key: cord-0294258-b7yj6ocm
authors: Fathi, A.; Dahlke, C.; Krahling, V.; Kupke, A.; Okba, N. M. A.; Raadsen, M. P.; Heidepriem, J.; Muller, M. A.; Paris, G.; Lassen, S.; Kluver, M.; Volz, A.; Koch, T.; Ly, M. L.; Friedrich, M.; Fux, R.; Tscherne, A.; Kalodimou, G.; Schmiedel, S.; Corman, V. M.; Hesterkamp, T.; Drosten, C.; Loeffler, F. F.; Haagmans, B. L.; Sutter, G.; Becker, S.; Addo, M. M.
title: Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome
date: 2022-02-15
journal: nan
DOI: 10.1101/2022.02.14.22270168
sha: 1de24fce95a3f975cff8c2f4581b386862888dce
doc_id: 294258
cord_uid: b7yj6ocm

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. A booster vaccination with MVA-MERS-S is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n=1) and the S2 subunit (n=3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials.

Emerging infections pose a major threat to public health. In recent years, outbreaks of severe acute respiratory syndrome (SARS), Ebola virus disease (EVD) and, currently, coronavirus disease 2019 have been declared public health emergencies of international concern. In response, international organizations such as the World Health Organization (WHO) and the Coalition of Epidemic Preparedness Innovations (CEPI) have developed guidance for research and development to increase pandemic preparedness, and swift and effective vaccine development plays a central role. It includes two strategic elements: The advancement of vaccine platforms, which can serve as blueprints for vaccine candidates against newly emerging pathogens, as well as the development of vaccine candidates against diseases likely to cause future epidemics, defined as priority diseases by the WHO 1 . Viral vectors represent promising vaccine platforms. They comprise recombinant attenuated or replication-deficient viruses that express gene sequences of the pathogen of interest. Vector vaccines are being evaluated as vaccine candidates against various emerging pathogens 2-4 , and have received licensure as vaccines against EVD 5,6 and COVID-19 7,8 . Modified Vaccinia virus Ankara (MVA) is a wellestablished replication-deficient poxviral vector that does not integrate into host cell DNA 9 . Nonrecombinant MVA has been licensed as a smallpox vaccine, and recombinant MVA has been studied as a vector vaccine candidate for multiple infectious disease and cancer indications 10 . It has been administered to over 120,000 individuals, including immunocompromised populations and children 6, 11 and has demonstrated a favorable safety profile 9,12 . In addition, a recombinant MVA-based vaccine has been licensed as part of a heterologous vaccination regimen against EVD 5 . and were transient (median duration 1 day, IQR 0-2.75). A detailed overview of AE frequency and grade can be found in Supplementary Table S2 .

As a result of the intensive study schedule, including early study visits and comprehensive laboratory analyses, we were able to closely assess hematologic changes and markers of organ function after booster vaccination. We observed transient changes in leukocyte counts with an increase by B:D1 and a decrease by B:D3 (Fig. 2a) , which could be attributed to a respective dynamic in neutrophil counts ( Fig. 2b) . Thrombocyte counts transiently decreased by B:D1 (Fig. 2c) . The changes of leukocyte, neutrophil and thrombocyte counts largely remained within the range of physiologic variation. A decrease in circulating lymphocytes on B:D1 (Fig. 2d ) as well as a discreet increase in plasma C-reactive protein (CRP) levels on B:D1 and B:D3 (Fig. 2e) were also observed. None of these changes were clinically significant, however, they indicate biologic activity of the vaccination and suggest cellular redistribution to other compartments. By B:D7, no hematologic changes or CRP elevation were observed as compared to baseline. All other measured biomarkers remained unchanged (i.e., markers of renal, hepatic, pancreatic, and cardiac organ function/injury, and electrolytes).

