Identification of natural killer markers associated with fatal outcome in COVID-19 patients.

Introduction: Increasing evidence has shown that coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunological response. Previous studies have demonstrated that natural killer (NK) cell dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of NK cell markers as a driver of death in the most critically ill patients. Methods: We enrolled 50 non-vaccinated hospitalized patients infected with the initial virus or the alpha variant of SARS-CoV-2 with moderate or severe illness, to evaluate phenotypic and functional features of NK cells. Results: Here, we show that, consistent with previous studies, evolution NK cells from COVID-19 patients are more activated, with the decreased activation of natural cytotoxicity receptors and impaired cytotoxicity and IFN-γ production, in association with disease regardless of the SARS-CoV-2 strain. Fatality was observed in 6 of 17 patients with severe disease; NK cells from all of these patients displayed a peculiar phenotype of an activated memory-like phenotype associated with massive TNF-α production. Discussion: These data suggest that fatal COVID-19 infection is driven by an uncoordinated inflammatory response in part mediated by a specific subset of activated NK cells.

The immunological effects of intradermal particle-based vaccine delivery using a novel microinjection needle studied in a human skin explant model.

For intradermal (ID) immunisation, novel needle-based delivery systems have been proposed as a better alternative to the Mantoux method. However, the penetration depth of needles in the human skin and its effect on immune cells residing in the different layers of the skin has not been analyzed. A novel and user-friendly silicon microinjection needle (Bella-muTM) has been developed, which allows for a perpendicular injection due to its short needle length (1.4-1.8 mm) and ultrashort bevel. We aimed to characterize the performance of this microinjection needle in the context of the delivery of a particle-based outer membrane vesicle (OMV) vaccine using an ex vivo human skin explant model. We compared the needles of 1.4 and 1.8 mm with the conventional Mantoux method to investigate the depth of vaccine injection and the capacity of the skin antigen-presenting cell (APC) to phagocytose the OMVs. The 1.4 mm needle deposited the antigen closer to the epidermis than the 1.8 mm needle or the Mantoux method. Consequently, activation of epidermal Langerhans cells was significantly higher as determined by dendrite shortening. We found that five different subsets of dermal APCs are able to phagocytose the OMV vaccine, irrespective of the device or injection method. ID delivery using the 1.4 mm needle of a OMV-based vaccine allowed epidermal and dermal APC targeting, with superior activation of Langerhans cells. This study indicates that the use of a microinjection needle improves the delivery of vaccines in the human skin.

Intradermal administration of the pneumococcal conjugate vaccine in mice results in lower antibody responses as compared to intramuscular administration.

INTRODUCTION: Several studies have shown that intradermal vaccination leads to improved immune responses. In addition, lowering vaccine doses will reduce costs and therefore potentially increase coverage. To determine whether intradermal delivery enhances the antibody responses against the 13-valent pneumococcal conjugate vaccine (PCV13), we compared intradermally and intramuscularly vaccinated mice. METHODS: Mice were immunized with PCV13, either intradermally or intramuscularly and CFU-counts in the nasal tissue were determined three or seven days after intranasal colonization with a serotype 4 clinical strain. Antibody concentrations against all thirteen polysaccharides were measured in blood and mucosal samples using a fluorescent-bead-based multiplex immunoassay. RESULTS: Antibody levels in both serum and mucosal samples were higher in the intramuscularly vaccinated group as compared to the intradermally vaccinated group. No protection against S. pneumoniae intranasal colonization was observed for either vaccination route. CONCLUSIONS: Intradermal vaccination was inferior to intramuscular immunization in inducing serotype-specific antibodies.

Memory CD4+ T-Cell Lymphocytic Angiopathy in Fatal Forms of COVID-19 Pulmonary Infection.

