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Protective mucosal immunity against SARS-CoV-2 after heterologous systemic prime-mucosal boost immunization | Nature Communications
Results . A systemic plasmid DNA prime significantly increases the mucosal immunogenicity of an intranasal adenoviral vector vaccine . In this first part of our study, we evaluated the immunogenicity of mucosally applied viral vector vaccines as a single shot vaccine or as a booster after an intramuscular plasmid DNA prime immunization. To this end, codon-optimized sequences encoding the full-length S and nucleocapsid (N) proteins of SARS-CoV-2 were inserted into pVax-1 expression plasmids (in the following designated as plasmid DNA vaccine) and into replication-deficient adenoviral vector vaccines based on serotype 5 (Ad5) or serotype 19a (Ad19a). BALB/c mice were immunized intranasally with the Ad5- or Ad-19a-based vaccines either without prior treatment or four weeks after an intramuscular plasmid DNA immunization with S- and N-encoding plasmids (Fig.? 1A ). Two weeks later, SARS-CoV-2 specific antibody responses were analysed in serum and BAL samples, whereas the local and systemic T cell responses were determined in lungs and spleens, respectively. Fig. 1: Humoral responses after intranasal immunization with Ad5- or Ad19a-based viral vector vaccines. A BALB/c mice were immunized intranasally with Ad5- or Ad19a-based vectors encoding the N and S protein of SARS-CoV-2 (2?×?10 6 infectious units per vector). Mice from the heterologous prime-boost groups were primed four weeks before by intramuscular injection of N- and S-encoding DNA plasmids (10??g per plasmid) followed by electroporation. Serum antibody responses were analysed thirteen days and mucosal immune responses in the BALs fourteen days after the mucosal immunization. Spike-specific IgG ( B ), IgG1 ( C ), and IgG2a ( D ) were assessed by a flow cytometric approach (dilutions: sera 1:400, BAL 1:100). Plaque reduction neutralization titres (PRNT75) were determined by in vitro neutralization assays ( E ). Bars represent group medians overlaid with individual data points; na?ve n ?=?4; DNA-Ad5 n ?=?5; other groups n ?=?6. Data were analysed by one-way ANOVA followed by Tukey’s post test ( B – D ) or by Kruskal–Wallis test (one-way ANOVA) followed by Dunn’s multiple comparison test ( E ). Statistically significant differences are indicated only among the different vaccine groups; p values indicate significant differences ( p ?Full size image In our flow cytometric assay 54 , spike-specific IgG, IgG1, and IgG2a could be easily detected in serum and BAL of animals treated with the prime-boost strategies, while antibodies in the BAL after a single dose of Ad19a or Ad5 were almost absent (Fig.? 1B–D ). Comparing the two adenoviral vectors as booster vaccines, the serotype 5 induced significantly higher levels of S-specific antibodies in the BAL, although the antibody levels in the sera were comparable for the both groups. This effect was confirmed in the IgG subclass analyses for both, IgG1 and IgG2a levels (Fig.? 1C, D ). Similar trends were also observed for N-specific antibody levels in sera and BALs (Supplementary Fig.? 1 ). In line with the amount of S-binding antibodies, profound virus neutralization was detected in sera and BAL samples from the groups DNA-Ad5 and DNA-Ad19a, while Ad5 or Ad19a alone did not induce significant levels of neutralizing antibodies (Fig.? 1E ). Given the differences in the local antibody levels, the IgA response in the BAL towards specific domains of the S protein were analysed in more detail by ELISA (Fig.? 2A–C ). These results confirmed that intranasal applications of Ad5-based vectors induce higher S-specific IgA levels than Ad19a-based vectors and these responses benefit from a systemic plasmid DNA prime. Furthermore, the vaccine-induced antibodies were directed against S1 including the receptor-binding domain (RBD) as well as against the S2 domain of the spike protein (Fig.? 2A–C ). Fig. 2: Mucosal, spike-specific IgA responses. BALB/c mice were vaccinated according to Fig.? 1A . BAL samples were tested for spike-specific IgA directed against the domains of S2 ( A ), S1 ( B ), or RBD ( C ) by ELISA (dilution: 1:10). Bars represent group medians overlaid with individual data points; na?ve n ?=?4; DNA-Ad5 n ?=?5; other groups n ?