Smallpox was eradicated world-wide by immunization with VACV delivered to skin with a bifurcated needle, a process known as skin scarification (s.s.)1-3
. We tested the hypothesis that rVACV immunization via s.s. is critical to the generation of superior protective immune responses. Using an established murine model of VACV s.s.4,5
, we immunized C57BL/6 mice with rVACV by either s.s. or hypodermic injection routes. We showed that mice vaccinated by s.s. with rVACV generated more IFN-γ-producing CD8+
T cells (), a superior recall IFN-γ response () and superior humoral responses (). We next immunized mice with rVACV by s.c. injection, with or without simultaneous epidermal disruption with a scarification needle (mock s.s.). Cellular and humoral responses were equivalent in these two groups, and were inferior to the rVACV-s.s. group (Supplementary Fig. 1a,b
online). Heat-inactivated (Δ) rVACV delivered via s.s. also failed to induce significant cellular or antibody response (Supplementary Fig. 1c,d
online). These data suggest that live VACV infection of disrupted epidermis is critical to superiority of s.s over other vaccination routes. We compared viral mRNA and protein expression following delivery of rVACV via s.s. and s.c routes. Despite equivalent initial viral loads (Supplementary Fig. 2a
online), s.s resulted in significantly higher viral gene expression at the inoculation site (Supplementary Fig. 2b,c
online). Epidermis and follicular epithelium were infected confluently after s.s., but not s.c. immunization (Supplementary Fig. 2c
online), resulting in a higher available antigen dose.
Inoculation with rVACV via barrier-disrupted epidermis generates significantly stronger cellular and humoral responses compared to s.c., i.d. and i.m. injection
We recently showed that, following rVACV scarification, antigen-specific T cells are imprinted with skin-homing markers in draining lymph nodes (dLN) and rapidly migrate into infected skin4
. We predicted that rVACV delivery via s.s. route would be highly protective against cutaneous challenge. Memory mice previously immunized with rVACV via different routes were challenged with a secondary rVACV infection to skin, and viral load at the challenge site was measured 6 d later. Mice immunized via s.s. had completely cleared virus from the infected site (> 6-log viral load reduction), while mice immunized by all other routes showed demonstratable but incomplete viral clearance (). These data indicate that s.s. rVACV vaccination induces fundamentally superior protective immunity in skin.
rVACV scarification generates superior protective immunity against cutaneous viral challenge that is mediated by skin-targeted TEM
The mode of protection against cutaneous challenge was distinct for i.p. and s.s. immunization. The moderate protection following i.p vaccination could be abrogated by T cell depletion and was entirely absent in antibody deficient μMT mice (). However, the s.s– induced protection against skin challenge remained intact in μMT mice, and was significantly compromised only after T cell depletion (). This is consistent with a significantly stronger recall T cell response following cutaneous challenge in s.s.-immunized mice compared to i.p.- immunized mice, in both normal and μMT mice (Supplementary Fig. 3
online). Therefore, T cell memory generated by rVACV s.s. immunization is both necessary and sufficient for the protection against cutaneous challenge.
We adoptively transferred OT-I transgenic T cells, specific for the Ovalbumin peptide SIINFEKL (Ova257–264
), 1 d before immunization with an rVACV that expresses Ova257–264
under an early gene promoter6
. OT-I cells were readily found in the skin of s.s. immunized memory mice, both prior to () and 4 d after () secondary cutaneous challenge, demonstrating efficient generation and recruitment of skin-homing TEM
cells by s.s. route. We have reported previously that both VACV-specific TEM
are generated after s.s. with rVACV4
. We next asked whether the protective skin immunity in s.s. immunized mice was mediated by TEM
already resident to skin before secondary challenge, or by newly activated TCM
from LN. T cell egress from lymphoid tissues was blocked by treating mice prior to and during cutaneous challenge with FTY7207,8
. FTY720 treatment induced pronounced lymphocytopenia via sequestration of lymphocytes in LN ()7,8
. Moreover, it led to a decrease of OT-I cells in skin and a concurrent accumulation of OT-I cells in dLN in cutaneously challenged mice (). Despite this, viral clearance from skin was unaffected by FTY720 treatment (). These results suggest that the skin-resident TEM
generated by the original rVACV skin scarification are highly effective in the control of virus upon subsequent cutaneous challenge. Activation of TCM
OT-I cells in dLN and their subsequent recruitment to skin after challenge was not required for the complete and rapid elimination of virus by 6 d. The importance of tissue resident TEM
is supported by recent studies of HSV infection9,10
. The efficient generation of skin-resident TEM
population was only achieved by rVACV immunization via s.s. route, as increased numbers of CD3+
T cells in skin tissues of s.s. immunized mice, but not mice in the injection groups, were demonstrated histopathologically both before and after secondary cutaneous challenge ( and Supplementary Fig. 4
online). These observations explain the superior protection against cutaneous challenge afforded by rVACV s.s. immunization.
