 years. The time elapsed between the last vaccination and first blood draw was 6.82 years (IQR 5.17, 8.67), while the time between the last vaccination and the second blood draw was 16.92 years (IQR 15.39, 18.43). Median time between blood draws was 9.42 years (IQR 7.63, 14.24). The sample size for some assays was reduced due to the limited availability of serum and PBMC samples for some subjects (see Table 2).

Table 1.

Study Cohort Demographics and Clinical Variables

Overall (n = 98)
Sex
 Female 55 (56.1%)
 Male 43 (43.9%)
Race
 White 97 (99.0%)
 Asian 1 (1.0%)
 Black or African American 0 (0.0%)
 American Indian or Alaskan Native 0 (0.0%)
 Native Hawaiian or Other Pacific Islander 0 (0.0%)
 Multiracial 0 (0.0%)
Ethnicity
 Non-Hispanic or Latino 97 (99.0%)
 Hispanic or Latino 1 (1.0%)
Age at First Vaccination (months)
 N 98
 Mean (SD) 15.88 (2.76)
 Median 15.21
 Q1,Q3 15.05, 15.89
 Range 11.99 – 36.04
Age at Second Vaccination (years)
 N 98
 Mean (SD) 8.30 (3.49)
 Median 7.67
 Q1,Q3 13.14, 17.21
 Range 11.48 – 19.13
Age at First Blood Draw (years)
 N 98
 Mean (SD) 15.13 (2.16)
 Median 14.79
 Q1,Q3 13.14, 17.21
 Range 11.48 – 19.13
Age at Second Blood Draw (years)
 N 98
 Mean (SD) 25.39 (4.22)
 Median 24.23
 Q1,Q3 21.87, 28.99
 Range 19.11 – 33.18
Time from Last Vaccination to First Blood Draw (years)
 N 98
 Mean (SD) 6.83 (2.52)
 Median 6.82
 Q1,Q3 5.17, 8.67
 Range 0.88 – 11.47
Time from Last Vaccination to Second Blood Draw (years)
 N 98
 Mean (SD) 17.09 (2.45)
 Median 16.92
 Q1,Q3 15.39, 18.43
 Range 11.83 – 25.60
Time between Blood Draws
 N 98
 Mean (SD) 10.25 (3.03)
 Median 9.42
 Q1,Q3 7.63, 14.24
 Range 6.92 – 14.65
Open in a new tab

Q1 and Q3 represent the first and third quartiles, respectively.

Table 2.

Comparison of Humoral Immune Response Variables in Healthy Young Adults over Time

Draw 1 (n = 96) Draw 2 (n = 96) Difference (n = 96) p-valuea
RV Serum Titer, (IU/mL) < 0.001
 N 89 96 89
 Mean (SD) 59.55 (45.31) 53.77 (49.46) −7.71 (30.35)
 Median 47.18 36.83 −6.68
 Q1,Q3 26.7, 79.81 21.68, 64.95 −22.26, 1.85
 Range 6.18 – 240.45 0.78 – 244.94 −77.1 – 121.24
RV Neutralizing Antibody Titer 0.018
 N 89 96 89
 Mean (SD) 68.59 (49.2) 62.39 (49.75) −6.17 (28.63)
 Median 53.11 47.5 −6.05
 Q1,Q3 35.13, 101.21 28.32, 86.35 −20.04, 6.52
 Range 11.09 – 283.77 3.98 – 348.65 −75.65 – 85.26
RV-specific SFU/2×105 cells 0.004
 N 44 96 44
 Mean (SD) 8.65 (9.21) 6.97 (7.61) −3.48 (7.16)
 Median 5.25 4.75 −2.50
 Q1,Q3 2.00, 11.50 2.00, 8.75 −5.63, 1.50
 Range −1.50 – 41.0 −2.00 – 36.50 −25.00 – 8.50
% RV-specific SFU/2×105 cells 0.447
 N 43 96 43
 Mean (SD) 2.03 (1.56) 2.26 (3.25) 0.08 (3.85)
 Median 1.81 1.62 −0.27
 Q1,Q3 0.93, 2.93 0.67, 3.72 −1.53, 0.64
 Range −0.90 – 6.99 −12.90 – 18.27 −13.85 – 14.97
Open in a new tab
a

Comparison between Draw 1 and Draw 2

Q1 and Q3 represent the first and third quartiles, respectively.

