Protective mucosal immunity against SARS-CoV-2 after heterologous systemic prime-mucosal boost immunization

Abstract

Several effective SARS-CoV-2 vaccines are currently in use, but effective boosters are needed to maintain or increase immunity due to waning responses and the emergence of novel variants. Here we report that intranasal vaccinations with adenovirus 5 and 19a vectored vaccines following a systemic plasmid DNA or mRNA priming result in systemic and mucosal immunity in mice. In contrast to two intramuscular applications of an mRNA vaccine, intranasal boosts with adenoviral vectors induce high levels of mucosal IgA and lung-resident memory T cells (T _RM ); mucosal neutralization of virus variants of concern is also enhanced. The mRNA prime provokes a comprehensive T cell response consisting of circulating and lung T _RM after the boost, while the plasmid DNA prime induces mostly mucosal T cells. Concomitantly, the intranasal boost strategies lead to complete protection against a SARS-CoV-2 infection in mice. Our data thus suggest that mucosal booster immunizations after mRNA priming is a promising approach to establish mucosal immunity in addition to systemic responses.

SciScore for 10.1101/2021.08.03.454858: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: The study was approved by an external ethics committee authorized by the Government of Lower Franconia and performed under the project license AZ 55.2.2-2532-2-1179.
Sex as a biological variable	Eleven weeks old, female K18-hACE2 mice (Jackson Laboratory, Bar Harbor, USA) were immunized as described before and infected four weeks after the boost immunization intranasally with 9×103 focus-forming units (FFU) of the SARS-CoV-2 strain BavPat1 in a total volume of 50 µl under light anaesthesia with inhaled isoflurane.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
HRP-coupled …

SciScore for 10.1101/2021.08.03.454858: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: The study was approved by an external ethics committee authorized by the Government of Lower Franconia and performed under the project license AZ 55.2.2-2532-2-1179.
Sex as a biological variable	Eleven weeks old, female K18-hACE2 mice (Jackson Laboratory, Bar Harbor, USA) were immunized as described before and infected four weeks after the boost immunization intranasally with 9×103 focus-forming units (FFU) of the SARS-CoV-2 strain BavPat1 in a total volume of 50 µl under light anaesthesia with inhaled isoflurane.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
HRP-coupled anti-mouse IgA (dilution 1:5,000, A90-103P, Bethyl Laboratories) detection antibodies were added for 1h at RT.	anti-mouse IgA suggested: (Bethyl Cat# A90-103P, RRID:AB_67140)
To analyse quantities of antigen-specific antibodies, 5×105 HEK 293T cells producing SARS-CoV-2 spike or nucleocapsid were incubated for 20 minutes at 4°C with the respective biological sample diluted in 100 µl FACS-PBS (PBS with 0.5% BSA and 1 mM sodium azide) to bind to spike protein on the surface, or in 100 µl permeabilization buffer (0.5% saponin in FACS-PBS) to bind to intracellular nucleocapsid protein.	antigen-specific suggested: (Miltenyi Biotec Cat# 130-092-105, RRID:AB_615062)
After washing with 200 µl buffer, specific antibodies were bound with polyclonal anti-mouse Ig-FITC (1:300, 4°C, 20 min incubation; BD Biosciences),	anti-mouse Ig-FITC suggested: None
SARS-CoV-2 infected cells were visualized using SARS-CoV-2 S-protein specific immunochemistry staining with anti-SARS-CoV-2 spike glycoprotein S1 antibody (Abcam) 90. Dennis.	anti-SARS-CoV-2 spike glycoprotein S1 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
To analyse quantities of antigen-specific antibodies, 5×105 HEK 293T cells producing SARS-CoV-2 spike or nucleocapsid were incubated for 20 minutes at 4°C with the respective biological sample diluted in 100 µl FACS-PBS (PBS with 0.5% BSA and 1 mM sodium azide) to bind to spike protein on the surface, or in 100 µl permeabilization buffer (0.5% saponin in FACS-PBS) to bind to intracellular nucleocapsid protein.	HEK 293T suggested: None
HEK293T-ACE2 cells stably expressing the human Angiotensin-converting enzyme 2 (ACE2) were transduced with the dilutions of the pseudotypes.	HEK293T-ACE2 suggested: None
For the detection of infectious virus in BAL and the lung, Vero E6 cells were seeded at 2×104 cells/well in a 96-well plate in 200 µl of D10 for confluent monolayer 24 h prior to infection.	Vero E6 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
The mRNA vaccine Comirnaty® encodes the stabilized prefusion S protein and is described elsewhere 83. Immunizations: Six to eight weeks old female BALB/cJRj or C57BL/6 mice were purchased from Janvier (Le Genest-Saint-Isle, France) and housed in individually ventilated cages in accordance with German law and institutional guidelines.	BALB/cJRj suggested: None C57BL/6 suggested: None
Eleven weeks old, female K18-hACE2 mice (Jackson Laboratory, Bar Harbor, USA) were immunized as described before and infected four weeks after the boost immunization intranasally with 9×103 focus-forming units (FFU) of the SARS-CoV-2 strain BavPat1 in a total volume of 50 µl under light anaesthesia with inhaled isoflurane.	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
Recombinant DNA
Sentences	Resources
Vaccines: Codon-optimized sequences for the N or the spike S protein of SARS-CoV-2 were cloned into the pVAX1 expression plasmid (ThermoFisher) optimized for DNA vaccinations referred to as pVAX1-SARS2-N and pVAX1-SARS2-S 81.	pVAX1 suggested: RRID:Addgene_137910)
For the production of pseudotyped reporter particles, HEK293T cells were transfected with the SIV-based self-inactivating vector encoding luciferase (pGAE-LucW), the SIV-based packaging plasmid (pAdSIV3), and the respective spike variant-encoding plasmid 86–88.	pGAE-LucW suggested: None
Software and Algorithms
Sentences	Resources
After further washing, samples were measured on an AttuneNxt (ThermoFisher) and analysed using FlowJo software (Tree Star Inc.)	FlowJo suggested: (FlowJo, RRID:SCR_008520)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Protective mucosal immunity against SARS-CoV-2 after heterologous systemic prime-mucosal boost immunization

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