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Smallpox vaccination induces a substantial increase in commensal skin bacteria that promote pathology and influence the host response

['Evgeniya V. Shmeleva', 'Department Of Pathology', 'University Of Cambridge', 'Cambridge', 'United Kingdom', 'Mercedes Gomez De Agüero', 'Maurice Müller Laboratories', 'Department For Biomedical Research', 'Universitätsklinik Für Viszerale Chirurgie Und Medizin Inselspital', 'University Of Bern']

Date: 2022-06

Interactions between pathogens, host microbiota and the immune system influence many physiological and pathological processes. In the 20 th century, widespread dermal vaccination with vaccinia virus (VACV) led to the eradication of smallpox but how VACV interacts with the microbiota and whether this influences the efficacy of vaccination are largely unknown. Here we report that intradermal vaccination with VACV induces a large increase in the number of commensal bacteria in infected tissue, which enhance recruitment of inflammatory cells, promote tissue damage and influence the host response. Treatment of vaccinated specific-pathogen-free (SPF) mice with antibiotic, or infection of genetically-matched germ-free (GF) animals caused smaller lesions without alteration in virus titre. Tissue damage correlated with enhanced neutrophil and T cell infiltration and levels of pro-inflammatory tissue cytokines and chemokines. One month after vaccination, GF and both groups of SPF mice had equal numbers of VACV-specific CD8 + T cells and were protected from disease induced by VACV challenge, despite lower levels of VACV-neutralising antibodies observed in GF animals. Thus, skin microbiota may provide an adjuvant-like stimulus during vaccination with VACV and influence the host response to vaccination.

Smallpox was caused by variola virus and was eradicated by widespread dermal vaccination with vaccinia virus (VACV), a related orthopoxvirus of unknown origin. Eradication was declared in 1980 without an understanding of the immunological correlates of protection, or knowledge of the effect of smallpox vaccination on the local microbiota. Here we demonstrate that intradermal infection of mice with VACV induces a ~1000-fold expansion of commensal skin bacteria that influence the recruitment of inflammatory cells into the infected tissue and enhance the size of the vaccination lesion. Antibiotic treatment reduced lesion size without changing virus titres. VACV vaccination induced robust protection from infection with microbiota-independent formation of antigen-specific CD8 + T cellular immunity. We also observed lower levels of VACV neutralising antibodies in genetically-matched germ-free (GF) mice in comparison with specific pathogen free (SPF) controls. Thus, dermal infection by VACV enhanced bacterial growth and these bacteria promote pathology and enhance the innate immune response. This finding has implication for dermal vaccination with live vaccines and may influence their immunogenicity.

In this study, the mouse intradermal model of VACV infection [ 18 ] was used to make a detailed investigation of the recruitment of inflammatory cells into the infected tissue. Previously, the cellular infiltrate in this model was found to contain abundant neutrophils and T cells and was markedly different to the response to respiratory infection [ 19 ]. Neutrophil recruitment is a hallmark of bacterial infection [ 20 ] and this led to the discovery here that the local skin microbiota expanded greatly following dermal VACV infection. Data from vaccinated antibiotic-treated or germ free (GF) mice showed that the pathology at the site of the viral infection is dominated by commensal bacteria, without influencing virus replication. Notably, the depletion of bacteria resulted in an alteration to the cellular infiltration into infected tissue and this might affect the subsequent adaptive immune response.

Vaccine development can be a slow and expensive process and understanding how to deliver antigens optimally to induce strong adaptive immunity remains poor. In particular, understanding how innate immunity leads to development of adaptive immunity is incomplete [ 13 , 14 ]. The activation of dendritic cells by innate immunity enhances antigen presentation and thereby improves the adaptive memory response [ 15 ], but innate immunity may also cause swift elimination of antigens, and so diminish adaptive immunity [ 16 ]. Since bacterial components are sometimes used as adjuvants to enhance innate immunity and vaccine efficacy [ 17 ], studying the role of microbiota following vaccination is important.

Secondary bacterial infection is sometimes a consequence of viral infection; classic examples are bacterial pneumonia following influenza virus, respiratory syncytial virus or parainfluenza virus infection [ 6 ]. These bacterial infections may be promoted by virus-induced tissue damage facilitating bacterial entry into tissue, loss of anti-microbial proteins, or virus-induced immunosuppression enabling invasion by commensal organisms. Bacteria causing these secondary infections are often derived from the host microbiota [ 7 , 8 ]. Bacterial infection has been described following infection by several poxviruses including avipoxvirus [ 9 ], cowpoxvirus [ 10 ], monkeypox virus [ 11 ] and variola virus [ 12 ] although this was not highlighted as a complication of smallpox vaccination [ 2 ].

Vaccinia virus (VACV) is the live vaccine that was used to eradicate smallpox, a disease caused by the related orthopoxvirus, variola virus, and declared eradicated by the World Health Organisation in 1980 [ 1 ]. During the smallpox eradication campaign, early vaccine batches were grown on the flanks of animals and, consequently, were not bacteriologically sterile. Vaccination was achieved by multiple skin puncture using a bifurcated needed containing a drop of vaccine suspended between the two prongs of the needle. Therefore, both the vaccine and the method of vaccination may have introduced bacteria into the vaccination site. Following vaccination a local lesion developed that healed in 2–3 weeks and left a characteristic vaccination scar. A careful study of smallpox vaccination outcomes in the USA described several serious dermal or neurological complications of vaccination [ 2 ], although bacterial infection of the vaccination site was not highlighted. Vaccination induced long-lasting humoral and cellular responses, although the precise immunological correlates of protection remain uncertain [ 3 ]. VACV encodes scores of proteins that block the innate immune response and induce local immunosuppression [ 4 ] and manipulation of these proteins can affect virulence and adaptive immunity [ 5 ].

These data do not distinguish between the deficiency of antibody-dependent immunity in GF animals per se and the decline in humoral immune responses due to a lack of stimulatory signals from microbiota. We attempted to address this by comparing vaccination of GF and S. aureus gnotobiotic mice, which were generated by mono-colonisation of GF animals with S. aureus strain NCTC 8325 two weeks prior to the i.d. vaccination with VACV. Unfortunately, mice reconstituted with S. aureus, differed from GF controls with lower body mass and reduced subcutaneous and visceral fat, possibly suggesting ongoing inflammation after colonisation with S. aureus. For these reasons, S. aureus colonised animals proved unsuitable for comparison.

(A-C): SPF or GF mice (n = 5) were injected i.d. with 10 4 PFU of VACV. SPF, AB-group received i.p. antibiotic treatment for 10 d from 1 d p.i. SPF, NoAB group received i.p. injections with PBS. Spleens and serum samples were obtained at 34 d p.i. (A) Absolute number of splenic CD8 + and CD4 + T cells. Bars represent means. (B) Absolute number of VACV-specific splenic CD8 + T cells. Bars represent means. (C) VACV-neutralising antibody responses determined by plaque-reduction neutralisation test. IC50, half maximal inhibitory concentration. Bars represent means. All experiments (except with GF mice) were performed twice and representative data from one experiment are shown. The experiments with GF mice were performed once and in a facility separate from SPF experiments. (D-F): GF and SPF groups of mice (n = 5 per group) were vaccinated (“vac”) i.d. in both ears with 10 4 PFU of VACV WR per ear. “Naive” groups (n = 3–5) were not vaccinated. AB and NoAB groups were treated as in (A-C). One month p.i. groups were challenged i.n. with (D, E) 3 × 10 6 PFU or (F) 6 × 10 6 PFU of VACV WR. Data represent the weight of each mouse compared to the weight of the same animal before challenge (d 0). The percentages for each group are means with SEM.

To assess the role of microbiota in the generation of VACV-specific adaptive immunity, we measured VACV-specific CD8 + T cell numbers and anti-VACV neutralising antibodies in samples obtained from SPF groups, with or without antibiotic treatment, and GF mice one month after vaccination. Absolute numbers of splenic CD4 + and CD8 + T cells, as well as VACV-specific CD8 + cells, were similar in all three groups of animals ( Fig 7A and 7B , and S7 Fig ). Also, neither absolute numbers of splenocytes, all CD45 + , nor CD45 + non-T cells differed significantly in vaccinated SPF and GF mice ( S8 Fig ). However, the levels of VACV neutralising antibodies were 3-fold lower in GF animals than in either group of SPF animals ( Fig 7C ). Despite the reduced antibody response in the vaccinated GF mice, all groups showed protection from disease upon intranasal challenge with a lethal dose of VACV ( Fig 7D–7F ). Each SPF group showed a small (~10%) transient weight loss after challenge before recovery. These data indicate that the GF mice may have an impaired capacity to generate neutralising antibody following i.d. vaccination with VACV, but that this does not affect their capacity to develop protective immunity against re-challenge in this model.

SPF or GF mice (n = 5–7 per group per time point) were injected i.d. with 10 4 PFU of VACV. SPF, AB-group received antibiotic (ceftriaxone) i.p. daily from 1 d p.i.; SPF, NoAB group received i.p. injections with PBS. (A) Tissue levels of cytokines and chemokines were measured by multiplex assay (Luminex). Data are shown as the fold change from the baseline (untreated intact ear samples). Means are shown. The experiment was performed twice and data from one representative experiment are shown. (B) Tissue levels of cytokines and chemokines in GF and SPF groups at d 10 p.i. measured by multiplex assay (Luminex). Graphs show results two independent experiments. Dotted lines indicate the lowest standards (or highest standard for CCL7). The experiment with GF animals was performed once.

To analyse the local inflammatory environment further, we measured an array of cytokines and chemokines in the ear tissue at d 5, 8 and 12 p.i. in control and antibiotic-treated mice. Consistent with the smaller lesions and reduction in cellular infiltration, among 17 different inflammatory mediators measured, there were reductions of IFNɣ, TNFα, CCL2, CCL4, CCL7, CXCL1 and CXCL10 levels in the antibiotic-treated group compared to controls at d 8, and also in the amount of CCL4 and CCL7 at d 12 p.i. ( Fig 6A ). Similar differences were also evident when comparing infected GF mice with SPF mice. Levels of IFNɣ, TNFα, CCL2, CCL7, CXCL1 and CXCL10 were considerably diminished in the GF mice ( Fig 6B ). These data indicate that the presence of skin microbiota accelerates overall immune cell recruitment and cytokine/chemokine production in VACV-infected skin.

Two groups of SPF mice were infected i.d. with 10 4 PFU of VACV. From d 1 p.i. onwards, one group (AB) received antibiotic (ceftriaxone) i.p. daily. The second group (NoAB) received PBS i.p. (A) Numbers of myeloid and lymphoid cell of subpopulations were measured in ear tissues at 2, 5, 7 and 12 d p.i. (n = 5 per group per time point). DC & MΦ, dendritic cells and macrophages; Mon, monocytes, TCRγδ, TCRγδ + T cells; TCRαβ CD4, TCRγδ - CD4 + T cells; TCRαβ CD8, TCRγδ - CD8 + T cells. Medians are shown; p values were determined by the Mann-Whitney test, * = p<0.05, ** = p<0.01. (B) Spearman correlation analysis of lesion sizes versus numbers of neutrophils, Ly6C+monocytes, TCRγδ T cells or TCRαβ (TCRγδ - ) T cells recruited to site of infection. The experiment was performed twice and representative data from one experiment are shown.

Next, we compared the local immune response to VACV infection in ear tissue of antibiotic-treated and untreated SPF animals. Recruitment of immune cells was low on d 1–4 p.i. ( Fig 5A ) but there was a sharp increase of Ly6C + inflammatory monocytes on d 5, followed by large leukocyte infiltration at d 7. Notably, the antibiotic-treated group showed a substantial reduction in the recruitment of multiple subpopulations of myeloid and lymphoid cells at d 7 p.i. ( Fig 5A ). By d 12 p.i., several subpopulations of leukocytes had declined, except for neutrophils in the NoAB-group, which continued to increase. During infection there was a notable positive correlation between the lesion size and the numbers of infiltrating neutrophils or TCRαβ T cells and this correlation did not hold for any other cell type ( Fig 5B ).

Specific pathogen free (SPF) or germ-free (GF) mice (n = 5–15 per group) were injected i.d. with 10 4 PFU of VACV WR. Group SPF AB was injected daily i.p. with antibiotic ceftriaxone from d 1 to 11 p.i. Group SPF NoAB received i.p. injections with PBS. (A) Images of haematoxylin and eosin (H&E) stained transverse ear sections 8 and 12 d p.i. Bars = 300 μm. Regions of necrotic tissue are labelled ‘a’ and regions of inflamed tissue labelled ‘b’. (B) Images of H&E stained transverse ear sections collected from GF mice 10 d p.i. Bars = 300 μm.

Histological examination of infected lesions showed that VACV-infected ear tissue from SPF mice contained significant cellular infiltrate and general signs of inflammation throughout the tissue as well as regions of necrosis at day 8 p.i. ( Fig 4A ). At day 12 p.i. the majority of the ear tissue in the NoAB-group of animals was necrotic whereas there was substantially less necrosis and tissue damage in the AB-group ( Fig 4A ). A striking difference in histological changes after VACV infection was observed when comparing GF and SPF mice. Infected ears of the GF animals had significantly reduced cell infiltration and only very mild tissue necrosis compared with infected ears from SFP mice ( Fig 4B ). Collectively, these observations indicate that skin microbiota promote lesion development after VACV infection and the smaller lesion sizes and lack of pathological tissue changes in GF and antibiotic-treated groups were due to the absence of microbiota or antibiotic-induced suppression of bacterial expansion.

Next the virus titres in infected tissue with or without antibiotic treatment were determined. Notably, despite considerable differences in lesion size, the viral titres in infected ear tissues were unaltered ( Fig 3C ). Seeding of the ear tissues obtained from AB-animals on blood agar plates did not result in bacterial growth, which might be explained by the presence of antibiotic in the tissue extracts that prevent bacterial growth, rather than by the total absence of bacteria from infected lesion. This indicated that: i) antibiotic treatment has no impact on virus replication, and ii) lesion size was influenced by the presence of bacteria rather than virus titre.

Topical administration of antibiotic creams on the infected ears also resulted in a reduction of lesion size following VACV infection ( S5 Fig ). However, it was not possible to control the dose administered because the animals tend to groom themselves and each other, leading to ingestion of the applied creams. Antibiotic treatment can influence antiviral immunity when administered orally [ 26 ] and so to minimise the influence on gut microbiota, the antibiotic was administered i.p. for all our experiments. To avoid changes in microbiota prior to infection, antibiotic treatment was only started from d 1 p.i. Throughout ceftriaxone treatment, the health status of animals and their leukocyte composition in peripheral blood, spleen and bone marrow were unaltered ( S6 Fig ).

Specific pathogen free (SPF) or germ-free (GF) mice (n = 5–15 per group) were injected intradermally with 10 4 PFU of VACV. SPF, AB animals received antibiotic i.p. from 1 d p.i.; SPF, NoAB animals received i.p. injections with PBS. (A-B) Ear lesion sizes of AB and NoAB-groups (A) or GF or SPF mice (B). Means and SEM are shown. Statistical analysis by two-way RM ANOVA test. (C) VACV titres in ear tissue p.i. PFU, plaque-forming units. NS–non-significant by Mann-Whitney test. Experiments (except for those with GF mice) were performed at least twice and representative data from one experiment are shown. The experiments with GF mice were performed once.

To investigate the influence of skin microbiota on lesion formation, a broad-spectrum bactericidal antibiotic (AB, ceftriaxone) was administered intraperitoneally (i.p.) over 13 d after VACV infection to suppress bacterial growth. C57BL/6 specific-pathogen-free (SPF) mice were infected i.d. with VACV, and one group received antibiotic treatment (SPF, AB-group), while another received phosphate-buffered saline (SPF, NoAB-group). Antibiotic treatment caused a significant reduction in lesion size after infection with VACV strain WR, a widely used laboratory strain of VACV ( Fig 3A ). A similar observation was made following infection with VACV strain Lister, a strain used widely for smallpox vaccination in humans ( S4 Fig ). To confirm that this effect was due to the presence of bacteria, rather than a non-specific consequence of antibiotic treatment, genetically-matched germ-free (GF) mice were infected i.d. with VACV strain WR. These mice showed delayed lesion formation and had a three-fold reduction in lesion size compared to SPF animals ( Fig 3B ).

Ear tissues were collected from SPF mice before (intact) and at 2, 5, 8 and 12 d p.i. with 10 4 PFU of VACV and at d 8 post PBS injection (mock), n = 15 per group per time point. Next generation sequencing was performed for DNA samples extracted from tissue. (A) Principal Component Analysis (PCA) of microbiome of genus taxon. PCA was conducted including all taxon with a minimum abundance of 0.1%. Ellipses represent a 40% confidence interval around the cluster centroid. (B) Relative abundance of most prevalent taxa of bacterial genera. (C) Heatmap of bacterial genera with hierarchical clustering. Bacterial genera with a minimum abundance of 1% in at least five samples was used for creation of the heat map. Five clusters are colour-coded with red, blue, green, violet and orange. “D”–day post injection.

Analysis of bacterial sequences at the family, genus and species levels showed significant changes in the composition of skin microbiota following infection with VACV ( Fig 2 , S2 and S3 Figs). Although mock-infection itself resulted in moderate modification of skin microbiome, the most significant changes occurred at d 5, 8 and 12 p.i., especially d 8 and 12, which was clearly demonstrated by principal component analysis ( Fig 2A , S2B and S3A Figs) as well as multivariate beta diversity analysis ( S1 Table ). The relative abundance of some dominant genera in intact skin such as Sporosarcina and Staphylococcus decreased at late times post VACV infection, while the proportion of Streptococcus, Enhydrobacter and Corynebacterium increased ( Fig 2B ). Perturbation of skin microbiome also occurred within the same genus, for instance VACV infection resulted in a rise of relative abundance of Staphylococcus aureus, a well-known opportunist, over other species of Staphylococcus ( S3B Fig ). While averaged data visualised as stack plots cannot represent in full the observed microbiota shift, heatmaps ( Fig 2C , S2A and S3C Figs) illustrate that the majority of d 8 and 12 VACV-infected samples had very pronounced dominance of ~1–3 bacterial genera, which differs from normal microbiota. However, specific dominant taxa varied from sample to sample. Therefore, there is no particular type of bacterium that spread favourably after VACV infection. Instead, random different bacteria are increased in infected samples. Thus, VACV infection leads to changes in the skin microbiome and the expansion of opportunistic bacteria.

To investigate the identity of the bacteria present during infection and to compare this with the microbiome in uninfected skin, 16S ribosomal RNA gene (rRNA) sequencing was performed. So that bacteria from the skin surface and within lesions was included, total genomic DNA was extracted directly from whole VACV-infected and non-infected ear tissues and next generation sequencing of the amplified V4 region of bacterial 16S rRNA was performed. Sequencing was carried out from material extracted directly from infected tissues without prior culturing to avoid bias introduced by the culturability of bacteria that might be present.

To investigate if there is an expansion of bacteria after i.d. infection with VACV, bacterial colony-forming units (CFUs) in infected tissue were quantified. CFUs in VACV-infected tissue was at least 100-fold greater than mock-infected tissue at all time points ( Fig 1C ) and at the peak at 10 d p.i. was about 1000-fold greater than control ( Fig 1D ). Given that the virus inoculum was bacteriologically sterile, these results suggest increased growth of commensal bacteria after VACV infection, possibly due to local virus-induced immunosuppression. Furthermore, as not all bacteria can grow under the experimental conditions (oxygenated blood agar) used in our experiments, the bacterial presence is likely to be underestimated and so larger than demonstrated in our study. Notably, the time of maximum number of bacteria and neutrophils correlated with maximum lesion size, suggesting a link between skin microbiota and the severity of pathology.

C57BL/6 SPF mice were injected i.d. with 10 4 PFU of VACV strain WR or PBS (mock) and ear tissues were collected at different times post injection. (A) Absolute numbers of different myeloid cells present in ear tissues at d 9 p.i. (n = 5 per time point). DC & MΦ: dendritic cells and macrophages; Eosin: eosinophils; Mon: monocytes; Neutr: neutrophils. Medians are shown. (B) Absolute numbers of neutrophils infiltrating ear tissues at different times p.i. Mock: mock-infected control, 5 d post intradermal injection of PBS (n = 3–5 per time point). Medians are shown. (C) Bacterial colonies grown from homogenised ear tissues and their 10-fold serial dilutions seeded on blood agar. (D) Bacteria colony-forming unit (CFU) counts of ear samples (n = 3). Results of CFU counts for mock samples at 7 d post PBS injection were obtained from a separate experiment. Means and SEM are shown. The experiments were performed at least twice and representative data from one experiment are shown.

Here the local innate and cellular immune responses to i.d. vaccination with VACV were characterised in detail, including quantifying subpopulations of infiltrating myeloid cells throughout infection. Flow cytometry showed a substantial increase in CD45 + CD3 - CD5 - CD19 - NK1.1 - CD11c - Siglec-F - Ly6G + cells (neutrophils) in ear tissue at all times p.i. ( Fig 1A and 1B ). Along with the surface markers, the morphological characteristics of this population (spherical ~8–10 μm cells with highly segmented nuclei) confirmed their identity as neutrophils ( S1 Fig ) [ 23 ]. The number of neutrophils increased up to 10 d p.i., and by d 5 had increased ~130 fold compared to mock-infected tissue ( Fig 1B ), coinciding with the first appearance of a dermal lesion. Neutrophils are part of the innate immune system and are front-line effector cells for defense against bacterial infection [ 20 ]. Their abundance in VACV-infected tissue suggested that bacterial co-infection might be present.

Intradermal (i.d.) infection of the mouse ear pinnae with VACV strain Western Reserve (WR) results in the development of a skin lesion at the site of infection that mimics smallpox vaccination in man. A lesion appears at d 6 post infection (p.i.), increases gradually to about d 10 and resolves by d 21 [ 18 ]. The model has been used to study the contribution of individual VACV proteins to virulence and immunogenicity [ 21 , 22 ].

Discussion

Host microbiota influence many physiological and pathological processes throughout life, including pre- and postnatal development, aging, metabolism and immunity [27,28]. Commensal organisms play an important role in the defense against pathogens, not only by preventing microorganisms from colonising epithelial niches, but also by influencing homeostasis, maturation and regulation of the immune system [24,29]. During infection by bacteria or viruses, host microbiota may either promote or diminish pathology [30,31] and the ability of commensals to influence inflammation via regulation of innate immune responses suggests that microbiota could be manipulated for host health benefit [24,29,32].

This study reports a 1000-fold increase in local skin microbiota after i.d. vaccination with VACV (Fig 1). This expanded bacterial load dominates the development of skin lesions following vaccination (Fig 3 and S5 Fig) and enhances the innate immune response by increasing leukocyte infiltration and local cytokine/chemokine (Figs 5 and 6). Notably, the formation of the antigen-specific CD8+ T cell response was found to be microbiota independent (Fig 7). Antibody-mediated immunity, on the other hand, was reduced 3-fold in GF animals (Fig 7), although further investigation is needed to determine the degree to which this observation is microbiota-dependent.

Multiple factors provide skin barrier function and control microorganisms on the skin surface including acidic pH, high-salt, antimicrobial proteins (defensins, cathelicidins, peptidoglycan recognition proteins, ribonucleases and psoriasin), free fatty acids, ceramide and filaggrin [8,33,34]. Keratinocytes and sebaceous glands, which synthesise these substances [35,36], are highly susceptible to VACV infection [37]. VACV-induced shut-off of host protein synthesis in infected cells [38] may diminish production of antimicrobial substances and therefore induce changes in local microbiota. Here, VACV infection is shown to induce large changes in skin microbiota, especially later after infection (Fig 2, S2 and S3 Figs), with changing dominance of different bacterial taxa. These dominant taxa varied between infected animals, indicating colonisation by random opportunistic bacteria, a common feature for secondary bacterial infections [39].

The ability of bacterial commensals to rapidly expand has been demonstrated previously; for instance, the doubling time of S. aureus in human nasal cavity is 2–4 h [40], which suggests a bacterial increase of 60-2000-fold in 24 h is possible (not accounting for bacterial death and assuming sufficient nutrient availability). The bacterial growth rate in a host organism depends on multiple factors including immune status of the host. During experimental sepsis the speed of bacterial expansion can be more than 100-fold per 24 h, when the immune system loses control of the infection [41]. Here we observed up to 100-fold increase of bacterial presence within the first 24 h post VACV-infection (Fig 1C and 1D), which can be explained by the fact that VACV is a very potent suppressor of the local innate immune response [5]. Further bacterial expansion over two weeks post i.d. VACV infection was only 10-fold (Fig 1C and 1D) possibly slowing down due to nutrient limitations and, after 5 dpi, the activation of the immune system and leukocyte infiltration (Figs 5 and 6), which limits bacterial growth and increases their death.

Although increased bacterial, but not viral, load correlated with enhanced lesion size, these increased bacteria were not sufficient for lesion formation, because bacteria increased 1–5 d p.i., before lesions appeared on d 6 (Figs 1C, 1D, 3A and 3B). Furthermore, GF mice still developed lesions, although these were delayed and diminished (Fig 3B). Inflammation was also reduced in GF animals following dermal scarification of VACV into the mouse tail [42]. VACV infection may disrupt skin integrity due to VACV-induced cytopathic effect [43] or cell motility [44] that enabled bacteria to enter tissue and proliferate. Local immunosuppression induced by VACV proteins that block innate immunity [4,5] including production of glucocorticoids by a VACV-encoded steroid biosynthetic enzyme [45,46] might facilitate further bacterial growth. Notably, a VACV strain lacking this enzyme induced smaller dermal lesions [22]. In the future, it would be interesting to measure microbiota after infection by this and other VACV mutants lacking specific immunomodulators, because the lesion sizes depend on the VACV strain used for infection [22], and are influenced by viral immunomodulatory proteins, without any direct impact on viral replication.

These observations with VACV have similarity to those made with the trypanosome Leishmania major in which skin lesions were smaller in GF mice than SPF mice [24]. That study also demonstrated the importance of the skin resident immune system in the progression of inflammation [24]. Inflammation after exposure to pathogens can be caused and/or aggravated by opportunistic commensals [25]. Bacterial invasion provides potent activators of innate immunity that promote cytokine/chemokine production and immune cell recruitment [47]. Consistent with this, larger lesions were associated with more neutrophils and T cells (Fig 5B), and increased tissue cytokines and chemokines (Fig 6). Interestingly, depletion of Ly6G+ cells results in delayed lesion healing post epicutaneous infection with VACV [37], consistent with the involvement of neutrophils in bacterial clearance and the resolution of inflammation. However, whether bacterial infection per se [48] or immune system activation by bacteria [49] induce tissue damage is unknown. During SARS-CoV-2 infection, increased neutrophil counts correlate with severe pathology and hyper-inflammation. Specifically, neutrophils are elevated in the second week from the onset of symptoms and are higher in severe COVID-19 in comparison with moderate disease [50–53]. Similarly, the presence of neutrophils predisposes to enhanced respiratory syncytial virus infection [54]. In our study, the massive recruitment of immune cells in infected tissue coincided with the appearance of the skin lesion. 16S rRNA gene sequencing did not reveal an expansion of a particular bacterial taxa, suggesting that the enhancement of the innate immune response does not seem to be associated with specific bacteria; rather, various types of bacteria are able to spread in the lesions (Fig 2C, S2A and S3C Figs) and induce inflammation. Therefore, three factors may influence lesion development after i.d. vaccination with VACV: 1) virus-induced tissue damage, 2) tissue destruction by invading bacteria, and 3) immunopathology. Lesion size in this i.d. model is also influenced by the age and strain of mice, and strain of VACV [22].

Bacterial lipopolysaccharides, flagellin, DNA and toxins are used as adjuvants to stimulate innate immunity and vaccine immunogenicity, mainly via enhanced antibody production [17]. Consistent with that, the innate immune response to vaccination of GF mice was lower than for genetically-matched SFP animals (Fig 6B), and whereas VACV-specific CD8+ T cell memory did not depend on microbiota, the humoral immune response was impaired in GF mice compared with SPF animals (Fig 7A–7C). However, necessarily experiments on GF mice and SPF mice were performed in separate animal facilities, which might influence the observed differences in VACV-neutralising antibodies. Nonetheless, no reduction of anti-VACV antibodies in antibiotic-treated SPF animals was seen (Fig 7C), despite reduced cytokine/chemokine levels. This maybe because antibiotic treatment does not eliminate bacteria, but limits their growth and spread, and therefore, bacteria and bacterial pathogen-associated molecular patterns (PAMPs) are still present at lower levels. These might be sufficient to provide the adjuvant-like stimulus required for a robust adaptive immune response. Others also reported reduced antibody responses in both GF and antibiotic-treated animals [55,56]. However, these studies used animals pretreated with antibiotic per os to induce alterations in the gut microbiome, while we administrated antibiotics i.p. only from 1 d after infection by VACV. Previous studies suggested that the influence of microbiota on T-cell memory may depend on the inoculation route or pathogen [55]. The influence of host microbiota on vaccination efficacy is a relatively new area of investigation and differing results have been reported [57–60]. Indeed, vaccine immunogenicity is influenced by multiple factors, such as age, genetics, previous exposures to various pathogens, assessment of distant or local microbiota, route of vaccination and the type of vaccine, making the design of experiments and interpretation of results challenging. Notably, in our study, the induction of CD8+ T cell memory following i.d. vaccination with VACV is microbiota-independent and not perturbed by the presence of bacteria in the skin, gut or elsewhere (Fig 7).

Although VACV-specific memory CD8+ T cells may have functional differences in the absence of microbiota [61], and despite reduced titres of VACV-specific neutralising antibodies, GF animals were protected as well as SPF mice from i.n. challenge with VACV one month (Fig 7D–7F). SPF mice that had been treated with antibiotic were also protected as well as control mice at 3 months after vaccination (S9 Fig). This reflects the solid immunity induced by vaccination in all groups and consequential resistance to challenge with a high dose of VACV. These conditions might not be suitable to reveal subtle differences in protection between the groups used, because the vaccination in our study may have resulted in the humoral or/and cellular adaptive immune response reaching a threshold, leading to full protection against challenge. Lower immunising doses or longer duration between vaccination and challenge might show differences. Nonetheless, this study illustrates the possible influence of skin microbiota for the generation of anti-VACV antibodies and this observation is important for vaccine development, as intradermal vaccination with other live vaccines might induce local expansion and changes in the composition of microbiota, and therefore, may influence the efficacy of vaccination.

In conclusion, this study demonstrates that i.d. vaccination with VACV induces substantial local bacterial infection derived from skin microbiota, which function as adjuvant to increase the innate immune response leading to greater skin inflammation. The enhanced bacteria induced formation of larger dermal lesions either by direct bacterial-induced cytotoxicity or immunopathology. Vaccination with VACV generates robust long lasting immune responses that eradicated smallpox from man [1] and can protect mice from lethal challenge [21,60]. As shown here, such protective immunity does not require additional stimuli from microbiota or other adjuvants. However, further activation of the innate immune system provided by skin microbiota might have a beneficial effect on the immunogenicity of other vaccines.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009854

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