These data prompted us to evaluate the efficacy of rVACV scarification for protection against a tumor challenge in skin. Mice immunized with rVACV expressing OVA257–264
via different routes were inoculated intradermally with the OVA-expressing B16 melanoma cell line MO511
. One week after MO5 implantation, tumor growth was evident at the injection site of unimmunized controls (), all of which experienced rapid tumor growth and became moribund within 1 month (). By 5 weeks after challenge, s.c.-immunized mice experienced 100% mortality, i.d.- and i.m.-immunized mice showed 75% mortality, and i.p.-immunized mice experienced 50% mortality (), with all surviving animals harboring large tumors (). In contrast, s.s.-immunized mice showed 100% survival by the end of the experiment (50 d after challenge) (). These results indicate that rVACV immunization via s.s. is highly effective in generating skin-targeted memory T cells recognizing antigens on cutaneous tumors.
Immunization with rVACV via s.s. route provided superior protection against cutaneous melanoma challenge
Smallpox, caused by Variola major virus, is transmitted primarily via respiratory droplets. To investigate how rVACV scarification also provides superior protection for respiratory tissues, we intranasally challenged rVACV memory mice, previously immunized by different routes, with lethal doses of pathogenic WR-VACV. Mice immunized via the s.c., i.d., or i.m. injection all showed significant weight loss after challenge, and were only partially protected from lethality (). In contrast, mice immunized by s.s. not only survived, but were completely protected from clinical illness (as judged by absence of weight loss) (). Vaccination via s.s. protected mice against the lethal respiratory challenge even at 3-log lower doses of rVACV vaccine (Supplementary Fig. 5
Immunization with rVACV as well as non-replicative MVA via s.s. route is highly effective to protect mice against lethal respiratory WR-VACV challenge
We used μMT immune mice or Wt immune mice depleted of T cells to explore the mechanism of this protection against lethal intranasal challenge. After rVACV s.s. immunization, both μMT immune mice and T cell-depleted WT immune mice were fully protected from illness and death, indicating that either T cell memory or antibodies were sufficient to provide complete protection against this lethal challenge (). Only T cell depletion in s.s. immunized μMT memory mice abrogated protection (). FTY720-treated s.s. immunized μMT memory mice were partially protected against the lethality of the intranasal challenge ( and Supplementary Fig. 6
). This suggests that protective TEM
lining upper respiratory mucosa are generated by s.s. immunization. The different clinical outcome after intranasal challenge between μMT memory mice with and without FTY720 treatment suggests that activation of TCM
in LN draining respiratory mucosa, and the subsequent recruitment of TEM
to the respiratory tract, are essential for full protection against respiratory infectious challenge. Collectively, these data indicate that even in the complete absence of an antibody response, VACV-specific TCM
together provide optimal protective immunity against lethal respiratory challenge in s.s. immunized mice.
Nationwide smallpox vaccination with VACV is precluded by an unacceptably high incidence of morbidity after vaccination, particularly in patients with atopic disorders12-18
. The non-replicating MVA is an attractive alternative to VACV with an impressive safety profile19-22
. MVA vaccines are typically delivered by i.m. injection. Skin scarification was not tested in MVA vaccination studies. We immunized C57BL/6 mice with MVA via s.s. This elicited cellular and humoral immune responses to VACV in a dose-dependent manner, and provided dose-dependent protection against lethal intranasal challenge with WR-VACV (Supplementary Fig. 7
online). Importantly, a single immunization with 2 × 106
plaque forming unit (pfu) MVA via s.s. route, but not i.m. injection, provided complete protection against morbidity and mortality in this model (). This is consistent with the significantly stronger immune responses following MVA s.s. immunization (Supplementary Fig. 8
Taken together, the data presented here establish that immunization with rVACV vaccines, including non-replicative strains, via superficially injured skin (i.e., s.s.), provides robust and anatomically flexible protection associated with a highly effective peripheral TEM
response without requirement for neutralizing antibody. The mechanism by which s.s. is so much more effective than other immunization routes is incompletely understood. VACV infection of epidermal keratinocytes after s.s. may trigger cascade of pro-inflammatory molecule production23-25
, as well as provide a higher dose of antigen, both of which may enhance the generation of optimal immunity. We hope that these insights will pave the way for the design of safe affordable and effective VACV-based immunization for infectious diseases and cancer.