Durability of rubella-specific humoral immunity after vaccination

In order to assess waning humoral immunity against rubella, we comprehensively analyzed rubella-specific humoral immune response outcomes (e.g., total IgG titer, neutralizing antibody titer, and B cell ELISpot response) in serum and PBMC samples from our study cohort. Rubella-specific neutralizing antibody titers were measured in duplicate for paired serum samples from each subject. A significant decrease in median IgG antibody titer was observed between the first blood draw and second blood draw (47.18 IU/mL vs. 36.83 IU/mL, p < 0.001) (see Table 2 and Figs. 1 & 2), although the majority of subjects (97%) remained above the accepted threshold for positive serostatus (> 10 IU/mL) [5, 39]. This decline in antibody titer was significantly associated (p < 0.0001) with the time elapsed since the last vaccination (see Fig. 3). Notably, antibody titers for three subjects had declined below 10 IU/mL by the second blood draw, while titers for an additional three subjects had significantly increased to > 200 IU/mL. We observed a similar decrease in median rubella-specific neutralizing antibody titer between the first blood draw and the second blood draw (53.11 vs. 47.49, p = 0.018) that was also significantly associated with time since the last vaccine dose (p = 0.002) (see Table 2 and Figs. 1 – 3).

Figure 1.

Figure 1.

Open in a new tab

Comparison of rubella-specific humoral immune response outcomes between blood draws. A) RV-specific serum titers (log2 scale) exhibited a statistically significant decrease (p < 0.001) between subjects at Draw 1 (n = 89) and Draw 2 (n = 96). B) Comparison of RV-specific neutralizing antibody titer (log2 scale) showed a statistically significant decrease (p = 0.018) between subjects at Draw 1 (n = 89) and Draw 2 (n = 96). C) Memory B cell ELISpot responses against RV exhibited a statistically significant decrease (p = 0.004) between subjects at Draw 1 (n = 44) and Draw 2 (n = 96). D) Memory B cell ELISpot responses represented as a percentage of total IgG-secreting B cells between subjects at Draw 1 (n = 43) and Draw 2 (n = 96). No statistically significant difference (p = 0.447) was observed between time points.

Figure 2.

Figure 2.

Open in a new tab

Pairwise analysis of rubella-specific humoral immune response outcomes between blood draws. Changes in serum antibody titer (A), neutralizing antibody titer (B), memory B cell ELISpot response (C), and the percentage of RV-specific memory B cells (D) are shown for paired samples between Draw 1 and Draw 2. Each data point is representative of an individual subject.

Figure 3.

Figure 3.

Open in a new tab

Correlations of rubella-specific humoral immune response outcomes with time elapsed since last vaccination. Each data point is representative of an individual subject. Declines in serum antibody titer (A, p < 0.001), neutralizing antibody titer (B, p = 0.002), and memory B cell ELISpot response (C, p = 0.009) were all significantly associated with time since the last vaccine dose. No significant association with time was observed for the percentage of RV-specific memory B cells (D, p = 0.45).

In addition to rubella-specific antibody titers, our study also assessed rubella-specific B cell ELISpot responses over time. B cell ELISpot responses were measured in paired PBMC samples from each subject. RV-specific B cell ELISpot responses declined significantly between the first and second blood draw (5.25 SFUs/2×105 cells vs. 4.75 SFUs/2×105 cells, p = 0.004), and the decline in ELISpot response was significantly associated with time elapsed since vaccination (p = 0.009) (see Table 2 and Figs. 1 – 3). Notably, the percentage of RV-specific SFUs/2×105 cells remained largely unchanged (1.8% vs. 1.6%, p = 0.45), as the decline in RV-specific response was proportional with a decline in overall B cell ELISpot response (see Table 2 and Figs. 1 & 2). Three subjects exhibited significant changes in the percentage of RV-specific B cell response (Figs. 1 & 2), which were attributed to repeatedly low measurements of total IgG-secreting B cells that skewed the representative percentages.

