Safety and Immunogenicity of an Accelerated Ebola Vaccination Schedule in People with and without Human Immunodeficiency Virus: A Randomized Clinical Trial

The safety and immunogenicity of the two-dose Ebola vaccine regimen MVA-BN-Filo, Ad26.ZEBOV, 14 days apart, was evaluated in people without HIV (PWOH) and living with HIV (PLWH). In this observer-blind, placebo-controlled, phase 2 trial, healthy adults were randomized (4:1) to receive MVA-BN-Filo (dose 1) and Ad26.ZEBOV (dose 2), or two doses of saline/placebo, administered intramuscularly 14 days apart. The primary endpoints were safety (adverse events (AEs)) and immunogenicity (Ebola virus (EBOV) glycoprotein-specific binding antibody responses). Among 75 participants (n = 50 PWOH; n = 25 PLWH), 37% were female, the mean age was 44 years, and 56% were Black/African American. AEs were generally mild/moderate, with no vaccine-related serious AEs. At 21 days post-dose 2, EBOV glycoprotein-specific binding antibody responder rates were 100% among PWOH and 95% among PLWH; geometric mean antibody concentrations were 6286 EU/mL (n = 36) and 2005 EU/mL (n = 19), respectively. A total of 45 neutralizing and other functional antibody responses were frequently observed. Ebola-specific CD4+ and CD8+ T-cell responses were polyfunctional and durable to at least 12 months post-dose 2. The regimen was well tolerated and generated robust, durable immune responses in PWOH and PLWH. Findings support continued evaluation of accelerated vaccine schedules for rapid deployment in populations at immediate risk. Trial registration: NCT02598388 (submitted 14 November 2015).

Compressed ED vaccine schedules are similarly less well studied.Safe accelerated vaccination schedules rapidly inducing peak immune responses could be advantageous for immunizing outbreak responders, similar to accelerated pretravel vaccination schedules for the prevention of hepatitis A and B, rabies, and tick-borne encephalitis [22].In a phase 1 study, the MVA-BN-Filo, Ad26.ZEBOV vaccine regimen induced higher and more frequent cellular immune responses and lower but similarly frequent humoral responses when administered over a 14-day period compared to a 56-day period in healthy adults [10].
As ED outbreaks primarily occur in regions of high HIV prevalence, and antibody responses to vaccines for other pathogens (e.g., hepatitis B) are lower in people living with HIV (PLWH), a thorough evaluation of ED vaccines in PLWH is relevant to the product's intended use [23].We, therefore, included a cohort of PLWH in a clinical trial to assess the safety and immunogenicity of the 14-day MVA-BN-Filo, Ad26.ZEBOV regimen in this population.Safety data from this pilot study (Part 1) informed progression to a larger, subsequent evaluation of accelerated Ebola vaccine regimens (Part 2).

Study Design
Part 1 of EBL2003/RV456 (NCT02598388) was conducted at a single center in the U.S., specifically the Walter Reed Army Institute of Research Clinical Trials Center, and Part 2 of the trial was conducted at six centers in sub-Saharan Africa; Part 1 results are presented here, with Part 2 results presented separately.Part 1 included people without HIV (PWOH) and PLWH, evaluating MVA-BN-Filo with Ad26.ZEBOV 14 days later.Randomization was conducted with a 4:1 ratio of vaccine to placebo, and study groups were enrolled in parallel.The study was conducted in accordance with the Helsinki Declaration and was approved by the Walter Reed Army Institute of Research Institutional Review Board.All participants provided written informed consent.The study protocol and statistical analysis plan are available in Supplements S1 and S2.

Participants
Participants were healthy and aged 18-70 years at randomization.PLWH were required to have chronic infection treated with stable antiretroviral therapy and CD4+ T-cell count > 200 cells/µL.Exclusion criteria included breastfeeding or pregnancy, prior ED, vaccination with a candidate Ebola vaccine, vaccination with a live-attenuated vaccine in the previous 30 days, inactivated vaccine receipt in the previous 14 days, and previous severe adverse reactions to vaccination.

Randomization and Masking
Participants were centrally randomized using computer-generated randomization with a block size of five.Study personnel (except those responsible for vaccine preparation) and participants were blinded to study vaccine allocation until all participants had completed at least the Day 380 visit (or discontinued earlier) and the database locked.Dispensing syringes were covered with masking tape.

