SARS-Cov-2: PreS-RBD vaccine induces multi-variant sterilizing immunity


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global COVID-19 pandemic. One possibility to control the pandemic is to induce sterilizing immunity through the induction and maintenance of neutralizing antibodies preventing SARS-CoV-2 from entering human cells to replicate in.

The currently available COVID-19 vaccines deviate from established vaccination technologies in that they rely on either the delivery of nucleic acid encoding the SARS-CoV-2 Spike protein (S) by adenovirus-based viral vectors, or by S-encoding mRNA into human cells, which then produce and release the S antigen triggering an immune response that resembles to some extent that following a SARS-CoV-2 infection 1.

Accordingly, the SARS-CoV-2-specific immune response induced by the aforementioned genetic vaccines is based on MHC class I- and MHC class II-mediated antigen presentation accompanied by S-specific CD4+ and CD8+ T cell responses and relatively short-lived S-specific, mainly IgG1 antibody responses 2-4 of which the levels and specificities may vary depending on the amount and quality of S antigen produced by the cells of the vaccinee 5.

The genetic SARS-CoV-2 vaccines could be made available relatively quickly because they by-pass the need of establishing a process for producing defined protein immunogens and thus represented an important first step towards reducing COVID-19-associated deaths. However, immunization with genetic SARS-CoV-2 vaccines has also been reported to be associated with adverse events such as thromboembolic events 6-8, myocarditis 9-11, anaphylactic reactions 12-13, neurological complications 14 and deaths considered to be associated with vaccination 15.

Furthermore, currently available SARS- CoV-2 vaccines are challenged by the emergence of novel SARS-CoV-2 variants of which certain seem to escape neutralization by vaccine-induced antibodies 16. One new variant B.1.1.529, named omicron, which was first identified in South Africa by the World Health Organization in the middle of November 2021 (WHO COVID-19 Dashboard is of particular concern because it has at least 21 mutations in the part encoding the S1 subunit and 15 amino acid exchanges in the receptor binding domain of the S protein, RBD, which is significantly more than previous variants of concern (VOCs) 17.

Indeed, omicron was detected in Asia in mid-November 18 and has since become a dominant VOC in all parts of the world 17. First reports already indicate that currently available COVID-19 vaccines may confer less protection against omicron 19-22 infection.

Antibodies directed against RBD are important for virus neutralization 23,24, correlate with protection in vaccinated subjects 25 and due to their ability to prevent the virus from entering the human cell and replicating in the host could be a key for obtaining sterilizing immunity 26.

However, it was found that approximately 20% of COVID-19 convalescent patients, although producing antibodies to other SARS-CoV-2 antigens and epitopes, do not sufficiently produce RBD-specific IgG antibodies to block the RBD-ACE2 binding and hence are poor- or non-responders to RBD 23,24.

Here, we report the construction, expression and purification, as well as the biochemical and immunological in vitro and in vivo characterization of a SARS-CoV-2 subunit vaccine PreS-RBD. PreS-RBD is based on a recombinant fusion protein consisting of the human hepatitis B virus (HBV)- derived PreS antigen, which by itself or as part of fusion proteins induces human HBV-protective immune responses 27-30 and two SARS-CoV-2 RBD domains attached to the N- and C-terminus of PreS.

According to the hapten-carrier principle discovered by Nobel laureate Baruj Benacerraf 31, the fusion of RBD to PreS aimed to increase the immunogenicity of RBD.

PreS-RBD was formulated with aluminum hydroxide (alum), an adjuvant which has been safely used both in vaccines against infectious diseases and in therapeutic allergy vaccines (i.e., allergen-specific immunotherapy, AIT) for decades 32.

AIT-induced allergen-specific IgG responses typically consist of rapidly evolving specific IgG1 responses and the late but sustained production of neutralizing allergen-specific IgG4 antibodies, which persist even years after discontinuation of treatment and leads to sustained protection of allergic patients from allergen-induced allergic inflammation 33-35.

Results obtained for the PreS-RBD subunit vaccine in this study suggest that PreS-RBD has several features, which make it an interesting SARS-CoV-2 vaccine candidate for inducing sterilizing immunity.


The first generation of COVID-19 vaccines introduced for global application mainly comprised genetic vaccines, inactivated whole virus vaccines but only one subunit vaccine which is in the final stage of receiving authorization 1,5 ( covid-19-candidate-vaccines). Available data for those vaccines which are used in mass immunization programs worldwide suggest that COVID-19 vaccines will indeed help to control the pandemic and reduce SARS-CoV-2 associated deaths.

However, the number of SARS-CoV-2 infections is steadily increasing, indicating that the virus is becoming endemic.

The SARS-CoV-2 subunit vaccine PreS- RBD reported by us here has several features, which may make it an attractive candidate vaccine addressing important needs in the field of COVID-19 vaccination that are not yet completely met by currently available SARS-CoV-2 vaccines.