Associations of rubella-specific humoral immunity with biological sex

As our cohort was comprised of both male and female subjects, we sought to analyze sex-based differences in rubella-specific humoral immunity. No significant differences were observed in rubella-specific total IgG (Draw 1: p = 0.92, Draw 2: p = 0.5) or neutralizing antibody titers (Draw 1: p = 0.28, Draw 2: p = 0.12) at either timepoint based on sex (see Table 3 and Fig. 4). Female subjects had higher average RV-specific B cell ELISpot responses compared to male subjects at both timepoints (see Table 3 and Fig. 4). Male subjects exhibited lower average B cell ELISpot responses than female subjects at the first blood draw (10.11 SFUs/2×105 cells vs. 7.18 SFUs/2×105 cells), but this difference was not statistically significant (p = 0.3). In contrast, RV-specific B cell ELISpot responses were significantly lower for male subjects by the second blood draw compared to female subjects (8.98 SFUs/2×105 cells vs. 4.24 SFUs/2×105 cells, p = 0.002). The overall percentage of RV-specific memory B cells was similar between male and female subjects at both timepoints (Draw 1: p = 0.6, Draw 2: p = 0.21) (see Table 3 and Fig. 4).

Table 3.

Sex-based Comparisons of Humoral Immune Response Variables over Time

Female (n = 55) Male (n = 42) Total (n = 97) p-valueb
RV Serum Titer (IU/mL)
 Draw 1 0.922
  N 52 38 90
  Mean (SD) 44.94 (2.22) 45.89 (2.06) 45.25 (2.14)
  Median 47.84 40.79 47.5
  Q1,Q3 24.76, 77.71 29.24, 88.65 26.72, 79.89
  Range 6.19 – 240.52 12.64 – 150.12 6.19 – 240.52
 Draw 2 0.502
  N 55 42 97
  Mean (SD) 35.51 (2.64) 40.22 (2.28) 37.53 (2.48)
  Median 37.27 38.59 37.27
  Q1,Q3 21.86, 65.34 22.94, 61.82 22.32, 64.89
  Range −1.28 – 245.57 7.16 – 235.57 −1.28 – 245.57
RV Neutralizing Antibody Titer
 Draw 1 0.282
  N 52 38 90
  Mean (SD) 49.52 (2.19) 58.89 (1.89) 53.45 (2.07)
  Median 52.71 53.82 53.08
  Q1,Q3 24.76, 93.05 37.53, 106.89 34.06, 99.73
  Range 11.08 – 284.05 13.93 – 218.27 11.08 – 284.05
 Draw 2 0.122
  N 55 42 97
  Mean (SD) 42.22 (2.36) 54.19 (1.93) 46.85 (2.19)
  Median 47.5 47.5 47.5
  Q1, Q3 23.75, 74.54 34.78, 89.26 28.64, 85.63
  Range 3.97 – 349.71 15.56 – 176.07 3.97 – 349.71
RV-specific SFU/2×105 cells
 Draw 1 0.297
  N 22 22 44
  Mean (SD) 10.11 (11.04) 7.18 (6.88) 8.65 (9.21)
  Median 5.50 5.25 5.25
  Q1,Q3 2.00, 15.38 3.25, 8.38 2.00, 11.50
  Range 0.00 – 41.00 −1.50 – 29.50 −1.50 – 41.00
 Draw 2 0.002
  N 55 42 97
  Mean (SD) 8.98 (8.76) 4.24 (4.51) 6.93 (7.58)
  Median 5.00 3.00 4.50
  Q1,Q3 3.25, 13.00 1.00, 5.88 2.00, 8.50
  Range −1.00 – 36.50 −2.00 – 16.50 −2.00 – 36.50
% RV-specific SFU/2×105 cells
 Draw 1 0.595
  N 21 22 43
  Mean (SD) 1.90 (1.78) 2.15 (1.36) 2.03 (1.56)
  Median 1.28 2.11 1.81
  Q1,Q3 0.52, 3.29 1.37, 2.52 0.93, 2.93
  Range 0.00 – 6.99 −0.90 – 5.89 −0.90 – 6.99
 Draw 2 0.207
  N 55 42 97
  Mean (SD) 2.62 (3.19) 1.78 (3.28) 2.26 (3.24)
  Median 1.58 1.79 1.64
  Q1,Q3 0.94, 4.02 0.56, 2.56 0.67, 3.70
  Range −4.35 – 18.27 −12.90 – 12.00 −12.90 – 18.27
Open in a new tab
b

Comparison between males and females at each blood draw

Q1 and Q3 represent the first and third quartiles, respectively.

