key: cord-0718656-plihbnsz
authors: Wolz, O.-O.; Kays, S.-K.; Junker, H.; Koch, S. D.; Mann, P.; Quintini, G.; von Eisenhart-Rothe, P.; Oostvogels, L.
title: Third dose of COVID-19 vaccine, CVnCoV, increased neutralizing activity against SARS-CoV-2 wild-type and Delta variant
date: 2022-02-24
journal: nan
DOI: 10.1101/2022.02.22.22271051
sha: 42fc918d770ba190ff8dab923205bffbe51a6e89
doc_id: 718656
cord_uid: plihbnsz

A third dose of CVnCoV, a former candidate mRNA vaccine against SARS-CoV-2, was previously shown to boost neutralizing antibody responses against SARS-CoV-2 wild-type in adults aged 18-60 and >60 years in a phase 2a clinical study. Here we report neutralizing antibody responses to wild-type and a variant of concern, Delta, after a third dose on day (D) 57 and D180. Neutralization activity was assessed using a microneutralization assay. Comparable levels of neutralizing antibodies against wild-type and Delta were induced. These were higher than those observed after the first two doses, irrespective of age or pre- SARS-CoV-2-exposure status, indicating that the first two doses induced immune memory. Four weeks after the third dose on D180, neutralizing titers for wild-type and Delta were two-fold higher in younger participants than in older participants; seroconversion rates were 100% for wild-type and Delta in the younger group and for Delta in the older group. A third CVnCoV dose induced similar levels of neutralizing responses against wild-type virus and the Delta variant in both naive and pre-exposed participants, aligning with current knowledge from licensed COVID-19 vaccines that a third dose is beneficial against SARS-CoV-2 variants.

19, including more than 5.7 million deaths, reported to WHO and nearly 9.9 billion vaccine doses have been administered worldwide [2] . SARS-CoV-2, an RNA virus, has genetically evolved over time resulting in the emergence of variants from different geographic regions [1] . Some variants with mutations that modify the SARS-CoV-2 spike protein, which is the antigen in authorized COVID-19 vaccines to date, are more resistant to the host neutralization response [3, 4] . The lineage includes three main subtypes (B1.617.1, B.1.617.2 and B.1.617.3), which contain diverse mutations in the receptor-binding domain of the SARS-CoV-2 spike protein. These mutations may increase the immune evasion potential of these variants, although results from some studies suggest cross-neutralization can occur [5] [6] [7] .

In May 2021 WHO classified the Delta mutant B.1.617.2 as a variant of concern, and in early January 2022 [8] , Delta was the most prevalent variant globally [5] . Delta infections can cause typical COVID-19 symptoms but with more severe disease than that caused by wild-type SARS-CoV-2, which is mostly observed in unvaccinated individuals, although some fully vaccinated individuals have also been infected [7] .

Recently we reported the results from three clinical trials assessing the safety, efficacy and immunogenicity of a first-generation COVID-19 vaccine candidate: CVnCoV [9] [10] [11] . CVnCoV was developed as a sequence-optimized unmodified mRNA vaccine using the proprietary RNActive ® technology platform. The mRNA, which encodes a stabilized form of spike-protein from the wild-type strain, is encapsulated in lipid nanoparticles. Clinical development of the first-generation CVnCoV vaccine has been stopped due to the concomitant development of improved second-generation vaccine candidates [12] . participants who had received 12 µg of CVnCoV on Day (D) 1 and D29, received a third dose (12 µg) on D57 (n=30, aged >60 years old) or on D180 (n=30, aged 18-60 years and n=15, aged >60 years).

Sera were collected before administration of the third dose on D57 and D180, and four weeks later, on D85, and D208, respectively. A microneutralization assay was performed in the laboratories of VisMederi Srl.

(Italy) to determine 50% neutralization titers for wild-type and the Delta variant [9] . The wild-type virus was Geometric mean titers (GMT) of neutralizing antibodies and their 95% confidence intervals (CI) were determined for the wild-type and Delta SARS-CoV-2 variant and summarized for each time point, according to age group and N-antigen serostatus (see below). Group seroconversion rates for neutralizing antibodies, defined as at least a four-fold increase in titers over baseline and geometric mean-fold rise (GMFR) in titers were calculated from baseline four weeks after the third dose (D85 for the D57 dose and D208 for the D180 dose).

To determine if participants were SARS-CoV-2 naïve or pre-exposed, sera were also tested at each time point for antibodies against the SARS-CoV-2 N protein using an ELISA (EI 2606-9601-2 G, EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany), since the vaccine does not contain the N protein.

