key: cord-0922235-z0octt2s
authors: Gattinger, Pia; Kratzer, Bernhard; Tulaeva, Inna; Niespodziana, Katarzyna; Ohradanova‐Repic, Anna; Gebetsberger, Laura; Borochova, Kristina; Garner‐Spitzer, Erika; Trapin, Doris; Hofer, Gerhard; Keller, Walter; Baumgartner, Isabella; Tancevski, Ivan; Khaitov, Musa; Karaulov, Alexander; Stockinger, Hannes; Wiedermann, Ursula; Pickl, Winfried F.; Valenta, Rudolf
title: Vaccine based on folded RBD‐PreS fusion protein with potential to induce sterilizing immunity to SARS‐CoV‐2 variants
date: 2022-04-15
journal: Allergy
DOI: 10.1111/all.15305
sha: 33cc90bca527871791d1dae5c6189508493f6f9f
doc_id: 922235
cord_uid: z0octt2s

BACKGROUND: 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. METHODS: We report the construction and in vitro and in vivo characterization of a SARS‐CoV‐2 subunit vaccine (PreS‐RBD) based on a structurally folded recombinant fusion protein consisting of two SARS‐CoV‐2 Spike protein receptor‐binding domains (RBD) fused to the N‐ and C‐terminus of hepatitis B virus (HBV) surface antigen PreS to enable the two unrelated proteins serving as immunologic carriers for each other. RESULTS: PreS‐RBD, but not RBD alone, induced a robust and uniform RBD‐specific IgG response in rabbits. Currently available genetic SARS‐CoV‐2 vaccines induce mainly transient IgG(1) responses in vaccinated subjects whereas the PreS‐RBD vaccine induced RBD‐specific IgG antibodies consisting of an early IgG(1) and sustained IgG(4) antibody response in a SARS‐CoV‐2 naive subject. PreS‐RBD‐specific IgG antibodies were detected in serum and mucosal secretions, reacted with SARS‐CoV‐2 variants, including the omicron variant of concern and the HBV receptor‐binding sites on PreS of currently known HBV genotypes. PreS‐RBD‐specific antibodies of the immunized subject more potently inhibited the interaction of RBD with its human receptor ACE2 and their virus‐neutralizing titers (VNTs) were higher than median VNTs in a random sample of healthy subjects fully immunized with registered SARS‐CoV‐2 vaccines or in COVID‐19 convalescent subjects. CONCLUSION: The PreS‐RBD vaccine has the potential to serve as a combination vaccine for inducing sterilizing immunity against SARS‐CoV‐2 and HBV by stopping viral replication through the inhibition of cellular virus entry.

comparing the numbers from fall/winter 2021 to those observed for the same period in 2020 (https://coron avirus.jhu.edu/map.html).

However, despite the fact that nearly 9.7 billion vaccine doses have been administered worldwide by this time, the number of weekly COVID-19 cases in 2022 has currently doubled compared with the same time in 2021 (https://coron avirus.jhu.edu/map.html).

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 IgG 1 antibody responses [2] [3] [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 toward 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] [7] [8] myocarditis, [9] [10] [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 int/) 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] [20] [21] [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] [28] [29] [30] and two SARS-CoV-2 RBD domains attached to the Nand 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 IgG 1 responses and the late but sustained production of neutralizing allergen-specific IgG 4 antibodies, which persist even years after discontinuation of treatment and leads to sustained protection of allergic patients from allergen-induced allergic inflammation. [33] [34] [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.

Detailed Materials and Methods can be found in the Appendix: Online Repository.