The immunopathological pulmonary mechanisms leading to Coronavirus Disease (COVID-19)-related death in adults remain poorly understood. Bronchoalveolar lavage (BAL) and peripheral blood sampling were performed in 74 steroid and non-steroid-treated intensive care unit (ICU) patients (23-75 years; 44 survivors). Peripheral effector SARS-CoV-2-specific T cells were detected in 34/58 cases, mainly directed against the S1 portion of the spike protein. The BAL lymphocytosis consisted of T cells, while the mean CD4/CD8 ratio was 1.80 in non-steroid- treated patients and 1.14 in steroid-treated patients. Moreover, strong BAL SARS-CoV-2 specific T-cell responses were detected in 4/4 surviving and 3/3 non-surviving patients. Serum IFN-γ and IL-6 levels were decreased in steroid-treated patients when compared to non-steroid treated patients. In the lung samples from 3 (1 non-ICU and 2 ICU) additional deceased cases, a lymphocytic memory CD4 T-cell angiopathy colocalizing with SARS-CoV-2 was also observed. Taken together, these data show that disease severity occurs despite strong antiviral CD4 T cell-specific responses migrating to the lung, which could suggest a pathogenic role for perivascular memory CD4 T cells upon fatal COVID-19 pneumonia.

Dichotomy in Neutralizing Antibody Induction to Peptide-Conjugated Vaccine in Squalene Emulsion Contrast With Aluminum Hydroxide Formulation.

W614A-3S peptide is a modified 3S motif of the HIV-gp41 (mutation W614A). We previously detected the presence of natural neutralizing antibodies directed against W614A-3S peptide (NAbs) in long-term non-progressor HIV+ patients. Here, we compared the efficacy of W614A-3S peptide formulated in either squalene emulsion (SQE) or in aluminum hydroxide (Alum) in inducing broadly-NAbs (bNAbs). Rabbit and mouse models were used to screen the induction of bNAbs following 4 immunizations. SQE was more efficient than Alum formulation in inducing W614A-3S-specific bNAbs with up to 67%-93% of HIV strains neutralized. We then analyzed the quality of peptide-specific murine B cells by single-cell gene expression by quantitative reverse transcription-PCR and single-cell V(D)J sequencing. We found more frequent germinal center B cells in SQE than in Alum, albeit with a different gene expression profile. The V(D)J sequencing of W614A-3S-specific BCR showed significant differences in BCR sequences and validates the dichotomy between adjuvant formulations. All sixteen BCR sequences which were cloned were specific of peptide. Adjuvant formulations of W614A-3S-peptide-conjugated immunogen impact the quantity and quality of B cell immune responses at both the gene expression level and BCR sequence.

LOX-1-Expressing Immature Neutrophils Identify Critically-Ill COVID-19 Patients at Risk of Thrombotic Complications.

Background: Lymphopenia and the neutrophil/lymphocyte ratio may have prognostic value in COVID-19 severity. Objective: We investigated neutrophil subsets and functions in blood and bronchoalveolar lavage (BAL) of COVID-19 patients on the basis of patients' clinical characteristics. Methods: We used a multiparametric cytometry profiling based to mature and immature neutrophil markers in 146 critical or severe COVID-19 patients. Results: The Discovery study (38 patients, first pandemic wave) showed that 80% of Intensive Care Unit (ICU) patients develop strong myelemia with CD10-CD64+ immature neutrophils (ImNs). Cellular profiling revealed three distinct neutrophil subsets expressing either the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the interleukin-3 receptor alpha (CD123), or programmed death-ligand 1 (PD-L1) overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1- or CD123-expressing ImNs is positively correlated with clinical severity, cytokine storm (IL-1ß, IL-6, IL-8, TNFα), acute respiratory distress syndrome (ARDS), and thrombosis. BALs of patients with ARDS were highly enriched in LOX-1-expressing ImN subsets and in antimicrobial neutrophil factors. A validation study (118 patients, second pandemic wave) confirmed and strengthened the association of the proportion of ImN subsets with disease severity, invasive ventilation, and death. Only high proportions of LOX-1-expressing ImNs remained strongly associated with a high risk of severe thrombosis independently of the plasma antimicrobial neutrophil factors, suggesting an independent association of ImN markers with their functions. Conclusion: LOX-1-expressing ImNs may help identifying COVID-19 patients at high risk of severity and thrombosis complications.

CD8+PD-L1+CXCR3+ polyfunctional T cell abundances are associated with survival in critical SARS-CoV-2-infected patients.

