mCuMVTT-RBM induces high levels of specific antibodies with high avidity against RBD and Spike proteins of SARS-CoV-2
Next, we investigated the immunogenicity of mCuMVTT-RBMin vivo . We have tested different doses and vaccination regimens (Fig. 2A). We concluded that priming with 100 µg on day 0 followed by a booster dose of 100 µg on day 28 induces the best antibody titers. Accordingly, BALB/cOlaHsd mice were s.c. immunized using this dose and regimen. No adjuvants have been added as the vaccine is self-adjuvanted with the prokaryotic ssRNA packaged during the expression process (14). The collected sera were tested for the induction of antibodies against RBD and spike protein of SARS-CoV-2. The results revealed a successful induction of anti-RBD IgGs; seven days following the priming dose which continued to increase steadily. The booster dose has significantly augmented the response by approximately ten-fold (Fig. 2B and C). Antibody response against the spike protein has also been detected following the priming dose albeit to a lower extend, compared to RBD. However, the response increased dramatically following the booster dose (Fig. 2D and E). The avidity of an antibody is defined as the binding strength through points of interaction with the cognate antigen. It can be quantified as the ratio of the dissociation constantK d for the intrinsic affinity over the one for the functional affinity of an antibody (24). Avidity and functional affinity terms can be exchanged in a looser sense (25). We have performed a modified immunoassay using 7M urea to detach low avidity antibodies. More specifically, we have compared the avidity of the antibodies in sera collected on day 42, after immunization with 100 µg on day 0 and boosting with 100 µg on either day 14 (D0/14 regimen) or 28 (D0/28 regimen). The results revealed that ~50% of the induced RBD-specific antibodies are of high avidity when using D0/28 vaccination regimen versus ~20% only for D0/14 regimen (p . 0.0008) (Fig. 2F-H). Similar findings have been seen for spike-specific antibodies (p . 0.0144) (Supplementary Fig.2 A-C). Some studies showed that neutralizing antibodies against SARS-CoV-2 wane relatively rapidly (26) and some patients may even completely lack long-lasting SARS-CoV-2 antibodies (27). It was therefore of interest to assess the longevity of antibodies induced after vaccination with mCuMVTT-RBM. Accordingly, we have tested RBD- and spike-specific IgG antibodies 134 days following priming. The results revealed that the antibody titers remained stable and comparable to the titer achieved on day 42 after boost (Fig. 2I and J).
mCuMVTT-RBM was tested in rabbits (males and females) to evaluate the immunogenicity and toxicity (tolerability) of the vaccine candidate. Rabbits were vaccinated intramuscularly (i.m.) on day 0 and received booster doses on days 14 and 28. Sera collected on day 42 have shown a strong increase in RBD-specific antibodies (around OD50 of 1:10000) (Supplementary Fig. 3). Toxicology studies demonstrated no evidence of clinical signs or systemic toxicity resulting from multiple administrations of mCuMVTT-RBM vaccine candidate. A mild influx of inflammatory cells at the injection site was observed.