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Dynamic single-cell RNA sequencing reveals BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation

COVID-19 continues to exact a toll on human health despite the availability of several vaccines. Bacillus Calmette Guérin (BCG) has been shown to confer heterologous immune protection against viral infections including COVID-19 and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model together with immune profiling and single cell RNA sequencing (scRNAseq). We observed that BCG reduced both lung SCV2 viral load and bronchopneumonia. This was accompanied by an increase in lung alveolar macrophages, a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. Single cell transcriptome profiling showed that BCG uniquely recruits immunoglobulin-producing plasma cells to the lung suggesting accelerated antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, and differentially expressed gene (DEG) analysis showed a transcriptional shift away from exhaustion markers and towards antigen presentation and repair. Similarly, BCG enhanced lung recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, with both cell-types also showing reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

2 Abstract: 26 COVID-19 continues to exact a toll on human health despite the availability of several vaccines.

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Bacillus Calmette Guérin (BCG) has been shown to confer heterologous immune protection 28 against viral infections including COVID-19 and has been proposed as vaccine against SARS-

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CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden 30 Syrian hamster model together with immune profiling and single cell RNA sequencing 31 (scRNAseq). We observed that BCG reduced both lung SCV2 viral load and bronchopneumonia.

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In this study, we employed the non-lethal, self-resolving golden Syrian hamster model to 106 evaluate the ability of intravenous BCG to protect against SCV2. We evaluated its impact on lung 107 viral titers, tissue pathology using histopathology, immunohistochemistry (IHC) and PET/CT   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2022. ; https://doi.org/10.1101/2022.03.15.484018 doi: bioRxiv preprint 6 hamsters on both D4 and D7 but was absent in all BCG-vaccinated hamsters on D4 and found in 130 only 25% on D7. Similarly, 100% of unvaccinated hamsters showed neutrophilic infiltration at D4 131 while only 50% of BCG-vaccinated hamsters showed this finding (Fig. 1D). In contrast BCG 132 vaccination showed a trend for increased macrophage infiltration (80% at D4) while only 60% of 133 unvaccinated animals showed this finding (Fig. 1E). Regarding the well-described SCV2-134 mediated lymphopenia, BCG vaccination was able to partially correct infiltrating lung lymphocytes 135 with BCG-vaccinated hamster lungs also showing significantly higher levels of lung lymphocytes 136 (CD3 positivity) both by traditional histology and immunohistochemistry (Fig. 1F, Fig. S1A), and 137 this was true for perivascular lymphocytic infiltration as well (Fig. S1B). As expected, all BCG 138 vaccinated hamsters showed granuloma formation, a phenomenon that was uniformly absent in 139 unvaccinated animals (Fig. S1C). Thus, prior BCG vaccination reduced SCV2-induced 140 bronchopneumonia with its concomitant neutrophilic infiltration while simultaneously preventing 141 lung lymphopenia and enhancing lung macrophage levels.

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We also evaluated the effect of BCG vaccination on lung viral load by measuring infectious viral 143 particles (TCID50) and viral RNA in lung homogenates. We observed a significant decrease in 144 TCID50 in BCG vaccinated animals at D4 (Fig. 1G), and a trend of reduced viral mRNA (Fig. 1H) 145 at this same time point. TCID50 and viral mRNA levels were significantly lower at D7 compared to 146 D4 and levels BCG-vaccinated animals did not differ from the unvaccinated ones likely because 147 of progressive disease resolution in both groups.
148 149 BCG vaccination blunts SCV2-mediated lung T cell lymphopenia, enhances macrophage 150 lung recruitment, and reduces lung infiltration by granulocytes. To assess overall cell 151 population fluxes in hamster lungs, we performed multicolor flow cytometry. We found that lung T 152 cell infiltration (CD3 + cells) was dramatically boosted by BCG vaccination in the absence of SCV2 153 (45% of live cells) compared to age-matched healthy animals (18%) ( Fig. 2A). In unvaccinated 154 animals challenged with SCV2, the total lung CD3 + population was reduced at D4 and D7 to 4-155 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2022. ; https://doi.org/10.1101/2022.03.15.484018 doi: bioRxiv preprint 7 6%; while BCG vaccinated, SCV2 challenged animals showed a trend to have nearly twice as 156 many CD3 + cells (10-12%) at these same time points (Fig 2A) supporting our histopathological 157 observations. This same effect of BCG preventing T cell depletion in the lung was present to an 158 even greater extent among lung CD4 + T cells (Fig. 2B). We did not observe this same pattern in 159 hamster spleens suggesting that the observed effects of BCG vaccination were limited to the site 160 of active infection in the lungs (Fig. S2 A-B). Similarly, BCG vaccination in the absence of SCV2 161 infection prompted a pronounced macrophage recruitment to the lung (55% of live cells) 162 compared to healthy (unvaccinated) animals (5%) (Fig. 2C). And while SCV2 infection led to a 163 modest macrophage lung recruitment by D7 (12% of live cells), previously BCG vaccinated 164 animals showed significantly elevated macrophage populations in the lungs (30% of live cells) on 165 both D4 and D7 (Fig. 2C). In contrast, for granulocytes, we found that SCV2-challenged, 166 unvaccinated hamster lungs revealed dramatic infiltration by polymorphonuclear leukocytes 167 (PMNs) on D4 (80% of live cells) and D7 (72%) (Fig. 2D) in accordance with the profound 168 bronchopneumonia observed (Fig. 1B-C). Pre-vaccination with BCG had the effect of limiting the 169 SCV2 mediated granulocytic infiltration to levels of 30% on D4 and 32% on D7 which were levels 170 essentially equivalent to those in the lungs of vaccinated, unchallenged hamsters. A modest 171 increase in macrophages at both D4 and D7 in the splenic compartment, not statistically 172 significant, was observed in BCG-vaccinated, SCV2-infected animals compared to unvaccinated 173 SCV2 infected animals (Fig. S2C). Splenic granulocytes trended to be lower in BCG vaccinated 174 animals compared to unvaccinated animals (Fig. S2D), similar to the observations in the lungs.