=?6. Data were analysed by one-way ANOVA followed by Tukey’s post test. Statistically significant differences are indicated only among the different vaccine groups; p values indicate significant differences ( p ?Full size image Next, we assessed the induction of cellular immune responses in the lung by the different vaccination schemes. Intravascular staining (iv-labelling) 55 was used to differentiate between circulating T cells present in the lung endothelium during sampling (iv?) and T RM (iv+). Since specific MHC-I multimers were not available at the time of this study, antigen-experienced T cells were identified by the expression of CD44 (gating strategy shown in Supplementary Fig.? 2 ). Similar to the humoral responses, CD44 + CD8 + T cells in the lung were most efficiently induced by the DNA-Ad5 scheme, although all treated animals mounted vaccine-induced cellular responses (Fig.? 3A ). The vast majority of lung CD8 + T cells were protected from the iv-labelling in all groups, and the most prominent T RM phenotype was CD103 + CD69 + (Fig.? 3B ). Antigen-specific CD4 + and CD8 + T cells were identified by ex vivo restimulation with peptide pools covering major parts of S and the complete N protein, respectively, followed by intracellular staining of accumulated cytokines (gating strategy in Supplementary Fig.? 3 ). The highest percentages of S-reactive CD8 + T cells were detected in the lungs of DNA-Ad5 treated animals with the majority of them predominantly producing IFNγ (Fig.? 4A ). Differences in the percentages of CD8 + T cells expressing IL-2 or TNF were less pronounced, and polyfunctional T cells positive for all four analytes including the degranulation marker CD107a were rarely found in all animals. In contrast, significantly elevated percentages of CD8 + T cells producing IFNγ or TNF as well as polyfunctional CD8 + T cells were detected in the spleens of DNA-Ad19a treated animals (Fig.? 4C ). Albeit at overall lower frequencies, the same observation was made for N-reactive CD8 + T cells in lungs and spleens (Supplementary Fig.? 4A, C ). Pronounced S- and N-specific CD4 + T cell responses were detected in all animals that received a prime-boost vaccination (Fig.? 4 and Supplementary Fig.? 4 ). In contrast to the CD8 + T cells, the majority of the CD4 + T cells were polyfunctional indicated by the simultaneous expression of IFNγ, TNF and IL-2. Again, immunization with the Ad19a-based vectors resulted in higher systemic responses measured in the spleen, whereas the mucosal response in the lung was more pronounced after delivery of Ad5-based vectors (Fig.? 4C, D , Supplementary Fig.? 4C, D ). Fig. 3: Tissue-resident memory T cell subsets in the lung. BALB/c mice were vaccinated according to Fig.? 1A . In absence of suitable MHC-I multimers, antigen-experienced CD8 + T cells were identified by CD44 staining ( A ). Intravascular staining was used to differentiate between circulating (iv+) and tissue-resident (iv?) memory cells. Tissue-resident phenotypes were assessed by staining for CD69 and/or CD103 within the iv-protected memory compartment ( B ). The gating strategy is shown in Supplementary Fig. 2 . Bars represent group means with SEM ( A ) or overlaid with individual data points ( B ); na?ve n ?=?4; DNA-Ad5 n ?=?5; other groups n ?=?6. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significant differences are indicated only among the different the vaccine groups; p values indicate significant differences ( p ?Full size image Fig. 4: Spike-specific T cell responses after intranasal immunization with Ad5- or Ad19a-based viral vector vaccines. BALB/c mice were vaccinated according to Fig.? 1A . Lung and spleen homogenates were restimulated with peptide pools covering major parts of S. The responding CD8 + ( A and C ) and CD4 + T cells ( B and D ) were identified by intracellular staining for accumulated cytokines or staining for CD107a as degranulation marker. The gating strategy is shown in Supplementary Fig. 3 . Bars represent group means overlaid with individual data points; na?ve n ?=?4 (exception: n ?=?3 in C and D ); DNA-Ad5 n ?=?5; other groups n ?=?6. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences are indicated only among the different vaccine groups; p values indicate significant differences ( p ?cell population positive for all assessed markers. Full size image Taken together, Ad5 proved a higher immunogenicity as mucosal vaccine vector compared to Ad19a and resulted in strong cellular and humoral immune responses against SARS-CoV-2 antigens if combined with an intramuscular plasmid DNA prime immunization. An intranasal boost following systemic mRNA vaccination potentiates mucosal antibody responses with pronounced neutralization breadth . Since mRNA vaccines are currently in use for mass vaccination campaigns in many countries, we wanted to compare the differential effects of a plasmid DNA or mRNA prime on the immunogenicity of a mucosal booster. Therefore, the previously described DNA-Ad5 scheme was compared to an mRNA prime (Comirnaty ? , Biontech/Pfizer) followed by an intranasal Ad5 boost (RNA-Ad5). Moreover, two vaccine groups that received two intramuscular injections with either mRNA (2x RNA) or an adenoviral vector (2x Ad5) reflecting current SARS-CoV-2 vaccination strategies were included (Fig.? 5A ). These experiments were performed in C57BL/6 mice to allow correlations to efficacy data in K18-hACE2 mice. Fig. 5: Humoral responses after homologous or heterologous prime-boost vaccination. A C57BL/6 mice received an intramuscular prime immunization with the spike-encoding DNA (10??g), Ad5-S (10 7 infectious units), or the mRNA vaccine, Comirnaty ? (1??g). Mice from the heterologous prime-boost groups were boosted four weeks later intranasally with Ad5-S (10 7 infectious units). The homologous prime-boost groups received a second dose of mRNA (1??g) or Ad5-S (10 7 infectious units) intramuscularly. Serum antibody responses were analysed 21 days and mucosal immune responses four weeks after the boost immunizations. Spike-specific IgG ( B ) were assessed by a flow cytometric approach (dilutions: Sera 1:800, BAL 1:20). BAL samples were tested for spike-specific IgA directed against RBD by ELISA ( C ). Plaque reduction neutralization titres (PRNT75) were determined by in vitro neutralization assays ( D ). Bars represent group medians overlaid with individual data points; sera all groups n ?=?8; BALs RNA-Ad5 n ?=?7, other groups n ?=?8 (out of two independent experiments). Data were analysed by one-way ANOVA followed by Tukey’s post test ( B and C ) or Kruskal–Wallis test (one-way ANOVA) followed by Dunn’s multiple comparison test ( D ). Statistically significant differences are indicated only among the different vaccine groups; p values indicate significant differences ( p ?Full size image Four weeks after the boost immunization, all vaccinated animals reached high levels of anti-S IgG in the serum (Fig.? 5B and Supplementary Fig.? 5 ). However, the anti-S IgG levels after the homologous RNA vaccination were significantly higher than in all other groups. Interestingly, this order does not reflect the anti-S response measured four weeks after the prime immunization. Here, the intramuscular injection of Ad5 induced the highest antibody levels, most probably by inducing more potent IgG2a responses than the RNA vaccine (Supplementary Fig.? 6 ). Contrary, the IgG levels detected in BALs were higher in the groups receiving the intranasal Ad5 boost vaccination (Fig.? 5B ). In addition, significantly increased local IgA antibody levels could be detected for both groups in a RBD-specific ELISA (Fig.? 5C ). On a functional level, the higher amounts of RBD-specific antibodies were mirrored by higher neutralizing capacities in the BALs of the groups DNA-Ad5 or RNA-Ad5 (Fig.? 5D ). Interestingly, the high amount of neutralizing antibodies in the sera were not significantly different among the vaccine groups independent of the route of the boost immunization. Since mucosal antibodies might be most important for preventing an initial infection and thereby transmission, we evaluated the protective capacity against SARS-CoV-2 variants in pseudotype-based virus neutralization assays (Fig.? 6 ). Here, the most robust and broadest responses were detected in the BALs of RNA-Ad5 treated animals with decreasing neutralizing potencies against spike proteins from SARS-CoV-2 lineages?D614G to B.1.1.7 (alpha variant)/P.1 (gamma variant) to B.1.351 (beta variant), and finally B.1.617.2 (delta variant). Interestingly, the RNA-Ad5 and DNA-Ad5 schemes resulted in comparable IC75 titres against alpha and delta, but DNA-Ad5 was less potent against the beta variant. This might reflect the different nature of the encoded S protein sequences. Finally, the solely systemic vaccination schedules provoked 4- to 32-fold lower titres of mucosal neutralization against D614G, alpha, beta, and gamma, whereas no neutralization of delta spike-pseudotyped reporter virus could be observed. These data underline the importance of mucosal immunizations in order to provide immediate neutralization of incoming virus at the entry site. Fig. 6: Neutralization of SARS-CoV-2 variants. C57BL/6 mice were vaccinated according to Fig.? 5A . BAL samples were analysed by pseudotype neutralization assays for the neutralization of different SARS-CoV-2 variants ( A – E ). Data points were shown for individual animals and bars represent group medians; RNA-Ad5 n ?=?7, other groups n ?