Figure 4.

Figure 4.

Open in a new tab

Sex-based comparison of rubella-specific humoral immune response outcomes. No statistically significant differences based on sex were observed in A) RV-specific serum IgG titers (log2 scale) at either Draw 1 (p = 0.922) or Draw 2 (p = 0.502); or B) RV-specific neutralizing antibody titers (log2 scale) at either Draw 1 (p = 0.282) or Draw 2 (p = 0.122). A statistically significant difference based on sex was observed for RV-specific memory B cell ELISpot response (C) at Draw 2 (p = 0.002) but not Draw 1 (p = 0.297). No statistically significant difference was observed in the proportion of memory B cells (D) at either Draw 1 (p = 0.595) or Draw 2 (p = 0.207) based on sex.

Discussion

Waning immunity has been associated with outbreaks of mumps in recent years [19, 22–24], and this has led to concerns over the durability of vaccine-induced immune responses against other diseases, including components of the MMR vaccine. Although vaccination against RV has been found to be safe and highly effective at preventing clinical disease, an increasing number of studies have shown that waning immunity against rubella may be more prevalent than previously reported [8, 15–18]. In light of these reports, our study sought to assess the durability of rubella-specific humoral immune responses (e.g., total IgG titer, neutralizing antibody titer, and memory B cell ELISpot) among a population of healthy, young adults who had previously received two documented doses of MMR vaccine.

Studies investigating the prevalence of RV-specific antibodies at the population level have reported high rates of seropositivity (90–100%) among immunized populations [7–13], and similar levels of RV-specific antibodies (95%) were observed in our cohort, although we did observe a statistically significant decrease in total IgG titers during the ~ 10-year period between blood draws. Rubella-specific IgG titers have been shown to decline at a moderate rate (2.6% per year) in the years following vaccination [18], and this is consistent with the 2.2% per year decrease observed in our cohort (47.18 IU/mL vs. 36.83 IU/mL, Figs. 1 & 2). Notably, the antibody titers for three subjects did wane below the currently accepted protective threshold (10 IU/mL) by the time of the second blood draw. Although RV-specific antibody titers may wane in the years following vaccination, current surveillance studies have found no evidence that waning immunity leads to an increased risk of disease or CRS [40], nor have outbreaks of rubella been documented in those who have received two doses of MMR vaccine. Still, our data suggest that a small portion of the population may become susceptible to RV infection even after successful vaccination, highlighting the importance of continued surveillance studies.

We also observed a statistically significant decrease in neutralizing antibody titer in our cohort (53.11 NT50 vs. 47.49 NT50, Figs. 1 & 2) that was similar to the observed decrease in total IgG titer. Neutralizing antibody titer is widely regarded as a more informative metric of protection against viral infection than total IgG titer, as it directly reports on the biological activity of virus-specific antibodies; however, as there is no established correlate of protection for RV based on neutralization assays, the clinical relevance of this data remains unclear. LeBaron et al. previously reported a significant decline in RV-specific neutralizing antibody titer in an adolescent cohort over a 12-year period following MMR vaccination, with 10% of the cohort found to be seronegative and 43% to have the lowest detectable antibody titer (the NT50 was 10) [15]. A similar study by Johnson et al. reported seropositivity by NT50 to decrease from 100% to 63% in a comparative pediatric study, suggestive of significant waning immunity against RV [17]. In contrast to these studies, our findings suggest that RV-specific neutralizing antibody titers wane at a more moderate rate in the years following vaccination, as they are much higher than the presumed correlate of protection (8 NT50) for RV [41].