In the SARS-CoV-2 naïve participants the neutralizing antibody GMTs against wild-type virus and the Delta variant increased on D85 and D208 from the pre-dose levels on D43, following a third dose on D57 and 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 24, 2022. ; https://doi.org/10.1101/2022.02.22.22271051 doi: medRxiv preprint D180, respectively ( Figure 1 , Table 1 ). The seroconversion rates and GMFRs were higher after the third doses than after the first two doses (Table 1) .

In participants aged >60 years GMTs against both wild-type and Delta on D208 after the third dose on D180 were higher than those on D85 after the third dose on D57 although the 95% CIs overlapped ( Figure 1 , Table 1 ). Seroconversion rates were higher after the third dose on D180 than after the third dose on D57 which was also reflected in the higher GMFRs from baseline titers suggesting a better response to the later dose (Table 1) .

The GMTs and GMFRs were higher in participants aged 18-60 years than in those >60 years of age after the third dose on D180, although the 95% CIs were overlapping. The seroconversion rates for wild-type were 100% for all participants, irrespective of age, and 100% and 90% for Delta in participants aged 18-60 years and >60 years, respectively (Table 1) .

Prior to the third dose on D180, the GMTs against wild-type and Delta were still detectable in the nine pre-exposed participants, but not in the naïve participants. On D208 the point estimates for GMTs against wild-type and Delta were higher in the pre-exposed participants than those in naïve participants ( Figure 1B ).

The data for the two pre-exposed participants who received a third dose on D57 have been included for completeness and show a tendency to higher GMTs compared with the naïve participants at the time points shown ( Figure 1A ). 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 recently reported that three doses of CVnCoV (12 µg) had an acceptable safety profile in both young and older adults and induced an increase in IgG and neutralizing antibody titers against the SARS-CoV-2 wild-type [11] . Here we compared the neutralizing antibody responses to the wild-type and Delta variant induced after a third dose of CVnCoV.

Four weeks after the third dose administered on D57 or D180, neutralizing antibody GMTs increased against both the wild-type and Delta variant in SARS-CoV-2 naïve participants above the levels observed on D43 after the first two doses. This demonstrates that the first two doses of CVnCoV had induced immune memory. The neutralizing antibody GMTs against Delta were lower than those against wild-type on D43 after the two doses, but reached similar levels, or higher as those for the wild-type after the third dose, demonstrating a robust immune response against Delta variant was induced. After the D57 dose, neutralizing immune responses against variants of concern, other than Delta, were also increased above those observed after 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 In participants aged >60 years, the D180 dose induced higher GMTs (although with overlapping 95%

CIs) and seroconversion rates for Delta than the D57 dose. The results suggest that a third dose administered at a later time-point is potentially more immunogenic than the earlier third dose, at least in individuals aged >60 years.

We compared the immune responses to CVnCoV by age because older individuals are at a higher risk of serious SARS-CoV-2 disease. In younger participants GMTs for wild-type and Delta were about two-fold higher than those in adults aged >60 years after two doses of CVnCoV and about 2.5-fold higher after 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

The copyright holder for this this version posted February 24, 2022. ; https://doi.org/10.1101/2022.02.22.22271051 doi: medRxiv preprint D180 dose. However, the GMTs were higher after three doses than after two in both younger and older individuals suggesting all participants benefitted from the third dose on D180. Immune responses are known to decline with age due to immunosenescence and this could explain the age-related observations in our study which have also been shown for other COVID-19 vaccines [4, 14] .

We previously reported that two doses of CVnCoV induced seroconversion in more than 60% of those who were immunologically naïve for SARS-CoV-2 although GMTs waned to baseline levels on D180 in our phase 1 and 2a studies [7, 11] . Here we observed that SARS-CoV-2 pre-exposed individuals still had measurable neutralizing antibodies against both wild-type and Delta up to six months after the first two doses.

This indicates that two doses of CVnCoV induced a more potent and longer-lasting immune response against the wild-type and Delta variant in pre-exposed individuals compared with SARS-CoV-2 naïve individuals.

In conclusion, a third CVnCoV dose induced strong neutralizing antibody responses against SARS-CoV-2 wild-type virus in adults aged 18-60 and >60 years demonstrating that the first two doses had induced immune memory. The third dose induced similar levels of neutralizing response against wild-type virus and the Delta variant in both naïve and pre-exposed participants. This is in alignment with the current knowledge from licensed COVID-19 vaccines that a third dose is beneficial against SARS-CoV-2 variants [15] [16] [17] .

Although the development of CVnCoV has stopped, these cross-neutralizing immune responses against SARS-CoV-2 variants are promising for the next generation SARS-CoV-2 vaccines which are based on the same mRNA platform and are being optimized for variants [18] . 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 24, 2022. ; https://doi.org/10.1101/2022.02.22.22271051 doi: medRxiv preprint

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