Objectives
The primary objectives were to assess the safety and immunogenicity of the MVA-BN-Filo, Ad26.ZEBOV regimen, as expressed by the number of participants with adverse events (AEs), and anti-EBOV GP antibody responses as measured by Filovirus Animal Non-Clinical Group (FANG) EBOV GP enzyme-linked immunosorbent assay (ELISA) at Day 36.A secondary objective compared the safety and tolerability of the regimen in PWOH and PLWH.Exploratory objectives included assessment of binding antibody responses at other time points, EBOV GP-specific CD4+ and CD8+ T-cell responses measured by intracellular cytokine staining (ICS), cross-strain neutralizing antibody responses, Fc-mediated EBOV GP-specific antibody effector functions, and changes in viral load among PLWH.

Safety Evaluations
Participants were observed for ≥30 min post-vaccination.Local and systemic solicited AEs were recorded for seven days following each vaccination.Safety blood tests were performed seven days after each vaccination.Unsolicited AEs and serious AEs (SAEs) were recorded from informed consent until Day 57 and study end, respectively.AEs were graded as 1-mild, 2-moderate, or 3-severe, according to the adapted Division of Microbiology and Infectious Diseases Toxicity Tables [24].Toxicity scales for clinical laboratory assessments were based on the U.S. FDA toxicity grading scale for healthy adults/adolescents enrolled in preventive vaccine trials [25].For PLWH, viral loads and CD4+ T-cell counts were measured at prespecified time points (Supplementary methods, Supplement S3).
Additionally, IL-4, IL-21, tumor necrosis factor-α (TNF-α), and CD154 were measured by ICS at the same time points and were used for combinatorial polyfunctionality analysis of antigen-specific T-cell subsets using the computational COMPASS package in R (version 4.2.3)(Supplementary methods, Supplement S3) [28].EBOV GP-specific antibody-dependent cellular phagocytosis (ADCP), antibody-dependent natural killer cell activation (ADNKA), as well as antibody-dependent complement deposition (ADCD) were measured (Supplementary methods, Supplement S3) [29].EBOV GP-specific antibody polyfunctionality was defined by positivity for all three Fc-mediated effector functions and neutralization.

Statistical Analysis and Sample Size
The sample of 75 participants, which included 60 who received active vaccine, was not based on formal statistical hypothesis testing.Nevertheless, if a specific AE was not observed, the one-sided 97.5% upper confidence limit of the true rate of this AE was <16.8% and <8.8% for a sample size of 20 (PLWH) and 40 (PWOH) active vaccine recipients, respectively.Additionally, this sample size permitted a thorough immunologic assessment of the accelerated regimen.Data were analyzed when all participants completed the study or discontinued prior to its end.Safety analyses for unsolicited events were performed on the full analysis set (those receiving ≥ 1 study vaccine dose).Analyses of solicited adverse events were based on participants in a full analysis set with recorded reactogenicity data in the database.The primary immunogenicity analysis set included all vaccinated participants who received both doses within the protocol-specified window and who had ≥1 evaluable post-vaccination immunogenicity sample.Data were analyzed descriptively without formal hypothesis testing.Spearman's correlation coefficients for binding antibody concentrations and neutralizing antibody titers were calculated on Day 36.Spearman's correlation coefficients for binding antibody concentrations and HIV viral load or CD4+ T-cell count were calculated on Days 15 and 36.PRISM version 9.4.1 (GraphPad Software, Boston, MA, USA) was used for ICS graphs.All other statistical analyses employed SAS version 9.2 (SAS Institute, Cary, NC, USA).

