Safety and Immunogenicity of a Revaccination With a Respiratory Syncytial Virus Prefusion F Vaccine in Older Adults: A Phase 2b Study

Abstract Background In the previous (parent) study, 2 doses of different formulations of an investigational vaccine against respiratory syncytial virus (RSVPreF3 OA) were well tolerated and immunogenic in older adults. This multicenter phase 2b extension study assessed safety and immunogenicity of a revaccination (third) dose of the 120 μg RSVPreF3-AS01E formulation. Methods In total, 122 older adults (60–80 years), previously vaccinated with 2 doses of RSVPreF3-AS01E formulations (containing 30, 60, or 120 μg RSVPreF3 antigen), received an additional 120 μg RSVPreF3-AS01E dose 18 months after dose 2. Vaccine safety was evaluated in all participants up to 6 months and immunogenicity in participants who received 120 μg RSVPreF3-AS01E doses until 1 month after dose 3. Results Similar to the parent study, mostly mild-to-moderate solicited adverse events and no vaccine-related serious adverse events or potential immune-mediated disorders were reported. Neutralizing titers and cell-mediated immune responses persisted for 18 months after 2-dose vaccination. Dose 3 increased RSV-specific neutralizing titers against RSV-A and RSV-B and median CD4+ T-cell frequencies. After dose 3, RSV-specific neutralizing titers but not CD4+ T-cell frequencies were below levels detected 1 month after dose 1. Conclusions Revaccination with 120 μg RSVPreF3-AS01E 18 months after dose 2 is well tolerated and immunogenic in older adults. Clinical Trials Registration NCT04657198; EudraCT, 2020-000692-21.

Respiratory syncytial virus (RSV) is a contagious seasonal virus causing respiratory tract infections in people of all ages [1,2].There are 2 main antigenic subtypes, RSV-A and RSV-B [1].The subtypes are cocirculating with alternating predominance across seasons, with a varying pattern [1,2].
RSV infections usually resolve without complications or sequelae in immune-competent persons [3].However, in older adults (OAs) aged ≥60 years, RSV can cause more serious respiratory illnesses (including lower respiratory tract disease) [3], especially in people with underlying medical conditions or those who are immunocompromised [4,5].In OAs, RSV infections thus lead to a significant disease burden [6], which was underestimated for a long time [7][8][9].According to a recent systematic review of data from high-income countries, the calculated pooled estimates of RSV acute respiratory infections in OAs aged ≥60 years were 1.62% (95% confidence interval [CI], .84%-3.08%) for attack rate, 0.15% (95% CI, .09%-.22%) for hospitalization rate, and 7.13% (95% CI, 5.40%-9.36%)for inhospital case fatality rate [8].Based on the described values and using the 2019 census data, the same review estimated that about 5 million cases of acute respiratory tract infection, half a million hospitalizations, and 33 000 in-hospital deaths of OAs could be attributed to RSV in 2019 [8].
The severity of RSV-associated disease in OAs has been ascribed to waning humoral and cellular immune responses (immunosenescence) that were induced by previous RSV infections [10][11][12][13][14][15].A protective immune response against RSV is orchestrated by antibodies (eg, immunoglobulin A [IgA] and IgG, neutralizing antibodies [nAb]), and lymphocytes (both cluster-of-differentiation-4-expressing [CD4 + ] and CD8 + T cells) that produce a variety of cytokines such as interleukins (ILs) and interferons (IFNs), resulting in viral clearance and protection [14].The RSV-specific immunity obtained after infection The Journal of Infectious Diseases is not long lasting and, even though most people have some level of postinfection immunity, this does not prevent subsequent RSV infections.Due to a higher disease burden in the vulnerable OA population, the waning immune responses lead to an increased risk for more severe disease in OAs.Thus, approaches to overcome waning immunity (eg, vaccination) can help avoid serious RSV-associated disease in OAs [16].
Several vaccines based on the prefusion conformation of RSV fusion protein (PreF) and using different delivery systems were recently evaluated in clinical studies [17][18][19][20][21][22][23][24][25][26].The RSV vaccine investigated in this study is based on PreF stabilized in its trimeric conformation (RSVPreF3) as the main antigen, and adjuvanted with AS01 E [18,27].In a previous phase 1/2 study (hereafter referred to as the parent study), different formulations of the RSVPreF3-based vaccine were administered 2 months apart to OAs aged 60-80 years [18].The vaccine formulation containing 120 µg of RSVPreF3 and adjuvanted with AS01 E (hereafter referred to as 120 μg RSVPreF3-AS01 E or RSVPreF3 OA) was selected for further clinical development, because it most potently induced humoral and cellular RSV-specific immune responses while retaining an acceptable safety profile in the parent study [18].An ongoing vaccine efficacy study demonstrated a consistently high efficacy (point estimate of 82.6% with a 96.95% CI, 57.9%-94.1%)for the 120 μg RSVPreF3-AS01 E formulation against RSV-related lower respiratory tract disease in OAs aged ≥60 years, thus meeting the primary study end point [27,28].The 120 μg RSVPreF3-AS01 E formulation has then been licensed for use in OAs in the United States and European Union [29].

