Immune response enhancement with GLS-5310 DNA primary vaccine against SARS-CoV-2 followed by administration of an mRNA vaccine heterologous boost

Abstract

Heterologous boost regimens are being increasingly considered against SARS-CoV-2. We report results for the 32 of 45 participants in the Phase 1 CoV2-001 clinical trial (Kim et al., Int J Iinfect Dis 2023, 128:112–120) who elected to receive an EUA-approved SARS-CoV-2 mRNA vaccine 6 to 8 months following a two-dose primary vaccination with the GLS-5310 bi-cistronic DNA vaccine given intradermally and followed by application of suction using the GeneDerm device. Receipt of EUA-approved mRNA vaccines after GLS-5310 vaccination was well-tolerated, with no reported adverse events. Immune responses were enhanced such that binding antibody titers, neutralizing antibody titers, and T-cell responses increased 1,187-fold, 110-fold, and 2.9-fold, respectively. This paper is the first description of the immune responses following heterologous vaccination with a DNA primary series and mRNA boost.

1. Introduction

The SARS-CoV-2 pandemic has witnessed the successful development of multiple vaccines against the virus. The vaccines are based on multiple platform technologies including inactivated virus, mRNA, protein nanoparticles, adenoviral vectored, and DNA that target the SARS-CoV-2 spike (S) protein. The vaccines are effective in preventing serious illness, however, vaccine-induced immunity appears to be time-limited, waning after 5 to 6 months [1], [2]. Moreover, successive waves of infection caused by emergent virus variants demonstrate resistance against vaccine and/or infection-induced antibodies against the S protein. For this reason, the World Health Organization has recommended that booster vaccinations be administered within 4 to 6 months following an initial vaccination series. Alternatively, vaccines that can induce significant T-cell responses may be advantageous as these correlate with less severe disease and more rapid viral clearance [3], [4], [5]. To date, cellular responses against SARS-CoV-2 proteins have remained largely unaffected by mutations in SARS-CoV-2 variants [6], [7].

In the United States and many other countries, booster vaccination programs are limited to mRNA vaccines. DNA vaccines, which are thermostable at refrigerated and controlled ambient temperatures mitigate the significant logistic challenges posed by cold-chain requirements associated with mRNA vaccines. Thus, the need for additional COVID-19 vaccine and booster options continues.

Heterologous vaccination regimens against SARS-CoV-2 that combine adenoviral and mRNA vaccines have demonstrated higher neutralizing titers than homologous regimens [8], [9] whereas T cell responses are not augumented [9]. Considering the high T cell responses that were observed following primary vaccination with GLS-5310 SARS-CoV-2 DNA vaccine, heterologous booster regimens may have potential benefit.

The CoV2-001 Phase 1 clinical trial enrolled 45 participants in an open-label, dose-escalation study to assess the safety and immunogenicity of the GLS-5310 Spike- and ORF3a-targeting bi-cistronic DNA vaccine administered with intradermal injection followed by application of the GeneDerm suction device (NCT04673149). As reported elsewhere [10], GLS-5310 vaccination was well tolerated. Binding antibody and T-cell responses were both dose-independent and remained stable throughout the 48-week follow-up period. We report here immune responses of the 32 study participants in the CoV2-001 study who elected to receive an mRNA vaccine during the latter stages of the trial when these vaccines became emergency-use-authorized (EUA) in South Korea. Vaccination with an mRNA vaccine after receipt of a primary series with GLS-5310 was well tolerated. Marked boosts to binding antibody titers, neutralizing titers, and T-cell responses, that were similar to or exceeded other primary vaccination-boost vaccination combinations, was observed.

2. Methods

The CoV2-001 clinical trial is a Phase 1 open-label, dose-escalation study of the GLS-5310 DNA vaccine administered by intradermal injection followed by application of suction to the injection site using the GeneDerm suction device conducted in Seoul, South Korea, during the early phase of the first wave of COVID-19. GLS-5310 is a bi-cistronic DNA vaccine that encodes for SARS-CoV-2 Spike and ORF3a antigens, with ORF3a having been added as an identified immunodominant SARS-CoV-2 T-cell antigen [11]. Study subjects were vaccinated twice with GLS-5310 as follows: Group 1a was vaccinated at 0 and 8 weeks with 0.6 mg/dose of GLS-5310, Group 1b at 0 and 8 weeks with 1.2 mg/dose, and Group 1c at 0 and 12 weeks with 1.2 mg/dose. Subjects were followed through 48 weeks from study enrollment. Immunologic assessments were scheduled for baseline (before the first vaccination), just prior to the second vaccination at either 8 or 12 weeks, 4 weeks following the second vaccination, and then at study weeks 24, 36, and 48.