We next assessed the effect of a booster vaccination with MVA-MERS-S on humoral immunogenicity, hypothesizing that it may enhance the magnitude of the MERS-specific immune response. Two distinct IgG ELISAs specific for the S1-subunit of MERS-CoV spike glycoprotein (MERS-CoV-S) were performed on B:D0, B:D7, B:D14 and B:D28, and included a control group of healthy, unvaccinated individuals with matching timepoints (Fig. 3) . All individuals who received the booster vaccination had no detectable antibody titers on B:D0 prior to vaccination, as measured in both the in-house (Fig. 3a ) and the EUROIMMUN (Fig. 3b ) ELISAs (in-house ELISA optical density (OD) 0.08 [95% confidence interval (CI) 0.03 -0.13], cut-off 0.5, all EUROIMMUN ELISA results see Supplemental Table S3 ). After booster vaccination antibody titers increased rapidly (by B:D7) and reached levels comparable to the All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in . All individuals (10/10) seroconverted by B:D14. No anti-MERS-CoV-S1 antibody titers were detected in the serum of the controls (n=2 in the in-house and n=4 in the EUROIMMUN ELISA, respectively).

Two neutralization assays, namely the virus neutralization tests (VNT) and the plaque reduction neutralization test assessing a ≥80% reduction of plaques (PRNT80), showed dynamics similar to the ELISA assays, in line with the strong correlation between binding and neutralizing MVA-MERS-Sinduced anti-MERS-CoV-specific antibody responses that has previously been reported 18 . NAb levels increased early after booster vaccination and reached maximum levels by B:D14 and B:D28 in PRNT80 ( Fig. 4a) and VNT (Fig. 4b) , respectively. These levels peaked earlier and exceeded the maximum levels observed after the initial vaccination regimen by more than ten-fold in both neutralization assays, 

To further characterize antibody generation to MERS-CoV-S-specific linear epitopes, we used microarrays mapping the proteome of MERS-CoV-S. Sera of all ten booster dose recipients were screened for IgA, IgM and IgG with 15-mer peptides spanning the whole MERS-CoV-S protein (two aa All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 15, 2022. ; https://doi.org/10.1101/2022.02.14.22270168 doi: medRxiv preprint lateral shift, 670 peptides in total) on D0, D28, D42, B:D0 and B:D28. Figure 5 shows a schematic of MERS-CoV-S (Fig. 5A ) with a heatmap of IgG binding to the individual MERS-CoV-S epitopes (Fig. 5b ).

While we did not observe significant binding of IgA and IgM antibodies to MERS-CoV-S epitopes (data not shown), we identified a significant induction of antibody responses to four immunodominant IgG epitopes on B:D28 as compared to baseline, namely amino acid (AA) sequences 535-553, 887-913, 1,225-1,247 and 1,333-1,353 on MERS-CoV-S ( Fig. 5c-f ). For AA 535-553 (Fig. 5c) , which consisted of three overlapping peptides (OLP), we observed a trend towards an increase of AA 535-553-specific IgG by D42 and a decline by B:D0, as well as a significant re-induction by B:D28 ( Supplementary Fig.   S1 ). Importantly, this epitope was located in the RBD of MERS-CoV-S (Fig. 5a) . Epitope 887-913 ( CoV spike protein-based immunofluorescence assay (IFA). We screened all vaccinees who had received the initial vaccination regimen with vaccinations on Days 0 and 28 (n=23) and negative All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 15, 2022. ; https://doi.org/10.1101/2022.02.14.22270168 doi: medRxiv preprint controls (n=6) at baseline (D0) and 14 days after the second vaccination (D42). We did not find antibody reactivity against SARS-CoV-1 spike protein, which served as the control antigen. Likewise, anti-MERS-CoV-S antibodies were not detected in controls and in D0 specimen from vaccinees, but in contrast, were significantly increased in LD as well as HD vaccinees on D42. Anti-MERS-CoV-S immunofluorescence titers on D42 strongly correlated with nAb as measured in PRNT80 Table S5 ).

We here investigated the safety and immunogenicity profile of an additional booster vaccination with the MVA-based vaccine candidate MVA-MERS-S against MEERS-CoV one year after primary immunization in a phase 1 clinical trial. Biologic monitoring demonstrated a good tolerability, and the reactogenicity of the booster vaccination appeared to be comparable to the initial primary immunization regimen 18 with regard to dynamics, type, and severity of AE.

We found that a late homologous booster of MVA-MERS-S potently increased insert-specific immunity. Anti-MERS-CoV-specific binding and neutralizing antibody titers increased in all participants, irrespective of initial LD or HD primary immunizations, and mean neutralizing antibody levels were up to 12-fold higher than the maximum levels observed after two vaccinations.