METHODS
This phase 2b extension study (NCT04657198) was conducted in accordance with the Declaration of Helsinki and the International Council for Harmonization requirements.The study was approved by institutional ethics committees.The participating OAs were enrolled at 7 centers in the United States, and 3 centers in Belgium.The study was open label as all participants received the same 120 μg RSVPreF3-AS01 E vaccine formulation.

Study Vaccine
The RSVPreF3 vaccine formulations have been described in detail [18].In this extension study, only the 120 μg RSVPreF3-AS01 E formulation was administered as the third dose.

Study Participants and Procedures
Eligible participants were healthy men and women who had received the 30-, 60-, or 120 μg RSVPreF3-AS01 E formulation in the parent study [18].Participants needed to be able and willing to comply with protocol requirements (as determined by investigator), and to have provided written informed consent prior to any study-specific procedures.Deviations from inclusion criteria were not allowed.Inclusion and exclusion criteria are listed in the Supplementary Material.
In the parent study [18], the OA participants were randomized to receive 2 doses of a given vaccine formulation or placebo on day 1 (also denoted as M0 time point) and day 61 (M2) (Figure 1).The follow-up period for OAs was up to 1 year after the second vaccination (M14).In this extension study, all OA recipients of the AS01 E -adjuvanted formulations were invited to receive the third vaccine dose containing the 120 μg RSVPreF3-AS01 E formulation at M20. Follow-up time was 6 months after dose 3 (M26).All vaccines were administered intramuscularly, into the deltoid region of the nondominant arm.
Because the present manuscript refers to findings of both the parent and extension studies, the timeline details are provided here for ease of reference.Time points M0-M14 refer to the parent study and included M0 (day 1, baseline, dose 1 administration), M1 (day 31, 1 month after dose 1), M2 (day 61, dose 2 administration), M3 (day 91, 1 month after dose 2), M8, and M14.This extension study includes time points M20 (dose 3 administration), M21 (1 month after dose 3), and M26 (end of study, 6 months after dose 3) (Figure 1).
Occurrence of adverse events (AEs) was recorded for all participants in the following periods: 4 days after dose 3 for solicited AEs (administration site [pain, redness, swelling] and systemic [fever]) AEs, 30 days after dose 3 for unsolicited AEs, and up to 6 months after dose 3 for AEs leading to study withdrawal, serious AEs (SAEs) and potential immunemediated disorders (pIMDs).
Blood samples for evaluation of humoral (approximately 20 mL) and cell-mediated immune (CMI) (approximately 25 mL) responses were collected at M20 and M21 time points only from participants in the 120/120 μg RSVPreF3-AS01 E group.Neutralizing titers against RSV-A and RSV-B were measured by neutralization assays, and RSVPreF3-specific IgG concentrations were determined using an in-house enzyme-linked immunosorbent assay (ELISA) [18].Frequencies of RSVPreF3-specific CD4 + and CD8 + T cells were evaluated using intracellular cytokine staining on peripheral blood mononuclear cells [18].