Our study also evaluated sex-based differences in the durability of humoral immune responses to rubella virus, as biological sex has been shown to influence adaptive immune response outcomes following vaccination [42, 43]. Although our statistical power was limited, total IgG titer and NT50 were statistically equivalent (total IgG: Draw1 p = 0.922, Draw 2 p = 0.502; NT50: Draw 1 p = 0.28, Draw 2 p = 0.12) between male and female subjects, respectively, and exhibited similar levels of decline between timepoints. Notably, B cell ELISpot responses were significantly higher on average in female subjects compared to male subjects at the second timepoint (8.98 SFUs vs. 4.24 SFUs, Fig. 4), with a greater decline in B cell response between timepoints observed for male subjects. This is consistent with our prior report of higher B cell ELISpot responses in older adult females following seasonal influenza vaccination [43]. The decline in RV-specific memory B cell responses was also observed for the combined cohort (8.65 SFUs vs. 6.97 SFUs, Figs. 1 & 2). Notably, no significant changes were observed in the percentage of RV-specific memory B cells between blood draws (1.8% vs. 1.6%, p = 0.45), suggesting that the total memory B cell population undergoes a proportional decline in both genders over time.

The results of our study suggest that RV-specific humoral immune responses wane with time after vaccination, but they do so at a moderate rate. Average IgG antibody titers for the study cohort remained above the threshold for protective immunity, although a statistically significant decline in total IgG titer was observed. In the absence of wild-virus circulation and subclinical boosting, waning immunity could place a small subset of the population at risk for RV infection, although current reports of rubella and CRS surveillance suggest no increased risk of disease [40]. The decline in RV-specific memory B cell responses—particularly among male subjects—is concerning, as a deficiency of both circulating antibodies and circulating memory B cells could severely compromise long-term protection against RV. The persistence of circulating antibody against RV in the years after vaccination is likely due to the induction of robust, long-lived plasma cells that establish residence in the bone marrow [44–47]. While we did not measure bone marrow-resident B cells in this study, this may represent an intriguing area of future research for understanding the cellular and molecular mechanisms governing the durability of vaccine-induced immune responses. In the absence of an established correlate of protection for NT50, it is difficult to interpret the clinical significance of the decline in neutralizing antibody titers observed in our cohort. Both of these areas warrant further study in order to fully define their association with RV-specific protective immunity.

Our study was limited by several factors. The study population was primarily Caucasian (97%), which is not representative of the overall U.S. population. Due to the initial recruitment of this cohort as part of another study, there was limited availability of serum and PBMC samples, which led to reduced sample size for some assays and a subsequent loss of statistical power. As these subjects were initially recruited several years after vaccination, we also did not have an opportunity to collect samples prior to or immediately after immunization to better assess early rates of waning of the immune response.

Despite these limitations, our study has several strengths. To our knowledge, ours is the first study to perform such a comprehensive evaluation of humoral immune response outcomes (i.e. IgG titer, NT50, B cell ELISpot) to assess waning immunity against RV in the years following vaccination. Furthermore, our study evaluated waning immunity to RV at one of the longest time points (~17 years) post-vaccination. Our cohort was also well-balanced with respect to biological sex (56.1% female vs. 43.9% male), allowing us to assess sex-based differences in waning immunity to rubella.

Collectively, our study provides further evidence that, while rubella-specific humoral immunity wanes over time, protective immunity is maintained among the majority of the population. The current correlate of protection for rubella is solely defined by RV-specific antibody titer and does not account for other metrics that contribute to humoral immunity (e.g., neutralizing antibody activity, memory B cell frequency). Additional comprehensive assessments of humoral immunity against rubella should be initiated to broadly define levels of protection against disease and to more clearly understand the mechanisms governing durable immunity following vaccination.

Acknowledgments

The authors would like to thank Caroline L. Vitse for her editorial assistance in preparing the manuscript, and Megan O’Byrne and Diane Grill for their contributions to statistical analysis. The work presented here was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award R37AI048793. Additional financial support was provided by funding to RBK through the Merck Investigator Studies Program. The content of this manuscript is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health or Merck Research Laboratories. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the United States Centers for Disease Control and Prevention.