Neutralizing antibodies were elicited in all individuals, even if they had previously failed to mount neutralizing antibodies following the original two-dose regimen. In a long-term follow-up of 7 individuals who received the booster vaccination we, furthermore, observed that MERS-CoV-specific antibody responses could still be detected 1.5 years after the third vaccination. Of note, we observed All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 15, 2022. ; https://doi.org/10.1101/2022.02.14.22270168 doi: medRxiv preprint that titers persistently exceeded the maximum titers observed after the initial two-vaccination regimen throughout the entire observation period (Weskamm et al., manuscript under review).

The licensed MVA vaccine against poxvirus is applied on Days 0 and 28. Consequently, this schedule has often been employed in the development of MVA-based vector vaccines 21 . Early secondary immunizations are chosen to maximize protective immunity in a timely manner, which is of particular importance in the context of outbreaks. However, additional and/or delayed booster vaccination may be required to increase the magnitude and duration of immunity and as a result, many established immunization regimens include a late booster vaccination in their regular schedule 22 .

Recently, the question of whether, when and how many booster vaccinations may be necessary for optimal protection has gained increased attention and has been a topic of intense debate in the context of SARS-CoV-2 vaccine development. Clinical trials and observational studies of booster vaccinations with distinct COVID-19 vaccines revealed acceptable safety profiles, which were comparable to those of primary immunization 23 . Booster vaccinations against SARS-CoV-2, given at least 5 months after the primary series, increased immunogenicity and efficacy as well as the breadth of immune responses and level of cross-protection from symptomatic infection with variants 24, 25 . A COVID-19 booster vaccination also substantially increased immunogenicity in immunocompromised and elderly individuals, who are at an increased risk for severe disease and may not mount sufficient and/or durable anti-SARS-CoV-2 immunity after primary immunization 26,27 . As a result, several national COVID-19 immunization guidances now recommend booster vaccinations 3-6 months after primary immunization 28,29 .

With regard to MVA-vectored vaccines, previous studies likewise support the rationale of implementing a booster vaccination. In a clinical Phase 1/2a trial of an MVA-based H5N1 influenza vaccine, a booster vaccination one year after an initial 28-day prime-prime regimen markedly increased binding and neutralizing antibody titers 30 . Booster vaccinations of MVA-vectored HIV vaccine candidates re-induced humoral and cellular insert-specific immunity when administered as late as 3-4 years after the last vaccination 31, 32 . Furthermore, a 56-day interval between vaccinations All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 15, 2022. ; https://doi.org/10.1101/2022.02.14.22270168 doi: medRxiv preprint led to an increased and more durable immune response compared to a 14-day interval in a non-human primate model of an MVA-based HIV vaccine, likely due to improved B-cell priming and innate immune responses 33 . These data further support the rationale of implementing booster vaccinations in immunization regimens with MVA-based vaccine candidates.

The identification of immunogenic epitopes is another essential aspect for vaccine development and perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 15, 2022. ; https://doi.org/10.1101/2022.02.14.22270168 doi: medRxiv preprint mice 20 . These data suggest that antibodies targeting epitope AA 1,225-1,247 induced by MVA-MERS-S may also play an important role in vaccine-conferred immunity.

The S2 subunit is highly conserved among betacoronaviruses. Immune responses targeted towards epitopes conserved among different coronaviruses may confer cross-reactivity and could be of relevance in delivering cross-protection. A study assessing the prevalence of anti-SARS-CoV-2 antibodies in an unexposed population did detect anti-S2-specific SARS-CoV-2 antibodies -while not detecting RBD-specific responses -as well as a strong correlation of anti-HcoV-OC43 spike protein antibodies and anti-SARS-CoV-2 S2-antibodies in a COVID-19 convalescent cohort 40 perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 15, 2022. ; https://doi.org/10.1101/2022.02.14.22270168 doi: medRxiv preprint female individuals could be included. Additionally, due to constraints in the study design, the booster vaccination was given in a relatively wide time window (12±4 months). Concerning the assessment of immunogenic epitopes, we could, furthermore, not identify conformational or discontinuous B-cell epitopes since we were only able to assess linear epitopes via microarray. The findings can be interpreted as proof-of-concept and will need to be validated in future studies involving a bigger and more representative cohort.