Study Objectives and End Points
Primary safety objectives were to evaluate the vaccine safety and reactogenicity in all participants up to 1 month after dose 3 in terms of occurrence of solicited AEs up to 4 days, and unsolicited AEs, SAEs, and pIMDs up to 30 days after dose 3. The primary immunogenicity objective was to evaluate humoral immune responses in the 120/120 μg RSVPreF3-AS01 E group of participants in terms of neutralizing titers against RSV-A and RSV-B up to 1 month after dose 3 (M21).
The secondary safety objective was to determine the safety of dose 3 in all participants until study end (ie, 6 months after dose 3) in terms of occurrence of SAEs and pIMDs.The secondary immunogenicity objective was to evaluate the humoral response in terms of RSVPreF3-specific IgG concentration and CMI response in terms of frequency of RSVPreF3-specific CD4 + T cells expressing at least 2 markers (among IL-2, CD40 ligand [CD40L], tumor necrosis factor-α [TNF-α] and IFN-γ) in the 120/120 μg RSVPreF3-AS01 E group of participants up to M21.Tertiary study objectives and end points are described in the Supplementary Material.

Statistical Analyses
Sample size for the parent study was previously presented in detail [18], and no additional estimations were done for this extension study.Analysis sets included enrolled set (participants who provided their informed consent to participate in the study), exposed set (participants who received dose 3), and per-protocol set (participants who received dose 3 with available immunogenicity data and without important protocol deviations including those leading to study exclusion [see Supplementary Material]).Safety was assessed on the exposed set, while immunogenicity was evaluated on the per-protocol set.
All data were analyzed using descriptive statistics.Categorical data were tabulated as the number and percentage of participants, while continuous data were described/plotted as mean with 95% CI or median with range (minimum and maximum).The geometric mean titers/concentrations (GMTs/GMCs) were computed as the antilogarithm of the arithmetic mean of the log 10 transformed titers/concentrations.Cutoff or lower limit of quantification (LLOQ) values for immunogenicity assays were: 18 estimated dilution 60 (ED60) (RSV-A nAb GMT), 30 ED60 (RSV-B nAb GMT), 25 ELISA units/mL (RSVPreF3specific IgG GMC), and 590/10 6 cells (CD4 + T-cell frequencies).Titers/concentrations below the assay cutoff were given an arbitrary value of half the assay cutoff, while those above the assay's upper limit of quantification (ULOQ) were assigned the ULOQ value.For calculations of the fold change in frequencies of CD4 + T cells expressing at least 2 markers, frequencies below the LLOQ were imputed to the LLOQ value.Missing or nonevaluable measurements were not replaced.

Demographic and Baseline Characteristics of Study Participants
In the parent study, 1005 OA participants received at least 1 vaccine/placebo dose [18].Of those, 302 received 1 and 291 received 2 doses of an RSVPreF3-AS01 E vaccine formulation (Figure 1) [18].
Until end of study (6 months after dose 3), 1 (2.6%) participant in the 30/120 μg RSVPreF3-AS01 E group, 2 (4.7%) participants in the 60/120 μg RSVPreF3-AS01 E group, and 1 (2.5%) participant in the 120/120 μg RSVPreF3-AS01 E group reported at least 1 SAE (Supplementary Table 2).None of the SAEs were considered vaccine related by the investigators.No AEs led to withdrawal from the study, and no pIMDs or deaths were reported in this study.