=?8 (out of two independent experiments). The dashed line indicates the lower limit of detection. Data were analysed by Kruskal–Wallis test (one-way ANOVA) followed by Dunn’s multiple comparison. Statistically significant differences are indicated only among the different vaccine groups; p values indicate significant differences ( p ?Full size image Lung-resident memory T cells are efficiently established by a mucosal boost but not by conventional mRNA vaccination . Next, we assessed the induction of systemic and resident T cell memory. Antigen-experienced CD44 + CD8 + T cells isolated from lung tissue were quantitatively most pronounced in the 2x RNA group (Fig.? 7B ). However, by analysing the contribution of tissue-resident (iv?) and vascular (iv+) compartments, a more complex picture emerged. The groups that received two systemic immunizations almost exclusively mounted circulating T cell memory (>95% iv+; Fig.? 7A, B ) and consistent to this, the predominant memory phenotypes were T EFF , T EM , and T CM (Fig.? 7C ). CD103 + CD69 + T RM were not established in the lungs of these animals. In complete contrast, the DNA-Ad5 immunized animals displayed mostly T RM but were lacking substantial numbers of circulating memory cells. Importantly, the RNA-Ad5 strategy induced the most comprehensive T cell memory consisting of both circulating subsets and CD103 + CD69 + T cells in the lung. Fig. 7: Circulating and tissue-resident memory T cell subsets in the lung. C57BL/6 mice were vaccinated according to Fig.? 5A . Antigen-experienced CD8 + T cells were identified by CD44 staining and intravascular staining was used to differentiate between circulating (iv-labelled) and tissue-resident (iv-protected) memory cells. Representative contour plots are shown in ( A ). B The total number of CD44 + CD8 + with the relative contribution of iv? and iv+ cells are summarized for each group. C Within the iv-labelled CD44 + CD8 + population, effector T cells (T EFF ; CD127 - KLRG1 + ), effector memory T cells (T EM ; CD127 + KLRG1 + ), and central memory T cells (T CM ; CD127 + KLRG1 ? CD69 ? CD103 ? ) were defined. Within the iv-protected population, T RM cells were defined as KLRG1 ? CD103 + CD69 + . The gating strategy is shown in Supplementary Fig. 2 . Bars represent group means overlaid with individual data points; all groups n ?=?4. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences are indicated only among the different vaccine groups; p values indicate significant differences ( p ?Full size image The analysis of spike-specific, cytokine producing CD8 + T cells showed a similar compartmentalization. Although the overall numbers of CD107a + , IFNγ + , and TNF + CD8 + T cells were highest in the lungs of the 2x RNA group, these cells were almost exclusively found in the vascular compartment (iv-labelled, Fig.? 8A–C ). The same is true for the homologous immunization with Ad5, albeit reaching much lower percentages of reactive cells. In line with the phenotypic analyses, RNA-Ad5 induced both systemic and local T cell responses, whereas DNA-Ad5 provoked mainly T RM . The trends observed for CD8 + T cell responses in the iv-labelled lung population were largely mirrored by the splenic responses (Fig.? 8D ), further underlining that the former population reflects circulating T cells present in the lung vasculature at the time of sampling. Spike-specific, tissue-resident CD4 + T cell responses were also effectively established by the mucosal boost strategies (Fig.? 9A, B ) and systemic IFNγ-producing CD4 + T cells in the spleen were induced by all vaccine schedules with two RNA shots being the most effective strategy (Fig.? 9D ). Fig. 8: Spike-specific CD8 + T cell responses. C57BL/6 mice were vaccinated according to Fig.? 5A . Lung ( B and C ) and spleen homogenates ( D ) were restimulated with a peptide pool covering major parts of S. The responding CD8 + T cells were identified by intracellular staining for accumulated cytokines or staining for CD107a as degranulation marker. A Representative contour plots showing IFNγ production in iv+ and iv? lung CD8 + T cells. The gating strategy is shown in Supplementary Fig. 3 . Bars represent group means overlaid with individual data points; all groups n ?=?4 (exception: n ?=?3 for DNA-Ad5 in D ). Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences are indicated only among the different vaccine groups; p values indicate significant differences ( p ?cell population positive for all assessed markers. Representative data from one out of three independent experiments with slightly different end time points are shown. Full size image Fig. 9: Spike-specific CD4 + T cell responses. C57BL/6 mice were vaccinated according to Fig.? 5A . Lung ( B and C ) and spleen homogenates ( D ) were restimulated with a peptide pool covering major parts of S. The responding CD4 + T cells were identified by intracellular staining for accumulated cytokines. A Representative contour plots showing IFNγ production in iv+ and iv? lung CD4 + T cells. The gating strategy is shown in Supplementary Fig. 3. Bars represent group means overlaid with individual data points; all groups n ?