The MVA-MERS-S vaccine candidate has now advanced to the next phase of clinical development and is currently assessed in a double-blind, two-center, randomized, placebo-controlled Phase 1b trial (NCT04119440) supported by CEPI. This ongoing study will build on our data by using a larger cohort to evaluate different dose levels, distinct prime-prime intervals (28 vs. 56 days) and, specifically, a third vaccination one year after prime.

As a follow-up to the initial Phase 1 clinical trial of MVA-MERS-S, where a two-dose vaccine regimen was administered on Days 0 and 28 18 , we designed a proof-of-concept study to assess the safety, tolerability and immunogenicity of an additional third vaccination of MVA-MERS-S as a booster dose 12±4 months after prime immunization. All study participants who had received both primary vaccinations (Days 0 and 28, n=23) were invited to participate in this follow-up study. The full amended study protocol including all inclusion and exclusion criteria is provided in the Supplementary Information.

In addition, healthy unvaccinated individuals were recruited into an observational study and donated blood samples, which served as controls for immunogenicity assays.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 15, 2022. ; https://doi.org/10.1101/2022.02.14.22270168 doi: medRxiv preprint (PRNT) were measured using HuH-7 cells incubated with MERS-CoV isolate EMC/2012 and serial dilutions of vaccinees' sera. For the VNT, the geometric mean titer of four replicates was calculated and a reciprocal titer of 8 was considered a positive response. In the PRNT, neutralization levels were defined as the reciprocal values of a ≥80% reduction of plaques (PRNT80) and a titer of ≥20 was considered positive (one measurement per sample).

To assess antigenic peptides of MERS-CoV-S and identify B cell epitopes, we screened high-density 

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 15, 2022. ; https://doi.org/10.1101/2022.02.14.22270168 doi: medRxiv preprint

Presence of antibodies to HCoV-OC43, HCoV-229E, HCoV-HKU1, HCoV-NL63 and MERS-CoV was evaluated by CoV spike protein-based recombinant IFAs, as previously described 45 . SARS-CoV-1 spike protein was used as control antigen. Serial dilutions of vaccinee or control sera were evaluated, starting at a dilution of 1:40. A reciprocal titer of ≥40 was considered the cut-off for positivity. For statistical calculations and visualization, reciprocal titers <40 were set at a value of 20.

Descriptive statistics were used to summarize metric (number, mean with SD, minimum, maximum, and median with IQR) and categorical data (frequencies). Geometric means along with two-sided 95% CI were calculated using logarithmic transformations of OD values. To avoid overinterpretation of data, a non-normal distribution was assumed. Wilcoxon matched-pairs signed rank test and Mann-Whitney-U test were used to analyze paired and unpaired samples, respectively. Correlations were calculated using Spearman r. Statistical calculation of the p-value was two-sided and a p-value of ≤0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 

All data associated with this study are in the manuscript or the Supplementary Information.

Deidentified participant data collected during the trial can be shared through a data or materials transfer agreement. Requests from external researchers with respective research proposals should be sent to the corresponding author.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

The authors declare no competing interests.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in We observed an increase in leukocyte and neutrophil counts as well as CRP levels and a decrease in lymphocyte and thrombocyte counts on B:D1 compared to B:D0. These changes from baseline were transient and not clinically significant, but indicate biologic activity after vaccination. Boxes indicate 25-75 percentile; whiskers are min. to max.; medians are shown as horizonal lines within the boxes. ULN= upper limit of normal. LLN=lower limit of normal. *p<0.05, **p<0.005, differences assessed using Wilcoxon matched-pairs signed rank test. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 15, 2022. ; https://doi.org/10.1101/2022.02.14.22270168 doi: medRxiv preprint were measured on D0 (pre-vaccination) and D42 via IFA. LD and HD vaccinees are depicted in blue and red, respectively, unvaccinated controls in grey. A titer of <40 (dotted line) was considered negative. While we observed increased anti-MERS-CoV titers on day 42 compared to baseline, there was no significant increase of anti-HCoV titers. ***p<0.001, assessed via Wilcoxon matched-pairs signed rank test.

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We thank all study volunteers without whom this study would not have been possible. We thank Dr. WO2014045254 A3, no payments to author or institution). The funder of this publicly-funded

The amendment to the clinical trial was reviewed and approved by the Competent National Authority