DISCUSSION
With the world population aging, disease prevention and reduced disease burden are important focus points of public health care.RSV is a common pathogen that can lead to severe respiratory disease in the OA population.OAs are susceptible to developing infection-associated morbidities and may be unable to mount an effective protective response against RSV [4,16].An RSV vaccine tailored toward the OA population will thus need to maximize the elicited immune responses, to overcome age-related immunosenescence, and to protect OAs against RSV-associated disease [4,12].Together with the ongoing phase 3 trials [30,31], this extension study provides further insights into vaccine-induced immune responses in the OA population.
Prior to the first vaccination, the enrolled OA participants were seropositive for RSV-A and RSV-B nAb [18] due to previous exposure to RSV.Following the 2-dose vaccination in the parent study, both humoral (RSVPreF3-specific IgG GMCs, and RSV-A and RSV-B nAb GMTs) and CMI (frequencies of CD4 + T cells expressing at least 2 markers among IL-2, CD40L, TNF-α, and IFN-γ) responses were highest at 1 month after dose 1 (M1, day 31), without an added effect of RSVPreF3-based vaccine dose 2 (M3, day 91) [18].These immune responses remained above baseline until 12 months after dose 2 (M14), although at lower levels than measured at M1 [18].
The described RSV-specific antibodies and CD4 + T cells persisted until revaccination in this study (M20), although with  different kinetics.At the start of this extension study (M20), the IgG and nAb levels were lower than at M14 (parent study) but still higher than before dose 1 (M0).This is consistent with data reported for other RSV candidate vaccines [17,24,26,[32][33][34][35][36].Importantly, however, the third 120 μg RSVPreF3-AS01 E dose induced an increase in RSVPreF3-specific IgG and RSV-A and RSV-B nAb levels by approximately 2-fold at M21 compared to M20.These findings demonstrate that 120 μg RSVPreF3-AS01 E -induced antibody levels remain above baseline for at least 18 months after the second vaccination and can be increased again by administering a third vaccine dose.The observed boosting of antibodies to levels below those measured after the first vaccination appears to be a common observation in the RSV vaccine field [23,26,35] and, thus, not specific to the RSVPreF3-based vaccine.
The observed humoral responses were coupled with the induction of CD4 + T-cell immunity.An important finding was that the frequencies of CD4 + T cells expressing at least 2 markers did not decrease further between M14 (parent study) and M20.Additionally, as measured at M21, the CD4 + T-cell Figure 3. Percentage of participants reporting (A) at least 1 solicited AE (any, administration-site, and systemic adverse event) within 4 days, or (B) at least 1 solicited administration-site AE within 4 days, or (C ) at least 1 unsolicited AE within 30 days after vaccination with the third dose of the 120 μg RSVPreF3-AS01 E formulation (exposed set).The only collected systemic AE was fever, which was defined as body temperature ≥38°C (grade 3 fever was defined as temperature >39°C).Grade 3 erythema and swelling were defined as being >100 mm in diameter.No serious AEs, pIMDs, and deaths were reported within 30 days after dose 3. Participants received 2 doses of the AS01 E -adjuvanted vaccine formulation with 30, 60, or 120 μg of RSVPreF3 antigen in the parent study and a third dose of the AS01 E -adjuvanted vaccine formulation containing 120 μg of RSVPreF3 antigen in the extension study, indicated by 30/120-, 60/120-, and 120/120 μg RSVPreF3-AS01 E .Abbreviations: AE, adverse event; AS01 E , adjuvant system [18]; CI, confidence interval; RSVPreF3, prefusion conformation of the respiratory syncytial virus fusion (F) protein.
compartment was stimulated to the level comparable to that observed 1 month after dose 1 (M1 in the parent study).Similar to the parent study [18], the predominant T-cell response profile was CD4 + T-helper cells 1 (Th1; cells expressing at least IFN-γ), without detectable CD8 + T-cell responses.It therefore appears that the 120 μg RSVPreF3-AS01 E vaccine formulation induces stable CD4 + Th1-biased cellular immune responses, which persist for at least 18 months after the second vaccination and increase with revaccination.These findings suggest that T-cell memory induced by the primary schedule  [18]; only data for 120/120 μg RSVPreF3-AS01 E formulation were obtained in the present (extension) study.Syringe symbols represent vaccination.Fold increase indicates fold increase in GMT and GMC values at M20 (before dose 3 in extension study) and M21 (1 month after dose 3 in the extension study) compared to M0 (before dose 1 in parent study) as well as GMT and GMC fold increase at M21 compared to M20.Time points 0, 1, 2, 3, 8, and 14 designate M0 (day 1), M1 (day 31), M2 (day 61), M3 (day 91), M8, and M14 in the parent study, respectively.Neutralizing titers against RSV-B were not measured at M2. Data are plotted as mean values with 95% confidence intervals.Participants received 2 doses of the AS01 E -adjuvanted vaccine formulation with 120 μg of RSVPreF3 antigen in the parent study and a third dose of the AS01 E -adjuvanted vaccine formulation containing 120 μg of RSVPreF3 antigen in the extension study, indicated by 120/120 μg RSVPreF3-AS01 E .Abbreviations: AS01 E , adjuvant system [18]; ED60, estimated dilution 60; ELU, enzyme-linked immunosorbent assay units; GMC/ GMT, geometric mean concentration/titer; IgG, immunoglobulin G; M, month; nAb, neutralizing antibody; RSV-A and RSV-B, respiratory syncytial virus subtypes A and B; RSVPreF3, RSV fusion protein stabilized in its prefusion trimeric conformation.[18].Only data for 120/120 μg RSVPreF3-AS01 E formulation were obtained in the present (extension) study.Syringe symbols represent vaccination.The dashed horizontal line represents the assay cutoff value of 590.Fold increase and the corresponding horizontal lines indicate fold increase in frequencies of CD4 + T cells at M20 (before dose 3 in extension study) and M21 (1 month after dose 3 in the extension study) compared to M0 (before dose 1 in parent study), as well as fold increase in frequencies at M21 compared to M20.Time points 0, 1, 2, 3, 8, and 14 designate M0 (day 1, dose 1 vaccination), M1 (day 31, 1 month after dose 1), M2 (day 61, dose 2 vaccination), M3 (day 91, 1 month after dose 2), M8, and M14 in the parent study.Data are plotted as box and whisker plots with a median, interquartile range (Q1 and Q3, first and third quartile), minimum and maximum.Participants received 2 doses of the AS01 E -adjuvanted vaccine formulation with 120 μg of RSVPreF3 antigen in the parent study and a third dose of the AS01 E -adjuvanted vaccine formulation containing 120 μg of RSVPreF3 antigen in the extension study, indicated by 120/120 μg RSVPreF3-AS01 E .Abbreviations: AS01 E , adjuvant system [18]; CD4 + , cluster-of-differentiation-4-expressing; CD40L, cluster of differentiation 40 ligand; IFN-γ, interferon-γ; IL-2, interleukin 2; M, month; RSVPreF3, respiratory syncytial virus fusion protein stabilized in its prefusion trimeric conformation; TNF-α, tumor necrosis factor-α.
An immunological correlate of protection for RSV is not yet established.However, the strong humoral and CMI responses elicited by the RSVPreF3 OA vaccine might be indicative of vaccine efficacy, as recently demonstrated in a phase 3 study [27,28].
The safety findings of this extension study, in terms of solicited and unsolicited AE occurrences, are in line with previously published results [18].Although the low number of enrolled participants was a limitation, the third dose of 120 μg RSVPreF3-AS01 E was well tolerated when administered 18 months after dose 2 (AS01 E -adjuvanted, containing either 30, 60, or 120 μg of RSVPreF3).No deaths, pIMDs, nor vaccine-related SAEs were reported during the extension study period.
The limitations of this study were the relatively low number of participants and the short follow-up time.Also, because the RSVPreF3 OA vaccine has been authorized as a single dose regimen in OAs, the generalizability of the present data is limited.However, even though these data as such will not be applicable to how the vaccine is authorized for the OA population, this study provides valuable information on the safety of a booster dose, as well as on the profile and persistence of immune responses after vaccination.The ongoing phase 3 studies are currently evaluating the long-term vaccine efficacy and immune responses after different revaccination schedules with 120 μg RSVPreF3-AS01 E administered as a single dose in OAs [27,28].Study strengths include comprehensive immunogenicity evaluation and a close safety follow-up.
In conclusion, the third dose of the selected 120 μg RSVPreF3-AS01 E formulation administered 18 months after the second dose was well tolerated and induced an increase in both humoral and CMI responses.