Optional vaccination with an authorized COVID-19 vaccine was allowed any time following the study visit occurring 4 weeks after the second vaccination with GLS-5310. Subjects who elected to receive an authorized vaccine were to continue in the study to assess both safety and immune responses.

Of the 45 enrolled subjects, 32 elected to receive mRNA vaccination and this is an ad-hoc analysis of immune responses observed in those participants during study follow up. GMT of binding antibody responses were determined by enzyme-linked immunosorbent assay (ELISA) to the S antigen. Neutralization was determined by plaque reduction neutralization titer (PRNT₅₀), and T-cell responses were assessed by ELISpot. Details of the study were presented elsewhere [10].

The study was reviewed and approved by the Institutional Review Board of Korea University, Guro Hospital.

3. Results

Of the 45 participants enrolled into the CoV2-001 clinical trial, 32 (71.1 %) subsequently elected to be vaccinated with one of the two newly-authorized mRNA vaccines: 28 (87.5 %) received BNT162b2 and 4 (12.5 %) mRNA-1273. The first mRNA vaccine was administered prior to the week 24 study visit for 1 participant, the week 36 study visit for 13 study participants, and the week 48 study visit for 18 subjects (Fig. 1 ). The time to receipt of an mRNA vaccine after the first GLS-5310 vaccination averaged 250 days, and 188 days after the second GLS-5310 vaccination (Table 1 ). There were no reported adverse events following mRNA vaccination.

Fig. 1.

Pictorial representation of vaccination schema in CoV2-001 clinical trial. Scheduled study visits are shown within the bar along the x-axis. DNA vaccinations are shown as blue colored hypodermic syringes. The first mRNA vaccinations are depicted as green hypodermic syringes with the number of individuals denoted in parentheses. One subject received an mRNA vaccine prior to study week 24 with administration of their second vaccination after study week 24; for seven subjects the first mRNA vaccination occurred prior to study week 36 and the second mRNA vaccination after study week 36. One subject received an mRNA their first mRNA vaccination just prior to the week 48 study visit. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1.

Change in humoral and cellular responses following mRNA vaccination after GLS-5310 primary vaccination.

	Group 1a 0.6 mg 0–8 weeks (n = 12)	Group 1b 1.2 mg 0–8 weeks (n = 9)	Group 1c 1.2 mg 0–12 weeks (n = 11)	All groups (n = 32)
Days (range) to 1st mRNA vaccination from
1st DNA vaccination	262 (218–302)	251 (217–295)	236 (149–316)	250 (149–316)
2nd DNA vaccination	211 (183–248)*	194 (157–238)	159 (64–245)	188 (64–248)
Anti-spike IgG
Pre-boost: average titer  log10 GMT ± SD	249 2.394 ± 1.155	959 2.982 ± 1.119	209 2.322 ± 1.1324	342 2.534
Post-boost: average titer  log10 GMT ± SD	409,600 5.612 ± 0.275	290,552 5.445 ± 0.451	546,750 5.738 ± 0.357	405,920 5.608
Log change	3.218	2.463	3.417	3.074
Fold change	611	290	2,614	1,187
Neutralizing titers
Pre-boost: average titer  log10 GMT ± SD	10 1.000 ± 0.189	18 1.2548 ± 0.136	9 0.953 ± 0.433	11 1.055
Post-boost: average titer  log10 GMT ± SD	1381 3.140 ± 0.344	1169 3.068 ± 2.242	1170 3.068 ± 0.441	1245 3.095
Log increase	2.140	1.814	2.116	2.040
Fold change	138	65	130	110
T-cell
Pre-boost (SFU/106 cells)	985	1503	979	1128
Post-boost (SFU/106 cells)	2950	2299	4268	3220
Increase	1965	796	3290	2092
Fold change	3.0	1.5	4.4	2.9

*One subject in Group 1a only was vaccinated only once with the GLS-5310 DNA vaccine.