Our experience in engineering vaccines for allergen-specific immunotherapy shows that according to the hapten-carrier principle 31 one can enhance the ability of a given antigen/peptide to induce specific antibody responses upon immunization by fusing it to an unrelated carrier protein and providing it in more than one copy. [36] [37] [38] Accordingly, we designed a fusion protein (PreS-RBD) consisting of two RBD domains, one fused to the N-terminus and one fused to the C-terminus of the human hepatitis B virus (HBV)-derived PreS, the HBV surface antigen containing the binding site of HBV to the NTCP (sodium taurocholate co-transporting polypeptide) receptor on hepatocytes 39,40 ( Figure 1A) . We have previously found that a vaccine based on PreS-fusion proteins containing hypoallergenic peptides derived from the major timothy grass pollen allergens (i.e., BM32) induced a robust grass pollen allergen-specific IgG response protecting grass pollen allergic patients from grass pollen allergy 41, 42 (ClinicalTrials.gov Identifier: NCT02643641). On the contrary, BM32 and one of its components BM325 (i.e., VVX001) induced antibodies in vaccinated subjects preventing HBV infection in vitro 29, 30 and in vivo, as recently shown in a therapeutic vaccination study conducted in patients suffering from chronic HBV infection. 43 Synthetic genes coding for PreS-RBD and, for control purposes for RBD alone, were codon-optimized either for the expression in E. coli or in human cell lines. Expressed proteins were purified via Nickel affinity chromatography via a hexahistidine tag which was added to the recombinant proteins ( Figure 1A Figure 1C ).

The unfolded RBD also did not bind to the ACE2 receptor (data not shown).

In a next set of experiments, we characterized recombinant RBD and PreS-RBD proteins regarding their reactivity with a panel of antibody probes specific for PreS, RBD, and the His-tag ( Figure 1D -G). Figure 1D shows that E. coli-and HEK cell-expressed RBD and PreS-RBD reacted with different dilutions of anti-His antibodies (HEK cellexpressed PreS-RBD and RBD > E. coli-expressed PreS-RBD and RBD).

No reaction was observed when the primary anti-His antibody was omitted ( Figure 1D ). HEK cell-> E. coli-expressed PreS-RBD reacted with antisera raised against PreS-peptides and against E. coli-expressed PreS whereas recombinant RBD proteins did not ( Figure 1E ,F). No reaction was observed when the primary anti-PreS antibodies were omitted ( Figure 1E ,F). Next, we tested sera obtained from subjects before the SARS-CoV-2 pandemic (i.e., historic control sera) and sera obtained from COVID-19 convalescent patients for IgG reactivity with E. coliand HEK cell-expressed RBD and PreS-RBD proteins. Historic control sera showed no IgG reactivity to folded RBD and PreS-RBD, whereas a few sera (ie P003, P004, and P010) showed low reactivity to unfolded RBD and PreS-RBD ( Figure 1G , left). By contrast, sera from COVID-19 convalescent patients showed pronounced IgG reactivity to HEK cell-expressed PreS-RBD >RBD but no relevant reactivity to E. coliexpressed proteins ( Figure 1G , right). Only few sera (i.e., B013, I002)

showed very weak reactivity to the unfolded bacterially expressed proteins. No reactivity was observed when patients´ sera were omitted ( Figure 1G ).

The ability of folded PreS-RBD or RBD to induce antibody responses was investigated by immunizing rabbits, which allows studying the uniformity of the induced immune responses in out-bred animals.

The choice of out-bred animals is important because we found that approximately 20% of SARS-CoV-2-infected subjects did not mount RBD-specific antibodies and hence represented "RBD-nonresponders". 24 Four groups of three rabbits were immunized three times in 3-week intervals with 20 or 40 µg of alum-adsorbed RBD or two doses of alum-adsorbed PreS-RBD containing equimolar amounts of RBD. Figure 2 shows IgG levels measured by ELISA for three different dilutions of sera of the 12 rabbits. Even after three injections, one (rabbit #3) and two (rabbits #5, #6) out of the six RBD-immunized animals were non-and low-responders, respectively, whereas each of the six PreS-RBD-immunized rabbits developed robust and uniform RBD-specific IgG levels already after two injections ( Figure 2 ).

Immunization of a SARS-CoV-2 naive human subject with unfolded E.

coli-expressed PreS-RBD was started on 9 October 2020 by the subject ( Figure 3 ). In total, three subcutaneous injections were administered approximately 4 weeks apart. Figure 4 shows that immunization with Of note, also rabbit antibodies induced by immunization with the folded HEK cell-expressed Wuhan-hu-1 PreS-RBD protein cross-reacted with SARS-CoV-variants delta and omicron ( Figure S3B ).