Figure 2 .
Figure 2. Participant flow chart with (A) reasons for withdrawal and elimination from the exposed set and (B) per-protocol set.a Participant withdrawal (including consent withdrawal) was due to a reason other than an adverse event and/or solicited adverse event, migration from study area, loss to follow-up, or sponsor study termination.Participants received 2 doses of the AS01 E -adjuvanted vaccine formulation with 30, 60, or 120 μg of RSVPreF3 antigen in the parent study and a third dose of the AS01 E -adjuvanted vaccine formulation containing 120 μg of RSVPreF3 antigen in the extension study, indicated by 30/120-, 60/120-, and 120/120 μg RSVPreF3-AS01 E .M20 and M21 indicate study time points at month 20 (dose 3 vaccination) and month 21 (1 month after dose 3) in the extension study.Abbreviations: COVID-19, coronavirus disease 2019; M, month; RSVPreF3, prefusion conformation of the respiratory syncytial virus fusion (F) protein.
Abbreviations: min-max, minimum to maximum; No. (%), number and percentage of participants in a given category; OA, older adults; RSVPreF3, prefusion conformation of the respiratory syncytial virus fusion (F) protein.a The data have been published in Leroux-Roels et al [18].b Includes American Indian or Alaska Native and Asian participants; 30, 60, and 120 μg AS01 E , participants who received at least 1 dose of the AS01 E -adjuvanted vaccine formulation with 30, 60, or 120 μg of RSVPreF3 antigen in the parent study; 30/120, 60/

