Vaccine based on folded receptor binding domain‐PreS fusion protein with potential to induce sterilizing immunity to SARS‐CoV‐2 variants

Abstract

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.

Combined assessment of S‐ and N‐specific IL‐2 and IL‐13 secretion and CD69 neo‐expression for discrimination of post–infection and post‐vaccination cellular SARS‐CoV‐2‐specific immune response

“… 53 While COVID‐19 convalescent individuals generally showed negligible levels of IgG 4 antibodies against SARS‐CoV‐2 S‐ or RBD‐proteins, 26 low levels of IgG 4 anti S‐ and anti RBD‐antibodies could be detected in a subset of individuals vaccinated with mRNA SARS‐CoV‐2 vaccines. 61 , 62 The slow but sustained build‐up of IgG 4 levels is typical for this immunoglobulin isotype, 63 as also observed in allergen‐specific immunotherapy trials 64 in which IgG 4 increased only after a series of 5–7 injections with antigen over a prolonged period of time (1–2 years). This may explain why IgG 4 build‐up is rather slow, even if T cells elaborate its switch factor, IL‐13.…”

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine

“…Interim results from a phase I/II trial of an RBD-human IgG1 Fc vaccine (ancestral strain) with montenide oil-in-water adjuvant in a two-dose prime-boost schedule provided evidence of strong neutralising antibody responses with low levels of reactogenicity 41 . Another approach used to develop an RBD-based vaccine was to fuse two RBDs to a hepatitis B surface antigen (PreS) plus Alum, which generated robust responses in rabbits and in a single human volunteer 53 . Others have developed RBD monomeric vaccines with Alum or Alum-based adjuvants also administered as three-dose schedules 37, 54 one of which is in human clinical trials 54 .…”

“…RBD protein vaccines are in various stages of development and testing(https://covid19.trackvaccines.org/vaccines) 8,37,40,41,[49][50][51][52][53][54][55][56][57][58] , and one, ZF2001 8,50 , is now approved for emergency use in several countries, including China, Uzbekistan, Indonesia and Columbia (https://covid19.trackvaccines.org/vaccines/27/). This is a dimeric RBD vaccine where two RBD subunits are linked via an engineered single-chain construct and administered with Alum adjuvant as a three-dose schedule 8 .…”

Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin

“…In-Cell ELISA was then developed using DY999 substrate solution according to the manufacturer’s recommendations (R&D Systems, Minneapolis, MN USA) and measured at 450 nm (and at 630 nm to assess the background) using a Mithras multimode plate reader (Berthold Technologies, Bad Wildbad, Germany). To calculate percent inhibition of infection in each well, the following formula was used: 100 – [(X - average of ‘no virus’ wells)/(average of ‘virus only’ wells - average of ‘no virus’wells)*100], where X is the background-subtracted read for each well, as we did previously ( 50 , 51 ).…”