Since the volunteer had been previously vaccinated with the PreS-containing grass pollen allergy vaccine BM325 (i.e., VVX001) 43 (ClinicalTrials.gov Identifier: NCT03625934), a PreS-specific IgG response was detected as early as visit 1, which further increased during immunization with E. coli-expressed PreS-RBD and even more so after immunization with folded HEK cell-expressed PreS-RBD ( Figure 4C ). Figure S4 further shows that the IgG isotype dominated the RBD-specific antibody responses in the immunized subject and was accompanied by a low IgM response and an IgA response which peaked shortly after the beginning of the immunization. The

PreS-specific antibody response in the subject was dominated by IgG antibodies, some IgM responses but no relevant IgA response ( Figure S4 ).

The PreS protein contains at its N-terminus the binding site of HBV to its receptor NTCP on hepatocytes, 39 and, therefore, is a Figure S5 shows that due to previous vaccination with Figure S5 ).

We found that immunization with the PreS-containing grass pollen allergy vaccine BM32 induces a biphasic allergen-and PreSspecific IgG response which consists of an early IgG 1 followed by a late but sustained IgG 4 subclass response. 29, 30, 35, 42 The late and sustained IgG 4 response is considered to be responsible for the long-term protective effect of allergen-specific immunotherapy which persists for several years even after discontinuation of vaccination. 32 Comirnaty, BionTech/Pfizer) (Table S1, Figure S6 ). Four out of the nine randomly enrolled healthy vaccinated subjects (i.e., A287, A292, A077, and C019) mounted only low S-and almost no RBDspecific IgG responses ( Figure S6 ). The quantification of S1-specific antibody levels confirmed these results, showing that they had S1specific antibody levels below 200 BAU/ml ( Table 1 ) which were lower than those of the majority (i.e., eight out of ten) of COVID-19 convalescent subjects ( Table 1) . The RBD-specific IgG subclass response in subjects vaccinated with registered vaccines consisted mainly of an IgG 1 subclass response, little IgG 2 , almost no IgG 4, and no IgG 3 ( Figure 4D ). , which is accordance with our previous study. 24 Immunization with folded HEK cell-expressed PreS-RBD induced a strong and sustained IgG response against folded RBD and against proteins containing folded RBD (i.e., insect cell-expressed S and S1, HEK cell-expressed S1) ( Figure S7A , left part) whereas no IgG responses to sequential RBDderived peptide epitopes were detected ( Figure S7A, right part) . The RBD-specific IgG response was accompanied by an initially strong but only transient IgA response specific for folded RBD ( Figure S7B ). No relevant SARS-CoV-2-specific IgM responses were found throughout the immunization period ( Figure S7C ). Immunization with folded PreS-RBD boosted the IgG response against unfolded PreS-RBD which is attributable to PreS-specific IgG antibodies ( Figure S7A ). Of note, no antibodies specific for the nucleocapsid protein (NP) or for S2 lacking RBD were observed during the whole period of immunization and observation ( Figure S7A-C) . Figure S8A -C shows that high levels of IgG antibodies specific for folded RBD and to a lower extent to E.coli-expressed 

V 9 V 1 0 V 1 1 V 1 2 V 1 3 V 1 4 V 1 5 V 1 6 V 1 7 V 1 8 V 1 9 V 2IgG to RBD V 1 V 2 V 3 V 4 V 5 V 6 V 7 V 8 V 9 V 1 0 V 1 1 V 1 2 V 1 3 V 1 4 V 1 5 V 1 6 V 1 7 V 1 8 V 1 9 V 2 0 b u f f e