Conflicts of Interest

Dr. Poland is the chair of a Safety Evaluation Committee for novel investigational vaccine trials being conducted by Merck Research Laboratories. Dr. Poland offers consultative advice on vaccine development to Merck & Co., Medicago, GlaxoSmithKline, Sanofi Pasteur, Emergent Biosolutions, Dynavax, Genentech, Eli Lilly and Company, Janssen Global Services LLC, Kentucky Bioprocessing, AstraZeneca, and Genevant Sciences, Inc. Drs. Poland and Ovsyannikova hold three patents related to measles and vaccinia peptide research. Dr. Kennedy holds a patent on vaccinia peptide research. Dr. Kennedy has received funding from Merck Research Laboratories to study waning immunity to measles and mumps after immunization with the MMR-II® vaccine. Drs. Poland, Kennedy, and Ovsyannikova have received grant funding from ICW Ventures for preclinical studies on a peptide-based COVID-19 vaccine. All other authors declare no competing financial interests. These activities have been reviewed by the Mayo Clinic Conflict of Interest Review Board and are conducted in compliance with Mayo Clinic Conflict of Interest policies. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and was conducted in compliance with Mayo Clinic Conflict of Interest policies.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • [1].Banatvala JE, Brown DW. Rubella. Lancet. 2004;363:1127–37. [DOI] [PubMed] [Google Scholar]
  • [2].Vaccinology: An Essential Guide. 1 ed: Wiley-Blackwell; 2015. [Google Scholar]
  • [3].Sydnor E, Perl TM. Healthcare providers as sources of vaccine-preventable diseases. Vaccine. 2014;32:4814–22. [DOI] [PubMed] [Google Scholar]
  • [4].Plotkin SA. Rubella eradication. Vaccine. 2001;19:3311–9. [DOI] [PubMed] [Google Scholar]
  • [5].Lambert N, Strebel P, Orenstein W, Icenogle J, Poland GA. Rubella. Lancet. 2015;385:2297–307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Miller E, Cradock-Watson JE, Pollock TM. Consequences of confirmed maternal rubella at successive stages of pregnancy. Lancet. 1982;2:781–4. [DOI] [PubMed] [Google Scholar]
  • [7].Crooke SN, Haralambieva IH, Grill DE, Ovsyannikova IG, Kennedy RB, Poland GA. Seroprevalence and durability of rubella virus antibodies in a highly immunized population. Vaccine. 2019;37:3876–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Davidkin I, Jokinen S, Broman M, Leinikki P, Peltola H. Persistence of measles, mumps, and rubella antibodies in an MMR-vaccinated cohort: a 20-year follow-up. J Infect Dis. 2008;197:950–6. [DOI] [PubMed] [Google Scholar]
  • [9].Smits G, Mollema L, Hahne S, de Melker H, Tcherniaeva I, van der Klis F, et al. Seroprevalence of rubella antibodies in The Netherlands after 32 years of high vaccination coverage. Vaccine. 2014;32:1890–5. [DOI] [PubMed] [Google Scholar]
  • [10].Barlinn R, Vainio K, Samdal HH, Nordbo SA, Nokleby H, Dudman SG. Susceptibility to cytomegalovirus, parvovirus B19 and age-dependent differences in levels of rubella antibodies among pregnant women. J Med Virol. 2014;86:820–6. [DOI] [PubMed] [Google Scholar]
  • [11].Alsibiani SA. Rubella Immunity among Pregnant Women in Jeddah, Western Region of Saudi Arabia. Obstet Gynecol Int. 2014;2014:659838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Byrne L, Brant L, Reynolds C, Ramsay M. Seroprevalence of low rubella IgG antibody levels among antenatal women in England tested by NHS Blood and Transplant: 2004-2009. Is rubella susceptibility increasing? Vaccine. 2012;30:161–7. [DOI] [PubMed] [Google Scholar]
  • [13].Almuneef MA, Memish ZA, Balkhy HH, Otaibi B, Helmi M. Seroprevalence survey of varicella, measles, rubella, and hepatitis A and B viruses in a multinational healthcare workforce in Saudi Arabia. Infect Control Hospital Epidemiol. 2006;27:1178–83. [DOI] [PubMed] [Google Scholar]
  • [14].Okafuji T, Okafuji T, Nakayama T. Persistence of Immunity Acquired after a Single Dose of Rubella Vaccine in Japan. Jpn J Infect Dis. 2016;69:221–3. [DOI] [PubMed] [Google Scholar]