Results
The study was performed from 14 December 2015 to 14 December 2017.Of the 138 individuals screened, 63 were not eligible (60 did not meet inclusion/exclusion criteria and 3 were due to other reasons), and 75 participants were randomized and received ≥1 vaccine dose; 40 PWOH and 20 PLWH received the active vaccine, and 10 PWOH and 5 PLWH received placebo (Figure 1).The mean age was 44 years, and 37% were female.Most participants were Black/African American (56%) or White (41%; Table 1).Overall, 72/75 (96%) participants completed the study; three discontinued in the active vaccine group (one PWOH and one PLWH were lost to follow-up; one PLWH moved out of state; Figure 1).
Solicited AEs were predominantly mild-to-moderate (Figure 2; Supplementary Tables S1 and S2, Supplement S3).The most frequently reported local AE across groups was injection-site pain.Eight participants reported a total of nine grade 3 solicited local AEs, seven of which were erythema, all occurring after Ad26.ZEBOV vaccination.Headache, myalgia, and fatigue were the most frequently reported solicited systemic AEs after any vaccination/placebo.No grade 3 solicited systemic AEs were observed following MVA-BN-Filo or placebo, and no grade 3 fevers were reported following any vaccination/placebo.Three participants (all PWOH) reported grade 3 solicited systemic AEs following Ad26.ZEBOV receipt.No remarkable trends were noted in unsolicited AE reporting.One grade 3 unsolicited anemia AE following MVA-BN-Filo administration occurred in a participant without HIV (Supplementary Table S3, Supplement S3) and was considered unrelated to the study vaccine.Solicited AEs were predominantly mild-to-moderate (Figure 2; Supplementary Tables S1 and S2, Supplement S3).The most frequently reported local AE across groups was injection-site pain.Eight participants reported a total of nine grade 3 solicited local AEs, seven of which were erythema, all occurring after Ad26.ZEBOV vaccination.Headache, myalgia, and fatigue were the most frequently reported solicited systemic AEs after any vaccination/placebo.No grade 3 solicited systemic AEs were observed following MVA-BN-Filo or placebo, and no grade 3 fevers were reported following any vaccination/placebo.Three participants (all PWOH) reported grade 3 solicited systemic AEs following Ad26.ZEBOV receipt.No remarkable trends were noted in unsolicited AE reporting.One grade 3 unsolicited anemia AE following MVA-BN-Filo administration occurred in a participant without HIV (Supplementary Table S3, Supplement S3) and was considered unrelated to the study vaccine.Five SAEs were reported by four participants, all of which occurred >28 days postvaccination (Supplementary Table S4, Supplement S3) and were considered unrelated to the study vaccine.No deaths were reported.One participant did not receive dose 2 due to grade 1 leukocytosis.The event occurred in a participant without HIV post-vaccination with MVA-BN-Filo and was considered nonserious and possibly related to the study vaccine by the investigator.
All PLWH had HIV viral loads < 200 copies/mL at screening and at the final visit, with a single transient post-vaccination blip (Supplementary Table S5, Supplement S3).
Fifty-seven participants who received the MVA-BN-Filo, Ad26.ZEBOV active regimen (38 PWOH; 19 PLWH) and 15 placebo recipients fulfilled the criteria for inclusion in the per-protocol immunogenicity analyses set.
On Day 15, 4/38 (11%) and 2/19 (11%) PWOH and PLWH, respectively, displayed an EBOV GP-specific binding antibody response following the first active vaccination (Figure Five SAEs were reported by four participants, all of which occurred >28 days postvaccination (Supplementary Table S4, Supplement S3) and were considered unrelated to the study vaccine.No deaths were reported.One participant did not receive dose 2 due to grade 1 leukocytosis.The event occurred in a participant without HIV post-vaccination with MVA-BN-Filo and was considered nonserious and possibly related to the study vaccine by the investigator.
All PLWH had HIV viral loads < 200 copies/mL at screening and at the final visit, with a single transient post-vaccination blip (Supplementary Table S5, Supplement S3).
Fifty-seven participants who received the MVA-BN-Filo, Ad26.ZEBOV active regimen (38 PWOH; 19 PLWH) and 15 placebo recipients fulfilled the criteria for inclusion in the per-protocol immunogenicity analyses set.

Discussion
This study illustrates an accelerated two-dose heterologous Ebola vaccination schedule comprised of MVA-BN-Filo followed by Ad26.ZEBOV 14 days later is well tolerated and can elicit durable humoral and cellular responses against the EBOV GP in both PWOH and PLWH.Solicited AEs were generally mild-to-moderate with limited duration in both populations.Among PLWH, vaccination had no appreciable clinically significant impact on HIV viral suppression.
In the absence of efficacy data, immunobridging is used to infer a vaccine's protective effect by comparing immunogenicity in humans to the relationship between immunogenicity and survival after challenge in nonhuman primates (NHP).Although a mechanistic correlate of protection for this vaccine has not yet been identified, binding antibodies against the EBOV surface GP strongly correlate with survival post-challenge in a fully lethal EBOV Kikwit NHP challenge model [30,31].The Ad26.ZEBOV, MVA-BN-Filo 56-day regimen provided nearly 100% protection against infection in this model.The protective efficacy of this vaccine regimen was demonstrated via immunobridging, facilitating marketing authorization approval under exceptional circumstances by the EMA [18,19].
Compared to the 56-day regimen of the Ad26.ZEBOV, MVA-BN-Filo vaccines, the accelerated reverse regimen in this study elicited lower binding antibody concentrations 21 days post-dose 2; however, response rates were high, and antibody concentrations at 6 and 12 months post-dose 2 were similar [5,[7][8][9][11][12][13].As the immunobridging model is only informative for the 56-day regimen, the degree of protection against infection induced by this trial's accelerated schedule cannot be extrapolated.Also, the protective efficacy of these vaccine regimens may be underestimated in this highly stringent animal model [30].
A cross-strain evaluation of neutralizing antibody responses demonstrated robust and durable titers to the Ebola Zaire Kikwit and Makona strains with strong correlations to the binding antibody responses measured in the FANG anti-EBOV GP IgG ELISA employing the Kikwit EBOV GP.Cross-strain neutralizing antibody responses against the Bundibugyo virus were detectable in some participants but at low titers.Animal model results suggest that antibody-dependent cell-mediated cytotoxicity and ADCP could confer protection against EBOV [32][33][34].Therefore, effector antibody functions such as ADCP, ADNKA, and ADCD were evaluated, and these responses were generally durable and polyfunctional.An HIV vaccine regimen based on the same Ad26 platform has also been shown to induce polyfunctional antibody responses in both humans and NHP [35], and ADCP activity has been linked to protection against infection in a relevant simian-human immunodeficiency virus challenge model [36].Cellular responses have also been linked to protection in NHP models [37], and in this study, we observed both CD4+ and CD8+ T-cell responses.This trial's data are aligned with previous observations that vaccines can stimulate functional immune responses in well-controlled PLWH, even if the magnitude of response is decreased [38].Protection with this accelerated schedule is not yet established; however, recent data characterizing immune responses elicited by a Day 360 boost with either Ad26.ZEBOV or MVA-BN-Filo have shown a significant anamnestic response across heterologous regimens irrespective of the vaccination order or dosing interval of the primary regimen [10].Indeed, we observe that this accelerated vaccine regimen induced a polyfunctional antibody, as well as T-cell response, in both PWOH and PLWH, often persisting to 12 months.Taken together, these data suggest that a heterologous two-dose vaccination regimen with an accelerated MVA-BN-Filo, Ad26.ZEBOV schedule may establish immune memory that can be rapidly recalled by subsequent boosting or even pathogen exposure.
Study limitations include the relatively small sample size, which limited statistical power to compare HIV status groups.Additionally, women were slightly underrepresented in the enrollment (37%).Study results would have greater utility if the approved 56-day regimen was included for direct comparison.Finally, the specimen collection schedule was not optimized for defining early innate immune responses.

