Development of SARS-CoV-2 vaccines: should we focus on mucosal immunity?

Figures & data

Figure 1. Graphic description of the key aspects involved in the development of mucosal anti-SARS-CoV-2 vaccines that might result in enhanced COVID-19 immunity compared to parenteral vaccines, a hypothesis based in the precedents generated by evaluating vaccine candidates against SARS-CoV-1 and MERS-CoV.

Table 1. Key outcomes derived from the preclinical evaluation of MERS-CoV and SARS-CoV-1 mucosal vaccine candidates.

Target virus	Antigen	Route and immunization scheme	Immune response	Ref.
MERS-CoV and SARS-Cov-1	VRPs expressing the SARS-CoV-1 or MERS-CoV N protein (VRP-SARS-N, VRP-MERS-N, respectively)	Mice were primed and boosted (6–7 weeks after priming) with VRP-SARS-N, VRP-MERS-S, or VRP-GFP i.n. or s.c.	I.n. immunization induced protective CD4^+ T cell immune response in lungs and airways and provided protection against challenge with SARS-CoV-1 through local INF-γ production. I.n. immunization with VRP-MERS-N showed similar results. Vaccination with VRP-SARS-N or VRP-MERS-N enhanced virus clearance from HLA transgenic mice upon challenge. Immunization with VRP-MERS-N induced cross-reactive protection.	[17]
MERS-CoV	RLP3-GEM: RBD bound to GEM particles through a protein anchor RLP3	Female BALB/c mice were i.n. vaccinated and boosted twice at 3-week intervals. Mice immunized with PBS were used as controls.	RLP3-GEM adjuvanted with GEL01 significantly increased RBD-specific IgG antibodies inducing a Th1-polarized response. IgA antibody levels also resulted higher specially at the small intestine. Moreover, RLP3-GEM-GEL01 increased splenocyte proliferation and Th1 and Th2 cytokines secretion.	[18]
	ChAdOx1 encoding the full-length MERS-CoV S protein (ChAdOx1 MERS)	Transgenic lethal BALB/c mice hDPP4 were i.n. or i.m. immunized with ChAdOx1 MERS. ChAdOx1 encoding enhanced-GFP immunized mice were employed as controls.	I.n. immunization with ChAdOx1 MERS conferred protection against lethal viral challenge. No viral RNA was detected in lower respiratory tract in vaccinated mice.	[19]
	S protein expressed in MVA (MVA-S)	Dromedary camels were i.n. and i.m. vaccinated twice at 2-week intervals. Control animals received wildtype MVA or PBS.	Neutralizing MERS-CoV specific antibodies were detected in serum after immunization. MVA-S significantly reduced viral RNA transcripts excreted infectious virus upon MERS-CoV challenge when compared to controls.	[20]
	RBD of S protein fused with Fc of human IgG (RBD-Fc)	Female BALB/c mice were prime-vaccinated with RBD-Fc plus Poly(I:C) adjuvant for i.n. immunization or ISA51 adjuvant for s.c. Groups were boosted at 21 and 42 days after first immunization, and at the end of 3 and 6 months. Mice immunized with PBS plus adjuvants were used as negative controls.	I.n. vaccination was able to stimulate long-term humoral IgG antibody response in sera and lungs. Also, i.n administration induced stronger local mucosal immune response represented by high level of IgA in lung wash when compared with s.c. immunization. Moreover, Il-2 and TNF-γ producing T cells resulted higher in mice immunized i.n.	[21]
SARS-Cov-1	rADV encoding S protein (rADV-S)	Female BALB/c mice were immunized three times at 2-week intervals i.n., i.m. or s.l. with rADV-S. Control mice were immunized i.m. with rADV-Mock.	S.l. immunization induced significant levels of specific IgA antibodies in bronchoalveolar lavages and neutralizing antibodies in sera. Moreover, it induced significantly higher percentages of SARS S-specific CD8 + T cells in the lungs as compared to i.m. route. S.l. vaccination did not redirect rADV vector to the olfactory bulb unlike i.n. vaccination.	[12]
	Plasmid DNA encoding N protein (pVAXN) loaded biotinylated chitosan nanoparticles functionalized with bifunctional fusion protein (bfFp)	Female BALB/c mice were i.n. or i.m. immunized twice at 3-week interval. Mice received either pVAXN soluble, loaded into nanoparticles or bfFp targeted nanoparticles in the presence or absence of DC maturation stimuli (anti-CD40 mAb).	I.n. administration of bfFp targeted pVAXN loaded nanoparticles in combination with anti-CD40 mAb induced higher levels of systemic IgG compared to naked DNA vaccine formulation administered through i.n. or i.m. route. Furthermore, significantly higher mucosal IgA levels in nasal wash were detected following nasal administration when compared with the other groups.	[22]