  • [15].LeBaron CW, Forghani B, Matter L, Reef SE, Beck C, Bi D, et al. Persistence of rubella antibodies after 2 doses of measles-mumps-rubella vaccine. J Infect Dis. 2009;200:888–99. [DOI] [PubMed] [Google Scholar]
  • [16].Davidkin I, Peltola H, Leinikki P, Valle M. Duration of rubella immunity induced by two-dose measles, mumps and rubella (MMR) vaccination. A 15-year follow-up in Finland. Vaccine. 2000;18:3106–12. [DOI] [PubMed] [Google Scholar]
  • [17].Johnson CE, Kumar ML, Whitwell JK, Staehle BO, Rome LP, Dinakar C, et al. Antibody persistence after primary measles-mumps-rubella vaccine and response to a second dose given at four to six vs. eleven to thirteen years. Pediatr Infect Dis J. 1996;15:687–92. [DOI] [PubMed] [Google Scholar]
  • [18].Seagle EE, Bednarczyk RA, Hill T, Fiebelkorn AP, Hickman CJ, Icenogle JP, et al. Measles, mumps, and rubella antibody patterns of persistence and rate of decline following the second dose of the MMR vaccine. Vaccine. 2018;36:818–826. [DOI] [PubMed] [Google Scholar]
  • [19].Rasheed MAU, Hickman CJ, McGrew M, Sowers SB, Mercader S, Hopkins A, et al. Decreased humoral immunity to mumps in young adults immunized with MMR vaccine in childhood. Proc Natl Acad Sci USA. 2019;116:19071–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Bitzegeio J, Majowicz S, Matysiak-Klose D, Sagebiel D, Werber D. Estimating age-specific vaccine effectiveness using data from a large measles outbreak in Berlin, Germany, 2014/15: evidence for waning immunity. Euro Surveill. 2019;24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Kennedy RB, Ovsyannikova IG, Thomas A, Larrabee BR, Rubin S, Poland GA. Differential durability of immune responses to measles and mumps following MMR vaccination. Vaccine. 2019;37:1775–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Smetana J, Chlibek R, Hanovcova I, Sosovickova R, Smetanova L, Polcarova P, et al. Serological survey of mumps antibodies in adults in the Czech Republic and the need for changes to the vaccination strategy. Hum Vaccin Immunother. 2018;14:887–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Hamami D, Cameron R, Pollock KG, Shankland C. Waning Immunity Is Associated with Periodic Large Outbreaks of Mumps: A Mathematical Modeling Study of Scottish Data. Front Physiol. 2017;8:233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Lewnard JA, Grad YH. Vaccine waning and mumps re-emergence in the United States. Sci Transl Med. 2018;10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].He H, Chen EF, Li Q, Wang Z, Yan R, Fu J, et al. Waning immunity to measles in young adults and booster effects of revaccination in secondary school students. Vaccine. 2013;31:533–7. [DOI] [PubMed] [Google Scholar]
  • [26].McLean HQ, Fiebelkorn AP, Ogee-Nwankwo A, Hao L, Coleman LA, Adebayo A, et al. Rubella virus neutralizing antibody response after a third dose of measles-mumps-rubella vaccine in young adults. Vaccine. 2018;36:5732–7. [DOI] [PubMed] [Google Scholar]
  • [27].Haralambieva IH, Salk HM, Lambert ND, Ovsyannikova IG, Kennedy RB, Warner ND, et al. Associations between race, sex and immune response variations to rubella vaccination in two independent cohorts. Vaccine. 2014;32:1946–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Lambert ND, Pankratz VS, Larrabee BR, Ogee-Nwankwo A, Chen MH, Icenogle JP, et al. High-throughput Assay Optimization and Statistical Interpolation of Rubella-Specific Neutralizing Antibody Titers. Clin Vaccine Immunol. 2014;21:340–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Lambert ND, Haralambieva IH, Ovsyannikova IG, Larrabee BR, Pankratz VS, Poland GA. Characterization of humoral and cellular immunity to rubella vaccine in four distinct cohorts. Immunol Res. 2013;58:1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Lambert ND, Haralambieva IH, Kennedy RB, Ovsyannikova IG, Pankrantz VS, Poland GA. Polymorphisms in HLA-DPB1 are associated with differences in rubella-specific humoral immunity after vaccination. J Infect Dis. 2015;211:898–905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Haralambieva IH, Lambert ND, Ovsyannikova IG, Kennedy RB, Larrabee BR, Pankratz VS, et al. Associations between single nucleotide polymorphisms in cellular viral receptors and attachment factor-related genes and humoral immunity to rubella vaccination. PloS One. 2014;9:e99997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Haralambieva IH, Ovsyannikova IG, Kennedy RB, Zimmermann MT, Grill DE, Oberg AL, et al. Transcriptional signatures of influenza A/H1N1-specific IgG memory-like B cell response in older individuals. Vaccine. 