Our recombinant PreS-RBD fusion protein can be produced in large quantities and high purity through expression in mammalian cells such as HEK cells which is a process that is well established all over the world not only for vaccines but also for the production of vaccines and biologics.

Furthermore, HEK cells are able to express folded proteins with posttranslational modifications such as N-glycosylation as they occur in the natural protein. As previously reported 24, we demonstrate that it is important to obtain the immunogen/antigen as a structurally folded protein because immunization with unfolded PreS-RBD failed to induce RBD-specific antibodies that are necessary to inhibit the RBD-ACE2 interaction and to achieve virus neutralization.

We show that the determination of the structural fold of PreS-RBD can be performed by using biophysical methods such as circular dichroism (CD) spectroscopy analysis of the protein and/or by showing the reactivity of the recombinant antigen with IgG antibodies from COVID-19 convalescent patients which specifically react with the folded but not with the unfolded, E-coli-expressed PreS-RBD (Figure 1C,G).

PreS-RBD is a recombinant protein and thus it is possible to perform precise dose-finding studies to determine the optimal amount of the immunogen for vaccination which is not possible for genetic vaccines 5. In fact, it has been reported that the amount and quality of S protein produced upon genetic vaccination may vary considerable between the different genetic vaccines and depending on delivery of the S-encoding genetic information into the host cells 5.

S antigen produced after genetic vaccination does not only remain at the injection site but may become expressed at sites and in cells distant to the injection site and may even circulate in the vaccinated subject and via adherence to ACE2 in the circulation may contribute to some of the observed rare side effects associated with vaccination.

We have formulated the recombinant PreS-RBD by adsorption to aluminum hydroxide, an adjuvant which has been safely used in numerous vaccines for decades. In our pilot stability studies we found that approximately 90% of PreS-RBD is bound to aluminum hydroxide and thus the injected antigen remains to a large extent at the injection site (Gattinger and Valenta, unpublished).

Importantly, aluminum hydroxide-formulated PreS-RBD remains stable for months at +4°C and also storage at higher temperature does not seem to affect the stability and immunogenicity of the vaccine which is important for a vaccine to be distributed and used globally, especially in countries with low resources (data not shown).

Our study indicates that administration of two to three doses of a molar equivalent of 40 microgram of folded PreS-RBD induces a robust induction of RBD-specific antibody responses, which are accompanied by specific T cell responses and induction of B memory/plasma cell responses.

Results obtained in the herein immunized subject demonstrate that the RBD-specific antibody response consists mainly of an IgG response composed of an early IgG1 and late IgG4 response of which the latter was not observed with genetic COVID-19 vaccines (Figure 4d) so far.

The biphasic induction of RBD-specific (i.e., early IgG1 and late, sustained IgG4) is very similar to that of BM32, a therapeutic grass pollen allergy vaccine which contains recombinant fusion proteins consisting of PreS and allergen peptides 35. BM32 has been safely used for the treatment of grass pollen induced allergy in several clinical studies 41,42 ( Identifier: NCT02643641) and it has been shown that BM32-induced PreS-specific antibodies protect against HBV infections in vitro because they are directed against the N-terminal part of PreS containing the binding site of HBV for the NTCP receptor on human hepatocytes 29,30.

In fact, very recently, it has been shown that immunization of patients with chronic HBV infections with BM325 (i.e., VVX001) had induced HBV-neutralizing antibodies in vivo 43. Furthermore, PreS-RBD not only induced RBD-specific IgG antibodies but also PreS- specific antibodies reacting with the NTCP binding sites of HBV genotypes A-H and hence may protect also against HBV infections (Figure 4C, Figure S4, Figure S5).

However, the PreS-RBD fusion protein was made by us not only with the intention to induce SARS-CoV-2- and HBV- neutralizing antibodies but to use PreS also as a carrier protein to enhance the immunogenicity of RBD. In a previous study we found that approximately 20% of SARS-CoV-2-infected patients did not produce RBD-specific IgG antibodies 23,24.

Further studies are needed to investigate the reasons for RBD-unresponsiveness which may include genetic factors such as HLA-association of RBD-specific immune responses and/or different repertoires of specific T cells and B cells. RBD-specific antibodies are important for the induction of sterilizing immunity to SARS-CoV-2 because these antibodies prevent the virus from binding to its receptor ACE2 on human cells and thus are critically important for virus neutralization 22-24.

We therefore hypothesized that immunization with RBD alone will eventually not be sufficient to induce uniform and robust RBD-specific antibodies in an outbred population. Indeed, our hypothesis was supported by results obtained from the immunization of outbred rabbits with RBD alone and the PreS-RBD fusion protein.

In this study and in a previous study 24 we found that approximately 20-30% of rabbits failed to produce robust RBD-specific antibodies when they were immunized with RBD alone whereas all rabbits immunized with PreS-RBD produced uniform and robust RBD-specific antibodies.