V 1 V 2 V 3 V 4 V 5 V 6 V 7 V 8 V 9 V 1 0 V 1 1 V 1 2 V 1 3 V 1 4 V 1 5 V 1 6 V 1 7 V 1 8 V 1 9 V 2 0 b u f f e

unfolded PreS-RBD were also present in nasal secretions and tears obtained at visits 15 and 18. At these time points, also moderate levels of IgA antibodies specific for folded RBD could be detected whereas no SARS-CoV-2-specific IgM antibodies were found ( Figure S8B ,C). Table S2 provides an overview of the analysis of cellular immune responses during the immunization period (visits [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . Approximately 1 week after immunization with unfolded PreS-RBD (V2 and V5), we observed a doubling of the percentages of plasmablasts ( Figure S9 ). This increase of the percentage of plasmablasts was extremely strong 1 week after the first immunization with folded PreS-RBD at visit 11 when 13.1% were detected. Almost a doubling of plasmablasts was also observed 1 week after the second (i.e., visit 14) and the third (i.e., visit 18) immunization (Table S2, Figure S9 ). The plasmablasts showed mainly the phenotype CD19 + IgM-CD38 ++ CD21 − and CD10 − that had been observed also in another vaccination study. 47 The strong increase of plasmablasts 1 week after the first immunization with folded HEK cell-expressed PreS-RBD (Table S2, (Table S2) .

At visit 9, when the first immunization with folded PreS-RBD was performed no RBD and PreS-specific CD4 + and CD8 + T-cell responses were detected ( Figure S10A ). They became only detectable after immunization with folded PreS-RBD at visits 11, 14, 17, and 19 . The RBD-specific CD4 + and CD8 + T-cell responses had a similar magnitude as those observed in subjects vaccinated with registered SARS-CoV-2 vaccines ( Figure S10A,B) . Of note, RBD-specific T-cell responses were not always associated with RBD-specific IgG antibody responses. For example, subjects A286 and A291 had no relevant RBD-specific T-cell responses but had RBD-specific IgG antibodies and subject A077 showed RBD-specific T-cell responses but had no relevant RBD-specific IgG antibodies (Figures S6 and S10 ). Table 1 provides an overview of the development of S1-specific (Table S1 ) and sera from ten COVID-19 convalescent patients obtained approximately 8 weeks after SARS-CoV-2 infection were included in the assays for comparison. Immunization with unfolded E. coli-expressed PreS-RBD neither induced S1-specific IgG antibodies nor antibodies inhibiting the interaction of RBD and ACE2, and also, no virus-neutralizing antibodies were detected ( Table 1, visits 1-9). At visits 19 and 20 (i.e., 3 and 4 weeks after the third immunization), S1-specific IgG antibody levels exceeded 2700 BAU/ml in the PreS-RBD-immunized subject and were higher than the median S1-specific IgG antibodies in subjects vaccinated with licensed vaccines (i.e., 91.0-2853. 8 and 20 were 160 and 120, respectively, and thus also higher than the median VNT (10-320; median: 60) as determined in subjects vaccinated with licensed vaccines. Figure S11 shows the correlations between antibody levels specific for S1, and RBD, inhibitions of RBD binding to ACE2 and virus neutralization titers for those samples for which data pairs are available in Table 1 . Highly significant correlations were found for S1-and RBD-specific IgG ( Figure S11A ), S1-specific IgG and percentages inhibition of RBD to ACE2 binding ( Figure S11B ), VNT50 titers and percentages inhibition of RBD to ACE2 binding ( Figure S11C) and percentages inhibition of RBD to ACE2 binding and VNTs ( Figure S11D ).

The first generation of COVID-19 vaccines introduced for global ap- [22] [23] [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. 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 , [17] [18] [19] [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-RBDimmunized 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, that 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, 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 IgG 1 antibodies but also a second wave of late but sustained IgG 4 antibodies.

In fact, it is known from AIT that AIT-induced allergen-specific IgG 4 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 IgG 4 antibodies which actually are considered as non-inflammatory neutralizing antibodies. 51 Finally, we would like to comment on the safety of the PreS-RBDbased 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, aluminumadsorbed 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.

We thank Dr. Karin Stiasny and Prof. Elisabeth Puchhammer-Stöckl for virus neutralization tests and Jutta Hutecek for excellent technical assistance. We thank Dr. Julia Eckl-Dorna and Dr. Nicolas

Campion for providing nasosorption devices. RV dedicates this work to his mother on the occasion of her 93rd birthday and to the scientific community of the world in the hope that it will contribute to fight the SARS-CoV-2 pandemic. 

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