Anti-spike binding IgG antibody responses were determined by enzyme-linked immunosorbant assay (ELISA) against a recombinant, vaccine-matched full-length S protein (Acrobiosystems, Newark DE). Neutralizing antibody responses were determined by plaque reduction on Vero cells to determine the titer of serum that neutralizes infection at 50 % relative to controls (PRNT₅₀). T cell responses were determined by enzyme-linked immunosorbant (ELISpot) assay to determin interferon gamma (IFN-γ) responses against pools of 15-mer peptides spanning the full length S and ORF3a proteins. Full details of the immunologic assays are presented elsewhere [11].

Samples for immunologic assessment were obtained at pre-scheduled study visits and were not based on the timing of the optional receipt of an mRNA vaccination. Pre-mRNA vaccination values were determined from blood samples obtained at the study visit immediately prior to the first mRNA vaccination. Post-mRNA vaccination immunology assessments were determined for the study visit immediately following mRNA vaccination, provided the visit was at least 14 days following mRNA vaccination. Thus, “baseline” pre-mRNA study visits occurred an average of 33.9 days (median 27 days, range 0–77 days) prior to the first administration of an mRNA vaccine, and the post-mRNA vaccination study visit averaged 51.4 days (median 58.5 days, range 11–98 days) following mRNA vaccine administration.

Because receipt of an authorized SARS-CoV-2 vaccine was allowed, but not proscribed, in the protocol, the timing of mRNA vaccine administration was not predetermined. For 23 subjects, both mRNA vaccine administrations occurred between two study visits such that the post-mRNA study visit assessed the immune responses of both vaccinations. For eight subjects, the two mRNA vaccinations occurred before and after a planned study visit such that one of the study visits assessed the immunologic effects of a single mRNA vaccination and the next study visit the effects of the second mRNA vaccination, i.e. the mRNA vaccinations “straddled” a study visit. And one subject received their first mRNA vaccination late in the study, just prior to the week 48 study visit.

Immune responses to mRNA vaccination are summarized in Table 1. Overall, pre-mRNA vaccination IgG GMTs averaged 342, and post-mRNA vaccination GMTs averaged 405,920, corresponding to a 1,187-fold (3.07 log) change (Fig. 2 a). Neutralizing antibody GMTs pre-mRNA vaccination averaged 11.4 and post-mRNA vaccination 1244.8, corresponding to a 110-fold (2.04 log) change (Fig. 2b). Comparison between treatment groups showed no statistical difference for any immunologic parameter. For the eight subjects for whom mRNA vaccinations straddled a study visit, there was a small increase in binding antibody titers after the second mRNA vaccination relative to the first mRNA vaccination of 0.038 log (1.09-fold) and a 0.4 log (2.5-fold) increase in neutralizing titers.

Fig. 2.

B-cell immune responses pre and post-mRNA vaccination. Endpoint geometric mean titers (GMT) pre and post-vaccination with an EUA mRNA vaccine are shown for binding antibodies as determined by ELISA (A), and neutralizing antibodies as determined by PRNT50 (B). P values are: *** corresponds to p <.0005, **** coresponds to p <.0001.

T-cell responses increased to 3220 SFU/10 [6] cells from a pre-mRNA vaccination value of 1128, corresponding to a 2.9-fold change (Table 1, Fig. 3 a). As expected, there was an expansion of T-cell responses against antigen pools corresponding to the S1 and S2 spike subunits, whereas responses against ORF3a, which is not present in either mRNA vaccine, remained stable (Fig. 3b).

Fig. 3.

T-cell immune responses pre and post-mRNA vaccination. Total T-cell responses for each participant as determined by ELISpot are shown in (A). T-cell responses against individual peptide pools averaged across all study participants are shown in (B). P values are: *** corresponds to p <.0005, **** coresponds to p <.0001.

4. Discussion

There is an ongoing effort to investigate how best to construct and optimize booster regimens for vaccines against SARS-CoV-2 as the epidemic has continued. Booster programs have focused on augmentation of binding and neutralizing antibody responses. T-cell responses against SARS-CoV-2 are increasingly being recognized as correlating with more rapid viral clearance and better clinical outcomes [3], [4], [5]. Moreover, a study from the United Arab Emirates found that for returning expatriates who had SARS-CoV-2 infection over the following 6 months, approximately 75 % had non-reactive T-cells whereas greater than 99 % were seropositive and that high levels of antibodies resulted in a 6 % risk reduction for re-infection [12]. Importantly, T-cell responses against S and ORF3a proteins have remained conserved despite the emergence of newer variants [6], [7]. Notably, we have previously reported that GLS-5310 could completely protect hamsters from viral challenge with either the vaccine-susceptible wild-type SARS-CoV-2 or the vaccine-resistant Beta variant, despite the lack of evident neutralizing activity against the Beta variant [11].

