A Thermostable Oral SARS-CoV-2 Vaccine Induces Mucosal and Protective Immunity

Abstract

An ideal protective vaccine against SARS-CoV-2 should not only be effective in preventing disease, but also in preventing virus transmission. It should also be well accepted by the population and have a simple logistic chain. To fulfill these criteria, we developed a thermostable, orally administered vaccine that can induce a robust mucosal neutralizing immune response. We used our platform based on retrovirus-derived enveloped virus-like particles (eVLPs) harnessed with variable surface proteins (VSPs) from the intestinal parasite Giardia lamblia, affording them resistance to degradation and the triggering of robust mucosal cellular and antibody immune responses after oral administration. We made eVLPs expressing various forms of the SARS-CoV-2 Spike protein (S), with or without membrane protein (M) expression. We found that prime-boost administration of VSP-decorated eVLPs expressing a pre-fusion stabilized form of S and M triggers robust mucosal responses against SARS-CoV-2 in mice and hamsters, which translate into complete protection from a viral challenge. Moreover, they dramatically boosted the IgA mucosal response of intramuscularly injected vaccines. We conclude that our thermostable orally administered eVLP vaccine could be a valuable addition to the current arsenal against SARS-CoV-2, in a stand-alone prime-boost vaccination strategy or as a boost for existing vaccines.

Keywords: COVID-19; VLP (virus-like particle); mucosal immunity; oral vaccination; vaccine.

Figure 1

Structure and organization of the SARS-CoV-2 vaccine. (A) Linear diagram of the sequence and structure elements of the SARS-CoV-2 spike proteins used as immunogens. Structural elements include the S1 and S2 ectodomains derived from the original Wuhan variant (Swt) in which specific mutation were inserted. The native furin cleavage site was mutated (RRAR ➔ QQAQ) in all variants (Sst₁-Sst₅) to be protease resistant. Specific substitution (in red) and respective position were indicated. The spike variants with a CT modified to abolish ER retention (modCT) were also generated. (B) SARS-CoV-2 eVLP structure. Native or stabilized form of the SARS-CoV-2 spike (Swt or Sst) were pseudotyped on eVLP formed with the viral matrix protein MLV-Gag in association or not with the SARS-CoV-2 M proteins and the VSP from the intestinal parasite Giardia lamblia.

Figure 2

Immunogenicity of different variants of SARS-CoV-2 spike-eVLPs in mice after oral immunization. VSP-pseudotyped eVLPs displaying the following SARS-CoV-2 spike protein variants (Sst1, Sst2, Sst3, Sst4, Sst5) or wild-type sequence (Swt) were produced and used for oral immunization in mice as described in Methods. No S means eVLPs without spike. Values represent the IgG titer in blood of each animal and the horizontal line indicates the mean value. Stabilized Spike 1 (Sst1) displayed onto eVLPs elicited the higher titers and was selected for subsequent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 in comparison to control group (No S, n = 10).

Figure 3

Serum antibody responses to intramuscular administration of different vaccine formulations in hamsters. Serum IgG (left) and serum IgA (right) titers of hamsters unvaccinated (naïve) or vaccinated intramuscularly with different formulations. Wild-type spike (Swt), stabilized S as in Figure 1 (Sst) and the same in which the cytoplasmic tail was modified (SmodCT) were used to pseudotype eVLPs including or not SARS-CoV-2 M proteins and Giardia VSPs. Values represent the mean ± s.e.m. **p < 0.01; ***p < 0.001; ns, not significant; Mann Whitney test comparing hamsters immunized with eVLPs pseudotyped or not with Giardia VSPs (n=10).

Figure 4

Serum antibody responses to oral administration of different vaccine formulations in hamsters. Serum IgG (left) and serum IgA (right) titers of hamsters unvaccinated (naïve) or vaccinated orally with different formulations. Wild-type spike (Swt), stabilized S as in Figure 1 (Sst) and the same in which the cytoplasmic tail was modified (SmodCT) were used to pseudotype eVLPs including or not SARS-CoV-2 M proteins and Giardia VSPs. Values represent the mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.001; Mann Whitney test comparing hamsters immunized with eVLPs pseudotyped or not with Giardia VSPs (n=10).

Figure 5

Bronchoalveolar lavage IgA responses in hamsters vaccinated intramuscularly or orally. Bronchoalveolar lavage IgA titers of hamsters unvaccinated (naïve) or vaccinated intramuscularly (left) or orally (right) with different formulations. Wild-type spike (Swt), stabilized S as in Figure 1 (Sst) and the same in which the cytoplasmic tail was modified (SmodCT) were used to pseudotype eVLPs including or not SARS-CoV-2 M proteins and Giardia VSPs. Values represent the mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant; Mann Whitney test comparing hamsters immunized with eVLPs pseudotyped or not with Giardia VSPs (n=7 to 10).

Figure 6

Neutralizing antibodies against SARS-CoV-2 entry induced in hamsters vaccinated intramuscularly or orally. Neutralizing antibody titers of intramuscularly (left) and orally (right) vaccinated animals with selected eVLP formulations and control animals (naïve). Values represent the mean ± s.e.m. ***p < 0.001. Mann Whitney test comparing hamsters immunized with eVLPs pseudotyped or not with Giardia VSPs (n = 10).

Figure 7

Bronchoalveolar IgA after an oral boost of intramuscularly vaccinated hamsters. Animals from Figure 5A , immunized by i.m. injection of VLPs pseudotyped with stabilized spike and VSP and formed in the presence or absence of SARS-CoV-2 M protein, were then boosted (+, squares) or not (-, cercles) with the same VLPs by oral route. Values represent the mean ± S.E.M. ***p < 0.001; ns, not significant; Mann Whitney tests comparing hamsters immunized with eVLPs formed with M or not and comparing hamsters orally boosted or not (n= 10).

Figure 8

Vaccinated hamsters challenged with SARS-CoV-2. Hamsters either orally or intramuscularly vaccinated with eVLPs expressing VSP and stabilized S with the addition or not of the M protein (eVLPs Sst/VSP, up; eVLPS Sst/M/VSP, down) were challenged intranasally with purified SARS-CoV-2 virus. Every two days, the weight and general status of the unvaccinated animals (open squares), the orally (closed cercle) or intramuscularly vaccinated (open cercle) were monitored and recorded. Additionally, intramuscularly vaccinated animals orally boosted with the same formulations were included and monitored (closed squares). Values represent the mean of three independent determinations made 1 h apart ± SD for each animal (n= 10/group).