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Single cell transcriptional profiling in lung cells in BCG vaccinated and unvaccinated 177 hamsters. To obtain a higher resolution of pulmonary immune responses linked to BCG 178 immunization during SCV2 infection, hamster lungs were examined using droplet-based single 179 cell RNA sequencing (10X Genomics) during the peak (day 4) and resolution (day 7) phases of 180 COVID-19. BCG-vaccinated, unchallenged hamster lungs were also studied. A total of 13 181 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   (Table   191 S2). These included lymphoid, myeloid, and non-immune cell types (Fig 3A-C). These cell 198 infection in vaccinated and unvaccinated hamsters. We were able to identify 10 distinct 199 lymphocyte cell types (Fig. 4A, S4A-B). Among the CD4 + T cells in hamster lungs, we were able 200 to distinguish Th1, Th17, Treg (regulatory T cells) and Tmem (memory T cells) cells primarily 201 based on Ifng, Itgb7, Foxp3, and Lef1 expression, respectively ( Fig. S4C-D). CTLs (cytotoxic T 202 cells) showed high Gzmk and Gzma but low Cd4 expression. Plasma cells were identified by 203 their elevated expression of Jchain and Mzb1 (Fig. S4C-D). As may be seen in CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   found that Th1 cells in unvaccinated, SCV2-challenged showed evidence exhaustion markers 228 ( Fig 4C) and a high degree of mitochondrial respiratory gene expression and translation (Fig 4D), 229 consistent with mitochondrial dysfunction in T cells that have been previously reported (32). In 230 contrast, in Th1 cells from BCG-vaccinated, SCV2 challenged animals, pathways related to T cell 231 activation, T cell immunity, and antigen processing were upregulated suggesting greater 232 functional immunity (Fig. 4E).

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In contrast, in Th1 cells from non-vaccinated, SCV2-challenged hamsters, we found DEGs that 245 were associated with markers of exhaustion and ongoing viral infection (Fig. 4F,G). Markers of   (Fig. 5A, Fig. S6A-B). Alveolar macrophages (AMs) are relatively 259 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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We identified two clusters of AMs, the first identified by the classic AM marker Siglecf (Siglecf+ 261 AMs) and the second identified by the presence of the scavenger receptor Marco (Marco+ AMs).

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BCG vaccination has been shown to induce recruitment of Siglecf+ AMs to the lung in healthy 263 human volunteers (42), and consistent with this, while we observed virtually no Siglecf+ AMs in 264 healthy hamster lungs, this population was strongly induced by BCG vaccination (Fig. S5C). In 265 contrast, scavenging Marco+ AMs were present in high numbers in healthy hamster lungs, and 266 BCG vaccination caused ~2-fold increased in this population (Fig. S5C). Unvaccinated, SCV-267 infected hamsters showed a high degree of depletion of both categories of AMs, but prior BCG 268 vaccination served to prevent the loss of both AMs from the lung (Fig 5A, S5C).

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Interstitial macrophages (IM) are more inflammatory than AMs and are recruited from the 271 periphery during lung infection (43,44). In addition to traditional IMs, we identified a second 272 population which strongly expressed Isg15 (Isg15+ macrophages) indicating that they are highly 273 responsive to IFN I and hence likely to be hyperinflammatory. Both IMs and Isg15+ macrophages 274 were virtually absent in healthy hamster lungs (Fig. S5C) and in unvaccinated hamsters, but both 275 of these cell types were recruited to the lungs in high numbers following SCV2 challenge.

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Interestingly, prior BCG-vaccination significantly blunted this recruitment (Fig 5A, S5C). In   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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Non-immune cells are modulated by BCG vaccination. Since we did not enrich for immune 358 cells prior to scRNAseq analysis, we were able to investigate non-immune cell responses. We 359 were able to identify 5 types of non-immune cells (AT2, endothelial, ciliated, AT1, and fibroblasts) 360 in hamster lungs. We identified both type I alveolar cells (AT1 cells), which are non-replicative,

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This study is the first to show that intravenous BCG protects against SCV2 infection in hamsters.

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We found a significant reduction of the SCV2 viral titer in BCG vaccinated animals at the peak of

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. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  infected with SCV2 and were sacrificed 30 days post-vaccination to serve as controls (Fig. 1A).       (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made      accession code GSE162208. We processed the data using the similar strategy described above 616 to filter low quality cells. Then MapQuery function in Seurat (v4.0.4) was performed to identify cell 617 types using our own data reference. In brief, we transferred cell type labels from our reference 618 data. Second, we integrated reference with query by correcting the low-dimensional embeddings.

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. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 15, 2022. ; https://doi.org/10.1101/2022.03.15.484018 doi: bioRxiv preprint 26 Finally, we projected the query data onto the UMAP structure of the reference, which allowed us 620 to compare the abundance of the same cell type between datasets.     (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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