Prior studies have compared the immune responses of homologous versus heterologous booster vaccination administered at least 4 months following a primary vaccination series [13], [14]. A general finding from these studies was that post-booster immune responses were relatively insensitive to the order of vaccine type as primary series or booster [13], [14]. Specifically, post-booster vaccination antibody and T-cell responses were similar regardless of whether individuals received an mRNA primary series followed by an adenoviral booster or whether they received an adenoviral primary followed by an mRNA booster [13], [14]. While T-cell responses have been reported to be greater for heterologous regimens [8], [15], [16], the magnitude of response was insensitive to the order in which vaccines were administered [16]. One additional consideration that may affect vaccine effectiveness, especially against newer variants, is that while there was a greater clonal expansion of memory B-cells following heterologous vaccination (as a primary series) with a ChAdOx1 prime and mRNA second vaccination [15] and higher binding avidity [8], mRNA boosting focused the circulating B-cell response to the prefusion S protein receptor binding domain [17].

Comparing the immune responses seen here to other studies is limited by the fact that methods and laboratories differ. With that caveat in mind, qualitatively, post-booster binding and neutralizing antibody responses would appear to be analogous to reported homologous or heterologous booster regimens that included an mRNA vaccine [13], [14]. Moreover, T-cell responses following GLS-5310 vaccination would appear to be many fold greater at baseline and post-booster responses relative to other vaccine combinations [14], [18].

This report is also limited as it is a post-hoc analysis of those who received an mRNA boost following a primary DNA vaccination series. Since this was not a planned study endpoint and was completely optional for participants, the timing for assessments of immune responses pre- and post-mRNA vaccination were based only around those study visits proscribed in the CoV2-001 clinical trial protocol. Thus, there was a wide range of time between the pre-mRNA vaccination blood sampling and mRNA vaccine administration, as well as a range of time between mRNA vaccination and the post-vaccination study visit. We had previously noted that T-cell responses remained stable through 48 weeks [10]; therefore, it is likely that there is minimal effect on pre-mRNA vaccination values in this study. Also, post-vaccination visits for most occurred more than two weeks after mRNA vaccination when immune responses are considered maximal [13] such that our results may underestimate peak post-boost immune responses.

In summary, mRNA vaccination following a primary vaccination series with the GLS-5310 DNA vaccine administered with GeneDerm suction device was well tolerated and markedly boosted pre-mRNA immune responses, exhibiting post-vaccination responses greater than reported homologous or heterologous regimens. However, maximal immune responses were observed with a single vaccination. We are currently conducting a study to evaluate the safety and immunogenicity of GLS-5310 administered as a single booster vaccination to individuals who had previously received either an mRNA or Ad26 primary vaccination series, with or without subsequent booster vaccination (NCT05182567).

5. Funding

Study support was provided by the Korea Health Technology R&D Project through Korea Health Industry Development Institute (KHIDI), Ministry of Health and Welfare, Republic of Korea (grant HQ20C0040).

6. Ethical approval

The study was reviewed and approved by the institutional review board at Korea University Guro Hospital, Seoul, Republic of Korea. All study participants provided written informed consent prior to enrollment. Research was conducted in accordance with the Code of Ethics pursuant to the Declaration of Helsinki.

7. Role in study

Study design, conceptualization, oversight, and data interpretation WJK, JYS, CCR, HL, SL, BK, DK, SS, YKP, JNM. Manuscript writing, review, editing: WJK, CCR, SBK, IK, KM, JNM. Study investigators: WJK, JYS, JGY, HS, HJH. Immunologic analysis: HL, AG, YO, JEP, BJ, JEL. Statistical analysis: SS, CCR, JNM.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The following authors are employees of GeneOne Life Science, Inc., the sponsor of the study and the developer of the GLS-5310 DNA vaccine against COVID-19 that is the subject of the paper. Christine R Roberts, Hyojin Lee, Areum Gil, Yeeun Oh, Ji eun Park, Bohyun Jeon, Ji Eun Lee, Deborah Kane, Sagar B Kudchodkar, Kar Muthumani, Young K Park *, Ijoo Kwon, Joel N Maslow *, * Young K Park and Joel N Maslow additionally own stock or stock options in GeneOne Life Science Inc. with a > $10,000 USD.

Data availability

Data will be made available on request.