Figure 4 .
Figure 4. Humoral immune responses in terms of RSV-A and RSV-B nAb GMTs (ED60) and RSVPreF3-specific IgG GMCs (ELU/mL) in the 120/120 μg RSVPreF3-AS01 E group (per-protocol set).Part of these data (until M14) have been published in the parent study[18]; only data for 120/120 μg RSVPreF3-AS01 E formulation were obtained in the present (extension) study.Syringe symbols represent vaccination.Fold increase indicates fold increase in GMT and GMC values at M20 (before dose 3 in extension study) and M21 (1 month after dose 3 in the extension study) compared to M0 (before dose 1 in parent study) as well as GMT and GMC fold increase at M21 compared to M20.Time points 0, 1, 2, 3, 8, and 14 designate M0 (day 1), M1 (day 31), M2 (day 61), M3 (day 91), M8, and M14 in the parent study, respectively.Neutralizing titers against RSV-B were not measured at M2. Data are plotted as mean values with 95% confidence intervals.Participants received 2 doses of the AS01 E -adjuvanted vaccine formulation with 120 μg of RSVPreF3 antigen in the parent study and a third dose of the AS01 E -adjuvanted vaccine formulation containing 120 μg of RSVPreF3 antigen in the extension study, indicated by 120/120 μg RSVPreF3-AS01 E .Abbreviations: AS01 E , adjuvant system[18]; ED60, estimated dilution 60; ELU, enzyme-linked immunosorbent assay units; GMC/ GMT, geometric mean concentration/titer; IgG, immunoglobulin G; M, month; nAb, neutralizing antibody; RSV-A and RSV-B, respiratory syncytial virus subtypes A and B; RSVPreF3, RSV fusion protein stabilized in its prefusion trimeric conformation.