	Inactivated SAR-CoV-1 plus PIKA a stabilized derivative of Poly (I:C) adjuvant	Female BALB/c mice were immunized three times with 2-week intervals through either an i.n. or i.p. route with inactivated SARS-CoV-1 or plus PIKA adjuvant	I.n. immunization adjuvanted with PIKA, induced significantly higher sIgA titer in intestine, saliva and vaginal washes when compared to i.p. Also, it induced IgA specific antibodies in serum unlike i.p. vaccination. PIKA was obligatorily for inducing neutralizing activity in serum after i.n. administration.	[23]
	SARS-CoV-1 virus-like particles (SARS-CoV VLPs)	Female BALB/c mice were i.n. or i.p. immunized with SARS-CoV-1 VLPs, either alone or mixed with CpG ODN or the non-CpG control. Mice received four immunizations at 2-week intervals.	I.n. immunization induced significantly higher IgA antibody levels in saliva, feces and genital tract when compared to i.p. immunization. Neutralization titer in i.n. vaccinated groups resulted higher in serum and genital tract washes than that from i.p. vaccinated groups.	[24]
	DNA encoding S protein (pci-S) forming a complex with PEI (PEI/pci-S)	Female BALB/c mice were i.n. immunized four times at 2-week intervals with either PEI/pic-S complex, pic-S or pic-mock.	Immunization with PEI/pci-S complexes elicited high level of SARS-CoV-1 S-specific-serum IgG antibody with a Th2 dominant response. Specific PEI/pci-S complex also significantly increased specific IgA antibody response in lung wash, B cell proliferation and MHC class II complex. Moreover, percentage of IFN-γ, TNF-α and IL-2 producing cells were higher.	[13]
	S protein expressed in MVA (MVA-S) and in MVTT (MVTT-S)	For mucosal vaccination study, Female BALB/c mice were immunized twice at 3-week interval via i.n., i.o., i.r. and i.m.	MVTT-S induced higher levels of neutralizing antibodies than MVA-S when inoculated via either i.n. or i.o. routes. Furthermore, these levels were higher than those induced via the i.m. route. When realized a s.c. pre-exposure to wild-type VTT, the efficacy of i.n. or i.o. vaccination was still better than i.m. inoculation.	[14]
	N protein co-administered with the mucosal adjuvant MALP-2 or expressed by the highly attenuated MVA (MVA-NC)	BALB/c mice were priming with NC protein add mixed with a MALP-2 derivative by i.n. route and boosting with MVA–NC by i.m. route or with NC + MALP-2.	When immunizing animals by i.n. route with NC plus MALP-2 followed by MVA–NC via i.m., a Th1-dominant response was stimulated, and NC-specific secretory IgA were detected in bronchoalveolar lavages. I.n. immunization with NC plus MALP-2 followed by either homologous or heterologous boost resulted in a significant increment in IL-2 secreting cells.	[25]
	rAAV expressing RBD SARS-CoV-1 S protein (RBD-rAAV)	For i.n. vaccination, female BALB/c mice were immunized with single prime dose or prime-boost doses at interval of 0.5 months. Meanwhile, for i.m. immunization, prime-boost interval was of 1.5 months.	I.n. vaccination induced stronger IgA immune response and local specific cytotoxic T cell response, through IL-2 and IFN-γ producing T cells. Moreover, i.n. immunization conferred slightly higher protection against virus challenge.	[15]
	Protollin-formulated S-protein	Female BALB/c mice were i.n. or i.m. immunized on days 0, 14 and 21. Alum-formulated S-protein was used for i.m. vaccination.	Antigen-specific lung IgA antibody levels resulted significantly higher in i.n. immunized mice. Also, it induced a Th1-polarized response. Mice immunized via i.n. had significant lower virus titer when compared to i.m. immunized mice.	[16]
	Attenuated Salmonella enterica serovar Typhi strain expressing N protein	Female BALB/c mice were immunized twice at 2-week interval via i.n., o.g., i.v. or i.p.	Mice i.n. immunized showed the highest levels of anti-N protein-specific IgG and IgG2a, inducing a Th1-polarized response supported also by the increase of IFN-γ-producing cells. Also, the highest levels of specific S-IgA were presented in mice i.n. vaccinated.	[26]

Abbreviations: N, nucleocapsid protein; RBD, receptor-binding domain; S, spike protein; VRPs, Venezuelan equine encephalitis replicon particles; GEM, gram-positive enhancer matrix; ChAdOx1, Replication-deficient simian adenovirus vaccine vector; MVA, modified Vaccinia virus Ankara; rADV, recombinant replication defective adenovirus vector; PEI, polyethylenimine; MVTT, replication-competent modified vaccinia Tian Tan vector; rAAv, recombinant adeno-associated virus; i.n., intranasal; i.m., intramuscular; i.p., intraperitoneal; s.l., sublingual; s.c., subcutaneous; i.o., intraoral; i.r., intrarectal; o.g., orogastric; i.v., intravenous.

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