This result can be explained by the hapten-carrier principle discovered by the Nobel laureate Benacerraf, who demonstrated that covalent coupling or fusion of a less immunogenic component (i.e., the hapten) to a protein carrier can enhance the immunogenicity of the hapten 31. We used this principle extensively for the construction of allergy vaccines based on allergen-derived peptides fused to PreS to enhance the immunogenicity of the allergen peptides 38.

Thus, the results obtained in this study are in agreement with our previous work performed in AIT. However, we noted an important difference between the PreS-based SARS-CoV-2 subunit-vaccine described in our study and previously described AIT vaccines based on PreS-fusion proteins. In order to induce antibody responses against folded RBD capable of neutralizing SARS- CoV-2 it was necessary to express PreS-RBD in eukaryotic cells, in particular in mammalian cells as folded protein whereas PreS-RBD expressed in E. coli as unfolded protein did not induce antibodies recognizing folded RBD which were capable of neutralizing SARS-CoV-2. By contrast, AIT vaccines based on unfolded PreS-fusion proteins induced IgG antibodies capable of recognizing folded natural allergens and preventing allergic patients IgE binding 38, 48.

Whether a vaccine based on PreS-RBD will be able to overcome RBD-non-responsiveness needs of course to be demonstrated in extensive vaccination trials. However, our initial data encourage to move into this direction.

The RBD-specific IgG antibodies induced in the human subject with PreS-RBD cross-reacted with RBD mutants and variants including even the highly mutated VOC omicron (Figure 4B, Figure S1, Figure S2, Figure S3) 17-20 suggesting that the PreS-RBD-based vaccine has the potential to cross- protect even against strongly mutated VOCs.

PreS-RBD contains two RBD domains, one fused to the N- and one fused to the C-terminus of PreS and it is therefore be quite easy to enhance the cross- protective effect by including RBDs from the two most divergent and most common SARS-CoV-2 VOCs in the PreS-RBD construct. This would have the advantage that the relevant epitopes of two SARS-CoV-2 VOCs can be included in only one antigen, which will allow addressing the challenge of emerging virus variants in a highly effective manner.

The RBD-specific antibodies induced in the PreS-RBD-immunized subject were found to block more strongly the binding of RBD to ACE2 than those obtained from subjects after full vaccination with currently available and licensed COVID-19 vaccines and from COVID-19 convalescent patients when determined by their median blocking activity (Table 1). These results were confirmed by testing the VNTs using two different virus neutralization assays, one measuring the production of virus antigen and the second determining the virus cytopathic effect.

In addition to the fact that folded PreS-RBD induces antibodies which block RBD-ACE2 binding and thus infection of the host cell, also other observations indicate, the folded PreS-RBD has features of a vaccine which could be used to induce sterilizing immunity against SARS-CoV-2 infections. One of these observations is that the RBD-specific antibodies were not only detected in serum but also in mucosal fluids (i.e., tear and nasal fluids) which are derived from the sites where the virus initially enters the body and infects host cells and initially replicates. A similar finding was obtained also for AIT vaccines which in fact block the docking of allergens to IgE antibodies bound to the effector cells of allergy at mucosal sites and thus prevent local allergic inflammation 49,50.

Another important finding was that immunization with PreS-RBD induced not only a first short-lived wave of specific IgG1 antibodies but also a second wave of late but sustained IgG4 antibodies. In fact, it is known from AIT that AIT-induced allergen-specific IgG4 antibodies persist in vaccinated  subjects for a long time and are therefore considered to be important for the long-term protective effect of AIT even after discontinuation of treatment 32,46.

Thus, PreS-RBD may have the potential to induce long-lasting sterilizing immunity against SARS-CoV-2 via induction of sustained production of RBD-specific IgG4 antibodies which actually are considered as non-inflammatory neutralizing antibodies 51.

Finally, we would like to comment on the safety of the PreS-RBD-based subunit vaccine. It is of course a limitation of this study that we do yet have data from extensive toxicity studies or vaccination trials in humans which will be the focus of next clinical studies. However, it should be mentioned that there were no adverse events observed in the immunized rabbits of which each has received so far five doses of the vaccine. There were also no adverse side effects observed in the immunized subject.

However, one may consider the huge experience with aluminum hydroxide- formulated vaccines which have been used safely for decades. In particular in AIT, aluminum- adsorbed vaccines are given often more than twenty times per year for several years 32 and the PreS-based allergy vaccine BM32 has been used safely extensively in clinical AIT trials 29,30,35,41,42 and also for vaccination against HBV 43.

In summary, we report the in vitro and in vivo characterization of a SARS-CoV-2 subunit vaccine which seems to have the potential of inducing sterilizing immunity to SARS-CoV-2 variants.

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