Conclusions
In summary, this is the first study to demonstrate safety and EBOV GP-specific immunogenicity in PLWH with an Ebola vaccination regimen of MVA-BN-Filo followed by Ad26.ZEBOV 14 days later.These results show that an accelerated vaccination schedule is well tolerated in both PWOH and PLWH and induces a broad array of durable humoral and cellular immune responses.These findings support continued consideration for accelerated regimen development for rapid deployment among outbreak responders and relevant global populations at immediate risk.Funding: This work was supported by Janssen Pharmaceuticals, who participated in the design and conduct of the study, safety monitoring, data analysis and interpretation, preparation, review, and approval of the manuscript, and the decision to submit the manuscript for publication.This study was also funded by JPM-CBRN Medical, who participated in the design of the study, safety

Figure 1 .
Figure 1.CONSORT diagram.Inf.U, infectious units; N, number of participants; PLWH, people living with HIV; PWOH, people without HIV; vp, viral particles.a Participants who discontinued vaccination could still complete the study.

Figure 1 .
Figure 1.CONSORT diagram.Inf.U, infectious units; N, number of participants; PLWH, people living with HIV; PWOH, people without HIV; vp, viral particles.a Participants who discontinued vaccination could still complete the study.

Figure 2 .
Figure 2. Solicited adverse events.(A) Solicited local adverse events; (B) Solicited systemic adverse events.Percentages reflect n/N, where n is the number of participants with one or more adverse events and N is the number of participants with available reactogenicity data after the given dose.Ad26, Ad26.ZEBOV; MVA, MVA-BN-Filo; PLWH, people living with HIV; PWOH, people without HIV.

Figure 2 .
Figure 2. Solicited adverse events.(A) Solicited local adverse events; (B) Solicited systemic adverse events.Percentages reflect n/N, where n is the number of participants with one or more adverse events and N is the number of participants with available reactogenicity data after the given dose.Ad26, Ad26.ZEBOV; MVA, MVA-BN-Filo; PLWH, people living with HIV; PWOH, people without HIV.

Table 1 .
Participants' demographic and baseline characteristics; full analysis set.
Supplementary Materials: The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/vaccines12050497/s1,Supplement S1: Study protocol, Supplement S2: Statistical analysis plan, Supplement S3: Supplementary material (Supplementary methods, Supplementary results, Supplementary Table S1: Solicited local AEs by worst severity grade [full analysis set], Supplementary Table S2: Solicited systemic AEs by worst severity grade [full analysis set], Supplementary Table S3: Unsolicited AEs [full analysis set], Supplementary Table S4: SAEs [full analysis set], Supplementary Table S5: HIV viral load: categorization of HIV viral loads in PLWH, Supplementary Table S6: EBOV GP-specific binding antibody responses in each study group, from baseline to study completion as measured by FANG anti-EBOV GP IgG ELISA, Supplementary