2016;34:3993–4002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Ovsyannikova IG, Salk HM, Larrabee BR, Pankratz VS, Poland GA. Single nucleotide polymorphisms/haplotypes associated with multiple rubella-specific immune response outcomes post-MMR immunization in healthy children. Immunogenetics. 2015;67:547–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Umlauf BJ, Pinsky NA, Ovsyannikova IG, Poland GA. Detection of vaccinia virus-specific IFNgamma and IL-10 secretion from human PBMCs and CD8(+) T cells by ELISPOT. Methods Mol Biol. 2012;792:199–218. [DOI] [PubMed] [Google Scholar]
  • [35].Haralambieva IH, Ovsyannikova IG, Kennedy RB, Goergen KM, Grill DE, Chen MH, et al. Rubella virus-specific humoral immune responses and their interrelationships before and after a third dose of measles-mumps-rubella vaccine in women of childbearing age. Vaccine. 2020;38:1249–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Chen MH, Zhu Z, Zhang Y, Favors S, Xu WB, Featherstone DA, et al. An indirect immunocolorimetric assay to detect rubella virus infected cells. J Virol Methods. 2007;146:414–8. [DOI] [PubMed] [Google Scholar]
  • [37].Haralambieva IH, Ovsyannikova IG, Kennedy RB, Poland GA. Detection and Quantification of Influenza A/H1N1 Virus-Specific Memory B Cells in Human PBMCs Using ELISpot Assay. Methods Mol Biol. 2018;1808:221–36. [DOI] [PubMed] [Google Scholar]
  • [38].Latner DR, McGrew M, Williams N, Lowe L, Werman R, Warnock E, et al. Enzyme-linked immunospot assay detection of mumps-specific antibody-secreting B cells as an alternative method of laboratory diagnosis. Clin Vaccine Immunol. 2011;18:35–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Skendzel LP. Rubella immunity. Defining the level of protective antibody. Am J Clin Pathol. 1996;106:170–4. [DOI] [PubMed] [Google Scholar]
  • [40].McLean HQ, Fiebelkorn AP, Temte JL, Wallace GS. Prevention of measles, rubella, congenital rubella syndrome, and mumps, 2013: summary recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. 2013;62:1–34. [PubMed] [Google Scholar]
  • [41].Plotkin SA. Correlates of protection induced by vaccination. ClinVaccine Immunol. 2010; 17:1055–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Klein SL, Flanagan KL. Sex differences in immune responses. Nat Rev Immunol. 2016;16:626–38. [DOI] [PubMed] [Google Scholar]
  • [43].Voigt EA, Ovsyannikova IG, Kennedy RB, Grill DE, Goergen KM, Schaid DJ, et al. Sex Differences in Older Adults' Immune Responses to Seasonal Influenza Vaccination. Front Immunol. 2019;10:180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [44].Andraud M, Lejeune O, Musoro JZ, Ogunjimi B, Beutels P, Hens N. Living on three time scales: the dynamics of plasma cell and antibody populations illustrated for hepatitis a virus. PLoS Comput Biol. 2012;8:e1002418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Brynjolfsson SF, Persson Berg L, Olsen Ekerhult T, Rimkute I, Wick MJ, Martensson IL, et al. Long-Lived Plasma Cells in Mice and Men. Front Immunol. 2018;9:2673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [46].Lightman SM, Utley A, Lee KP. Survival of Long-Lived Plasma Cells (LLPC): Piecing Together the Puzzle. Front Immunol. 2019;10:965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [47].Hammarlund E, Thomas A, Amanna IJ, Holden LA, Slayden OD, Park B, et al. Plasma cell survival in the absence of B cell memory. Nat Commun. 2017;8:1781. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES