as many lung TRM were generated with MVAOVA s.s. compared to direct pulmonary 23 immunization with MVAOVA, and both routes vaccination protected mice against lethal 24 pulmonary challenge with VACVOVA. Strikingly, MVAOVA s.s.-generated effector T cells 25 exhibited overlapping gene transcriptional profiles to those generated via direct pulmonary 26 immunization. Overall, our data suggest that heterologous MVA vectors delivered via s.s. are 27 uniquely well-suited as vaccine vectors for respiratory pathogens like COVID-19. In addition, 28 MVA delivered via s.s. could represent a more effective dose-sparing smallpox vaccine. 29 Vaccines against viral and bacterial pathogens have become a fundamental part of pediatric and 31 adult patient care 1-4 . Once ubiquitous diseases like smallpox, polio, measles, tetanus, and 32 diphtheria have either been eliminated or substantially reduced in incidence by vaccination in 33 most of the industrialized world. Vaccination against seasonal influenza has been more 34 challenging, and vaccination against HIV has proven elusive 5-7 . Vaccines against emerging 35 diseases like Ebola, SARS, and MERS and most recently COVID-19 are the subject of intense 36 interest and widespread activity [8] [9] [10] . Most vaccines are administered by intramuscular or 37 subcutaneous injection. While readily accessible, skeletal muscle tissue is poorly adapted to 38 initiating immune responses, as is subcutaneous adipose tissue 11 . In contrast, upper layers of the 39 skin are the site of continuous and multiple immune responses over a lifetime 12 . Smallpox 40 vaccination through skin with Vaccinia virus (VACV) has been uniquely successful 2,11 . 41 The eradication of smallpox by worldwide epicutaneous immunization with VACV was 42 the greatest public health achievement of the 20 th century 2 . Since that time, VACV has been 43 employed as a vaccine vector in many settings 13 . However, its use has been limited by 44 unacceptable morbidity, particularly in recipients who are immunocompromised 14 . More 45 recently, Modified Vaccinia Ankara (MVA), a replication-deficient variant of VACV, has come 46 into wider use 15 . Although it lacks ~10% of the parent genome 16 , it retains the immunogenicity 47 of the parent virus and has just been approved by the FDA as a modern alternative for smallpox 48 preventative vaccination 17 . Like VACV, it is also being widely used as heterologous vaccine 49 vector 18 . However, MVA and derivative vectors are almost invariably delivered intramuscularly 50 or subcutaneously 19 . 51 Several important features of smallpox vaccination deserve to be re-emphasized. In the 20 th 52 century, delivery of VACV i.m. was ineffective at conferring protection against smallpox. In contrast, development of a cutaneous "pox" lesions, achieved only after epicutaneous 54 immunization, was considered emblematic of successful protective vaccination, suggesting that 55 this mode of delivery was critically important 14 . In addition, smallpox vaccination was effective 56 in patients with agammagloblulinema, while VACV immunization had disastrous complications 57 in patients with T cell deficiency 20 . This suggested that T cells were critically important for 58 protective immunity 21, 22 . Finally, Variola virus is transmitted via respiratory droplets, suggesting 59 an oropharyngeal-pulmonary mode of transmission 23 . It is notable that murine models of 60 epicutaneous skin immunization with VACV generate memory T cell populations in both skin 61 and lung, and these lung memory T cells protect against lethal pulmonary challenge with this 62 virus 11 . Intramuscular immunization with VACV in these models did not yield comparable 63 protection. This suggests that protection against smallpox is at least in part mediated by T 64 cells 22, 24 , and that skin immunization is an effective means of generating protective memory T 65 cell populations in the lung 11 . 66 In the present study, we asked whether immunization with MVA was more effective and 67 more effective if delivered epicutaneously (s.s.) as compared to intramuscularly (i.m.). We also 68 asked whether skin immunization with an MVA vector generated populations of antigen specific 69 CD8 + T cells in lung as well as skin. In addition, epicutaneous immunization was compared to 70 intradermal, subcutaneous, and intramuscular immunization in generated protective immunity 71 against a lethal pulmonary challenge. Finally, we asked if T cell imprinting by skin draining and 72 lung draining nodes was similar. 
Doses from 10 4 pfu to 10 7 pfu of MVA were used for epicutaneous immunization (s.s.), 78 and after 7 days, lymph node and spleen T cells were harvested and stimulated in vitro with 79 VACV infected splenocytes, after which IFN-γ production was measured. All MVA doses led to 80 significant T cell IFN-γ production, with 10 6 and 10 7 pfu being equally potent (Fig. 1a,b) . Other 81 groups of mice were immunized with these doses, and after 30 days these mice were challenged 82 on the skin with VACV. After 6 days, biopsies of the immunized sites were taken and VACV 83 DNA was measured by PCR. All immunization doses led to diminished VACV DNA at the 84 infected site (compared to unimmunized controls), but 10 6 and 10 7 pfu immunization showed 85 superior protection (Fig. 1c) . Other groups of mice were immunized in an identical manner and 86 were subjected to lethal intranasal infection with VACV at day 30. All unimmunized mice 87 rapidly lost weight and succumbed to the infection. In contrast, 40% of 10 4 , 70% of 10 5 , and 88 100% of 10 6 and 10 7 pfu immunized mice survived the infection (Fig. 1d,e) . Thus, 10 6 pfu is the 89 lowest MVA dose that provides both strong T cell cytokine production as well as optimal 90 protective immunity against skin and pulmonary infection. 91 To test whether delivery of MVA to scarified skin could induce poxvirus-specific 92 immune responses, we inoculated C57BL/6 mice with MVA or Vaccinia Virus (VACV) by 93 scarification. By 7 days after inoculation, a pustular lesion resembling a "pox" reaction had 94 formed at the inoculation site in all the immunized mice. The pox lesions induced by MVA and 95 VACV skin scarification followed similar patterns of evolution (although with different size and 96 kinetics), from macules to papules to vesicles and finally into pustules which ruptured and healed 97 over time with scars (Extended data, Fig. 1) . MVA-induced pox reactions did heal more rapidly 98 than those induced by replication competent VACV (Extended data, Fig. 1) . To determine the safety of MVA in immunocompromised hosts, we next immunized immunodeficient Rag1 -/-100 mice with VACV and MVA, respectively, and followed the mice for several weeks. While both 101 groups of mice lost some weight over the first two weeks, MVA immunized mice rapidly 102 regained the weight and flourished over the next several weeks (Fig. 1g) . In contrast, 100% of 103 the VACV immunized mice developed progressive weight loss and expanding cutaneous lesions 104 of VACV infection, ultimately requiring euthanasia (Fig. 1f-h) Langerin DTA mice and more markedly diminished in Langerin DTR + DT mice (Fig. 1i) . At 112 day 30, skin TRM were significantly diminished in Langerin DTA mice and even more 113 diminished in Langerin DTR+DT mice (Fig. 1i) . This pattern was also true for T cells bearing 114 markers of TCM and TEM. These data suggest that both LC and langerin positive dermal DC play 115 an additive role in optimal antigen presentation of MVA-encoded antigens to T cells. 116 We next compared the anatomical route of vaccine delivery on the T cell response to 117 MVA vaccination. Using CFSE OT-1 loaded mice, MVAOVA was delivered by epicutaneous 118 infection (s.s.), or injected intradermally (i.d.), subcutaneously (s.c.), or intramuscularly (i.m.). 119 Draining lymph nodes were harvested at 60 hours and 5 days, and OT-1 cells were analyzed by 120 FACS. LN from s.s. immunized mice showed roughly 90% of OT-1 proliferating, and 60% 121 making IFN-γ, at 60 hours, with comparable numbers at 5 days (Fig. 2a,c) . Vaccination by i.d.
was less effective, with 71% of OT-1 cells proliferating and 33% making IFN-γ at 60 hours, with 123 modest improvement at 5 days post infection (Fig. 2a,c) . Both s.c. and i.m. showed poor OT-1 124 activation at 60 hours with some improvement at 5 days (Fig. 2a,c) . When lymph node or spleen 125 OT-1 cells were stimulated with antigen, significantly more IFN-γ was produced by OT-1 cells 126 from mice vaccinated via s.s. compared to other routes (Extended data, Fig. 2) . Vaccination via 127 i.d. was intermediate with regard to IFN-γ production, while s.c. and i.m. led to nearly four-fold 128 lower IFN-γ levels (Extended data, Fig. 2) . In terms of absolute numbers of OT-1 cells 129 generated, s.s. was superior to all modes of vaccination, with i.d. being second and both i.m. and 130 s.c. far less effective (Fig. 2b,d) . We next took OT-1 cells from the 5-day post-immunization 131 time point and performed transcriptional profiling on OT-1 cells generated after s.s., i.d., s.c., or 132 i.m., respectively. While there was some overlap, there were surprisingly many differences 133 between T cells generated by different routes of immunization, even at the same day post 134 immunization ( Fig. 2e, Extended data, Fig. 3) . Principal component analysis revealed that Teff 135 generated by s.s. and i.m. were transcriptionally quite distinct. T cells generated after s.s., i.d., 136 and s.c., were more similar but still quite distinct from one another. T cells generated by s.s. and 137 i.d. clustered closely but were still clearly not overlapping. Moreover, s.s generated most 138 abundant skin infiltrating cells at day 5 post immunization (Fig. 2g) . 139 We next examined memory OT-1 T cells generated at 45 days by these four routes of 140 immunization. With regard to TCM, s.s. generated the largest population of these cells, roughly 141 twice as many as i.m. (Fig. 3a) . The difference was even more striking when TEM were 142 examined; here, s.s. generated at least 3-fold more cells than did other modes of immunization, 143 with s.c. being least effective (Fig. 3b) . TRM were then examined, in both skin and lung. 144 Immunization via s.s. generated 3-fold more skin TRM, and more than twice as many TEM, with i.d. being the second most effective route (Fig. 3-f ). Because MVA is often delivered i.m., it is 146 important to note that the number of TRM generated by this route was more than 4-fold lower 147 than by s.s. (Fig. 3 c-f) . Transcriptional profiling showed that at 45 days, OT-1 TEM's still 148 showed non-overlapping PCA clusters from s.s, i.d., s.c., and i.m. immunized mice. In contrast, 149 TCM from the same mice showed transcriptional profiles that were more tightly clustered, 150 indicating that differences between the groups were minimal (Fig. 2e) . Skin TRM could not be 151 compared because insufficient 45-day TRM were generated by i.m. and s.c. immunization. 152 In subsequent experiments, we examined groups of mice vaccinated by these different 153 routes for their ability to respond to a lethal intranasal challenge of VACVOVA. Groups of ten 154 mice assayed 45 days after initial vaccination were subjected to intranasal challenge, and mice 155 were weighed daily after vaccination. Mice that lost >20% of body weight were sacrificed. contrast, mice vaccinated i.d., s.c., or i.m. lost substantial weight (Fig. 3g) , and while 60% of i.d. 159 vaccinated mice survived, only 40% and 30% of mice vaccinated i.m. and s.c., respectively, 160 survived (Fig. 3h) . These results are consistent with the superior production of different memory 161 T cell subsets after vaccination by s.s.. 162 We were struck by the capacity of skin immunization via s.s. to generate both skin TRM 163 and lung TRM. While skin and gut T cell trafficking have been studied extensively, lung T cell 164 trafficking has been studied less comprehensively. We immunized CFSE OT-1 loaded mice with 165 MVAOVA via three routes: s.s. to assess skin homing, intraperitoneally (i.p.) to assess gut 166 homing, and intra-tracheally (i.t.) to assess lung homing. At 60 hours, T cells were collected 167 from the respective draining lymph nodes (inguinal for skin, mesenteric for gut, and mediastinal for lung) and were sorted based on CFSE expression into cells that had not divided (P0) or had 169 divided once through five times (P1-P5; Fig. 4a ). Cells were subjected to transcriptional 170 profiling, and results were analyzed bioinformatically. By principal component analysis, P0 cells 171 from skin, gut, and lung homing nodes clustered near each other (Fig. 4b) . However, as early as 172 P1 and clearly by P2, OT-1 cells activated in different nodes diverged significantly in 173 transcriptional profile. In particular, OT-1 cells from mesenteric nodes were quite distinct from 174 OT-1 cells from inguinal and mediastinal nodes (Fig. 4b) . Interestingly, P1-P5 cells from 175 inguinal (skin draining) node clustered closely with P1-P5 cells from mediastinal (lung draining) 176 nodes, suggesting similar pathways involved in skin and lung homing imprinting (Fig. 4b) . 177 Excluding genes upregulated in all T cell groups, lung and skin homing T cells shared 178 upregulation of 150 genes, compared to 73 and 90 upregulated in only skin or only lung, 179 respectively (Fig. 4c, d) . In contrast, only 11 upregulated genes were shared between skin and 180 gut, and only 36 between lung and gut. Examination of chemokine receptors and integrin genes 181 showed homology between lung and skin, while gut immunization showed unique upregulation 182 of CCR9, α4 and β7 integrins (Fig. 4e) . These data suggest a very similar pattern of gene 183 expression of T cells activated in skin versus lung draining LN, and a pattern in gut draining LN 184 that is very different from lung and skin draining LN. 185 We next directly compared the capacity of skin (s.s.), lung (i.t.), and gut (i.p.) 186 immunization with MVAOVA to generate lung TRM. Mice were immunized by the above routes 187 and after 45 days, lung TRM were analyzed. As expected, lung immunization resulted in the 188 highest number of lung TRM, but skin immunization by s.s. generated more than half as many 189 TRM in lung (Fig. 4f) . In contrast, i.p. immunization resulted in less than 10% of the lung TRM 190 compared to lung immunization (Fig. 4f) . Like skin TRM, lung TRM were CD69 + , CD103 + , CD62L -, KLRG1 -, and expressed E and P selectin ligands (Extended data, Fig. 4) . A 192 companion cohort of mice were subjected to lethal intranasal challenge with VACVOVA. Mice 193 immunized i.t. or s.s. showed mild weight loss but 100% recovery and survival (Fig. 4g,h) . Mice 194 immunized i.p. showed more severe weight loss, and only 60% survived the infectious challenge 195 (Fig. 4g,h) . In another series of experiments, i.t. immunization was compared to s.s. 196 immunization with regard to generation of skin TRM. While s.s. was most efficient at generating 197 skin TRM, lung immunization via i.t. generated 50% of the skin TRM compared to s.s. Competing interests statement 291 The authors declare that they have no competing financial interests. 