=?4 (exception: n ?=?3 for DNA-Ad5 in D ). Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences are indicated only among the different vaccine groups; p values indicate significant differences ( p ?cell population positive for all assessed markers. Representative data from one out of three independent experiments with slightly different end time points are shown. Full size image In conclusion, only intranasal vaccination schedules were able to induce profound mucosal immunity in the respiratory tract consisting of neutralizing IgG, IgA, and lung T RM . Compared to DNA-Ad5, the RNA-Ad5 strategy provoked a more efficient neutralization of VOCs and established a comprehensive T cell immunity consisting of both T RM and circulatory T cells. Systemic and mucosal vaccine schedules effectively protect from experimental SARS-CoV-2 infection . In order to assess the protective efficacy of the vaccination strategies, human ACE2 transgenic mice (K18-hACE2) were immunized as described before and challenged four weeks after the boost immunization with 9?×?10 3 FFU of the SARS-CoV-2 strain BavPat1 as previously described 56 . Since the 2x Ad5 immunization was less immunogenic than the 2x RNA immunization, this group was replaced by another 2x Ad vaccination regime consisting of an intramuscular Ad19a prime followed by the established intranasal Ad5 boost (Fig.? 10A ). Seven out of eight unvaccinated control animals reached humane endpoints at day five indicating a severe and lethal course of the disease (Fig.? 10B ). They presented weight loss starting at day four post-infection with a concomitant increase of clinical signs (Fig.? 10C, D ). In contrast, all vaccinated groups were largely protected from weight loss, clinical signs of disease, and mortality (Fig.? 10B–D ). High levels of viral RNA in lung homogenates and BAL fluids were only detected in unvaccinated animals indicating efficient viral replication, while from the vaccinated animals only two of the 2x RNA group had viral RNA copy numbers in the lung above the detection limit (Fig.? 10E ). Similarly, infectious virus was retrieved from the lungs of unvaccinated animals but not from the immunized groups (Fig.? 10F ). Due to the nature of this challenge model, high viral RNA copy numbers were also detected in the brains of na?ve animals (Supplementary Fig.? 7 ). Although viral RNA was still detectable in the brains of most vaccinated animals, the copy numbers were reduced by 4–5 logs, and no significant differences among the vaccine groups could be seen. Fig. 10: Protective efficacy against SARS-CoV-2 infection. A K18-hACE2 mice (2x RNA n ?=?7, other groups n ?=?8) received an intramuscular prime immunization with the spike-encoding DNA (10??g) followed by electroporation, Ad19a-S (10 7 infectious units), or the mRNA vaccine, Comirnaty ? (1??g). Mice from the heterologous prime-boost groups were boosted four weeks later intranasally or intramuscularly with Ad5-S (10 7 infectious units). The 2x RNA group received a second dose of mRNA (1??g) intramuscularly. Four weeks after the boost immunization, mice were infected intranasally with 9?×?10 3 FFU SARS-CoV-2. All animals were monitored daily for survival ( B ), body weight ( C ), and clinical score ( D ). Curves in ( C ) and ( D ) represent group means with SEM. Animals reaching humane endpoints were euthanized and are marked by a cross at the respective time point. Viral RNA copy numbers were assessed in lung homogenates and BAL samples by qRT-PCR ( E ) and infectious virus was retrieved and titrated from lung homogenates ( F ). Data points shown represent viral copy number or virus titre of each animal with the median of each group, whereby circles indicate a survival of 5 days post infection and triangles indicates euthanized mouse according humane endpoints at day 4 (triangle pointing down) or day 5 (triangle pointing up). The dashed line indicates the lower limit of detection. Data were analysed by Kruskal–Wallis test (one-way ANOVA) and Dunn’s Pairwise Multiple Comparison Procedures as post hoc test in comparison to PBS control; p values indicate significant differences ( p ?Full size image Taken together, the mucosal boost strategies were able to fully prevent mortality and symptomatic disease upon experimental SARS-CoV-2 infection. The protective efficacy was equal to the current approved vaccination regimen consisting of two intramuscular injections of Comirnaty ? . .
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