The importance of the adaptive T cell response in the control and resolution of viral infection has been well established. However, the nature of T cell-mediated viral control mechanisms in life-threatening stages of COVID-19 has yet to be determined. The aim of the present study was to determine the function and phenotype of T cell populations associated with survival or death of patients with COVID-19 in intensive care as a result of phenotypic and functional profiling by mass cytometry. Increased frequencies of circulating, polyfunctional CD4+CXCR5+HLA-DR+ stem cell memory T cells (Tscms) and decreased proportions of granzyme B-expressing and perforin-expressing effector memory T cells were detected in recovered and deceased patients, respectively. The higher abundance of polyfunctional PD-L1+CXCR3+CD8+ effector T cells (Teffs), CXCR5+HLA-DR+ Tscms, and anti-nucleocapsid (anti-NC) cytokine-producing T cells permitted us to differentiate between recovered and deceased patients. The results from a principal component analysis show an imbalance in the T cell compartment that allowed for the separation of recovered and deceased patients. The paucity of circulating PD-L1+CXCR3+CD8+ Teffs and NC-specific CD8+ T cells accurately forecasts fatal disease outcome. This study provides insight into the nature of the T cell populations involved in the control of COVID-19 and therefore might impact T cell-based vaccine designs for this infectious disease.

Strategies for Immunomonitoring after Vaccination and during Infection.

Immunomonitoring is the study of an individual's immune responses over the course of vaccination or infection. In the infectious context, exploring the innate and adaptive immune responses will help to investigate their contribution to viral control or toxicity. After vaccination, immunomonitoring of the correlate(s) and surrogate(s) of protection is a major asset for measuring vaccine immune efficacy. Conventional immunomonitoring methods include antibody-based technologies that are easy to use. However, promising sensitive high-throughput technologies allowed the emergence of holistic approaches. This raises the question of data integration methods and tools. These approaches allow us to increase our knowledge on immune mechanisms as well as the identification of key effectors of the immune response. However, the depiction of relevant findings requires a well-rounded consideration beforehand about the hypotheses, conception, organization and objectives of the immunomonitoring. Therefore, well-standardized and comprehensive studies fuel insight to design more efficient, rationale-based vaccines and therapeutics to fight against infectious diseases. Hence, we will illustrate this review with examples of the immunomonitoring approaches used during vaccination and the COVID-19 pandemic.

Immune Profiles Identification by Vaccinomics After MVA Immunization in Randomized Clinical Study.

Background: Our previous work has demonstrated the benefits of transcutaneous immunization in targeting Langerhans cells and preferentially inducing CD8 T-cell responses. Methods: In this randomized phase Ib clinical trial including 20 HIV uninfected volunteers, we compared the safety and immunogenicity of the MVA recombinant vaccine expressing HIV-B antigen (MVA-B) by transcutaneous and intramuscular routes. We hypothesized that the quality of innate and adaptive immunity differs according to the route of immunization and explored the quality of the vector vaccine-induced immune responses. We also investigated the early blood transcriptome and serum cytokine levels to identify innate events correlated with the strength and quality of adaptive immunity. Results: We demonstrate that MVA-B vaccine is safe by both routes, but that the quality and intensity of both innate and adaptive immunity differ significantly. Transcutaneous vaccination promoted CD8 responses in the absence of antibodies and slightly affected gene expression, involving mainly genes associated with metabolic pathways. Intramuscular vaccination, on the other hand, drove robust changes in the expression of genes involved in IL-6 and interferon signalling pathways, mainly those associated with humoral responses, and also some levels of CD8 response. Conclusion: Thus, vaccine delivery route perturbs early innate responses that shape the quality of adaptive immunity. Clinical Trial Registration: http://ClinicalTrials.gov, identifier PER-073-13.

New GMP manufacturing processes to obtain thermostable HIV-1 gp41 virosomes under solid forms for various mucosal vaccination routes.

The main objective of the MACIVIVA European consortium was to develop new Good Manufacturing Practice pilot lines for manufacturing thermostable vaccines with stabilized antigens on influenza virosomes as enveloped virus-like particles. The HIV-1 gp41-derived antigens anchored in the virosome membrane, along with the adjuvant 3M-052 (TLR7/8 agonist) on the same particle, served as a candidate vaccine for the proof of concept for establishing manufacturing processes, which can be directly applied or adapted to other virosomal vaccines or lipid-based particles. Heat spray-dried powders suitable for nasal or oral delivery, and freeze-dried sublingual tablets were successfully developed as solid dosage forms for mucosal vaccination. The antigenic properties of vaccinal antigens with key gp41 epitopes were maintained, preserving the original immunogenicity of the starting liquid form, and also when solid forms were exposed to high temperature (40 °C) for up to 3 months, with minimal antigen and adjuvant content variation. Virosomes reconstituted from the powder forms remained as free particles with similar size, virosome uptake by antigen-presenting cells in vitro was comparable to virosomes from the liquid form, and the presence of excipients specific to each solid form did not prevent virosome transport to the draining lymph nodes of immunized mice. Virosome integrity was also preserved during exposure to <-15 °C, mimicking accidental freezing conditions. These "ready to use and all-in-one" thermostable needle-free virosomal HIV-1 mucosal vaccines offer the advantage of simplified logistics with a lower dependence on the cold chain during shipments and distribution.

Mechanisms of innate events during skin reaction following intradermal injection of seasonal influenza vaccine.

The skin plays a crucial role in host defences against microbial attack and the innate cells must provide the immune system with sufficient information to organize these defences. This unique feature makes the skin a promising site for vaccine administration. Although cellular innate immune events during vaccination have been widely studied, initial events remain poorly understood. Our aim is to determine molecular biomarkers of skin innate reaction after intradermal (i.d.) immunization. Using an ex vivo human explant model from healthy donors, we investigated by NanoLC-MS/MS analysis and MALDI-MSI imaging, to detect innate molecular events (lipids, metabolites, proteins) few hours after i.d. administration of seasonal trivalent influenza vaccine (TIV). This multimodel approach allowed to identify early molecules differentially expressed in dermal and epidermal layers at 4 and 18 h after TIV immunization compared with control PBS. In the dermis, the most relevant network of proteins upregulated were related to cell-to-cell signalling and cell trafficking. The molecular signatures detected were associated with chemokines such as CXCL8, a chemoattractant of neutrophils. In the epidermis, the most relevant networks were associated with activation of antigen-presenting cells and related to CXCL10. Our study proposes a novel step-forward approach to identify biomarkers of skin innate reaction. SIGNIFICANCE: To our knowledge, there is no study analyzing innate molecular reaction to vaccines at the site of skin immunization. What is known on skin reaction is based on macroscopic (erythema, redness…), microscopic (epidermal and dermal tissues) and cellular events (inflammatory cell infiltrate). Therefore, we propose a multimodal approach to analyze molecular events at the site of vaccine injection on skin tissue. We identified early molecular networks involved biological functions such cell migration, cell-to-cell interaction and antigen presentation, validated by chemokine expression, in the epidermis and dermis, then could be used as early indicator of success in immunization.

Innate gene signature distinguishes humoral versus cytotoxic responses to influenza vaccination.

BACKGROUND: Systems vaccinology allows cutting-edge analysis of innate biomarkers of vaccine efficacy. We have been exploring novel strategies to shape the adaptive immune response, by targeting innate immune cells through novel immunization routes. METHODS: This randomized phase I/II clinical study (n=60 healthy subjects aged 18-45 years old) used transcriptomic analysis to discover early biomarkers of immune response quality after transcutaneous (t.c.), intradermal (i.d.), and intramuscular (i.m.) administration of a trivalent influenza vaccine (TIV season 2012-2013) (1:1:1 ratio). Safety and immunogenicity (hemagglutinin inhibition (HI), microneutralization (MN) antibodies and CD4, CD8 effector T cells) were measured at baseline Day (D)0 and at D21. Blood transcriptome was analyzed at D0 and D1. RESULTS: TIV-specific CD8+GranzymeB+(GRZ) T cells appeared in more individuals immunized by the t.c. and i.d. routes, while immunization by the i.d. and i.m. routes prompted high levels of HI antibody titers and MN against A/H1N1 and A/H3N2 influenza viral strains. The early innate gene signature anticipated immunological outcome by discriminating two clusters of individuals with either distinct humoral or CD8 cytotoxic responses. Several pathways explained this dichotomy confirmed by nine genes and serum level of CXCL10 were correlated with either TIV-specific cytotoxic CD8+GRZ+ T-cell or antibody responses. A logistic regression analysis demonstrated that these nine genes and serum levels of CXCL10 (D1/D0) best foreseen TIV-specific CD8+GRZ+ T-cell and antibody responses at D21. CONCLUSION: This study provides new insight into the impact of immunization routes and innate signature in the quality of adaptive immune responses.

Critical Role for Skin-Derived Migratory DCs and Langerhans Cells in TFH and GC Responses after Intradermal Immunization.

Intradermal delivery of antigen represents a potent route of immunization that involves multiple blood- and skin-derived dendritic cell subpopulations endowed with specialized functions and dynamics in their ability to prime naïve CD4+ T cells in the draining lymph nodes. However, their individual contributions to the generation of CD4+ T follicular helper (TFH) cells and germinal centers (GCs) remain to be understood. We found that intradermal immunization of mice with a particle-based vaccine induced robust TFH and germinal center B-cell responses in skin draining lymph nodes, which were completely abrogated when skin cell emigration was prevented. However, in this later condition, both lymph node-resident and blood-derived inflammatory cells access the antigen in the draining lymph nodes but are not able to induce TFH cell differentiation. Rather, only skin-derived dendritic cells up-regulated key genes related to TFH cell development in the draining lymph nodes. Depletion of Langerhans cells partially abrogated TFH and germinal center B-cell responses. Thus, after intradermal immunization, only skin-derived migratory dendritic cells, including Langerhans cells, permit the generation of TFH cells and germinal centers. Identifying the relative contributions of tissue and lymphoid organ dendritic cell subsets in generating humoral immune responses is of great importance for the development of tailored vaccines.

Characterization of pandemic influenza immune memory signature after vaccination or infection.

The magnitude, quality, and maintenance of immunological memory after infection or vaccination must be considered for future design of effective influenza vaccines. In 2009, the influenza pandemic produced disease that ranged from mild to severe, even fatal, illness in infected healthy adults and led to vaccination of a portion of the population with the adjuvanted, inactivated influenza A(H1N1)pdm09 vaccine. Here, we have proposed a multiparameter quantitative and qualitative approach to comparing adaptive immune memory to influenza 1 year after mild or severe infection or vaccination. One year after antigen encounter, severely ill subjects maintained high levels of humoral and polyfunctional effector/memory CD4⁺ T cells responses, while mildly ill and vaccinated subjects retained strong cellular immunity, as indicated by high levels of mucosal homing and degranulation markers on IFN-γ⁺ antigen-specific T cells. A principal component analysis distinguished 3 distinct clusters of individuals. The first group comprised vaccinated and mildly ill subjects, while clusters 2 and 3 included mainly infected individuals. Each cluster had immune memory profiles that differed in magnitude and quality. These data provide evidence that there are substantial similarities between the antiinfluenza response that mildly ill and vaccinated individuals develop and that this immune memory signature is different from that seen in severely ill individuals.

Longitudinal and integrative biomodeling of effector and memory immune compartments after inactivated influenza vaccination.

Most vaccines, including those against influenza, were developed by focusing solely on humoral response for protection. However, vaccination activates different adaptive compartments that might play a role in protection. We took advantage of the pandemic 2009 A(H1N1) influenza vaccination to conduct a longitudinal integrative multiparametric analysis of seven immune parameters in vaccinated subjects. A global analysis underlined the predominance of induction of humoral and CD4 T cell responses, whereas pandemic 2009 A(H1N1)-specific CD8 responses did not improve after vaccination. A principal component analysis and hierarchical clustering of individuals showed a differential upregulation of influenza vaccine-specific immunity including hemagglutination inhibition titers, IgA(+) and IgG(+) Ab-secreting cells, effector CD4 or CD8 T cell frequencies at day 21 among individuals, suggesting a fine-tuning of the immune parameters after vaccination. This is related to individual factors including the magnitude and quality of influenza-specific immune responses before vaccination. We propose a graphical delineation of immune determinants that would be essential for a better understanding of vaccine-induced immunity in vaccination strategies.

Recombinant retrovirus-derived virus-like particle-based vaccines induce hepatitis C virus-specific cellular and neutralizing immune responses in mice.

While the immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood, it is now admitted that an effective vaccine against HCV will need to induce both cellular and humoral immune responses and address viral heterogeneity to prevent immune escape. We developed a vaccine platform specifically aimed at inducing such responses against HCV antigens displayed by recombinant retrovirus-based virus-like particles (VLPs) made of Gag of murine leukemia virus. Both ex vivo produced VLPs and plasmid DNA encoding VLPs can be used as vaccines. Here, we report that immunizations with plasmid DNA forming VLPs pseudotyped with HCV E1 and E2 envelope glycoproteins (HCV-specific plasmo-retroVLPs) induce strong T-cell-mediated immune responses that can be optimized by using proper DNA delivery methods and/or genetic adjuvants. Additionally, multigenotype or multi-specific T-cell responses were observed after immunization with plasmids that encode VLPs pseudotyped with E1E2 derived from numerous viral genotypes and/or displaying NS3 antigen in capsid proteins. While homologous prime-boost immunizations with HCV-specific plasmo-retroVLPs or ex vivo produced VLPs induce a low level of specific antibody responses, optimal combination of plasmo-retroVLPs and VLPs was identified for inducing HCV-specific T-cell and B-cell responses as well as neutralizing antibodies. Altogether, these results have important meanings for the development of anti-HCV preventive vaccines and exemplify the flexibility and potential of our retrovirus-based platform in inducing broad cellular and humoral immune responses.

Neutrophils transport antigen from the dermis to the bone marrow, initiating a source of memory CD8+ T cells.

The bone marrow (BM) has been identified as a possible organ for T cell priming, yet the fundamental mechanisms of a polyclonal immune response in the BM remain unknown. We found that after intradermal injection of modified vaccinia Ankara virus, unexpected sources of newly primed polyclonal virus-specific CD8(+), but not CD4(+), T cells were localized in the BM and the draining lymph nodes (dLNs) prior to blood circulation. We identified neutrophils as the virus-carrier cells from the dermis to the BM. In both neutrophil-depleted and Ccr1(-/-) mice, virus-specific BM CD8(+) responses were lost. Myeloid antigen-presenting cells were required for BM CD8(+) T cell priming. A systems biology analysis of dLN and BM virus-specific CD8(+) T cells revealed distinct transcriptional and multifunctional profiles for cells primed in each organ. We provide direct evidence for how antigen is transported to the BM, providing a source of virus-specific memory CD8(+) T cells.

Cutting edge: Protective effect of CX3CR1+ dendritic cells in a vaccinia virus pulmonary infection model.

The protective host immune response to viral infections requires both effective innate and adaptive immune responses. Cross-talk between the two responses is coordinated by the chemokine network and professional APCs such as dendritic cells (DCs). In mice, subpopulations of myeloid DCs in peripheral tissues such as lungs and in blood express CX3CR1 depending on the inflammation state. We thus examined the host response of mice deficient in the chemokine receptor CX3CR1 to an intranasal vaccinia virus infection. CX3CR1-deficient mice displayed significantly more severe morbidity and mortality compared with control wild-type mice within 10 d following vaccinia virus infection. CX3CR1(-/-) mice had increased viral loads and a reduced T cell response compared with wild-type mice. Finally, an adoptive transfer of CX3CR1(+/+) DCs completely protected CX3CR1(-/-) mice to a previously lethal infection. This study therefore opens up the possibility of novel antiviral therapeutics targeting lung DC recruitment.

Intradermal immunization triggers epidermal Langerhans cell mobilization required for CD8 T-cell immune responses.

The potential of the skin immune system for the generation of both powerful humoral and cellular immune responses is now well established. However, the mechanisms responsible for the efficacy of skin antigen-presenting cells (APCs) during intradermal (ID) vaccination still remain to be elucidated. We have previously demonstrated in clinical trials that preferential targeting of Langerhans cells (LCs) by transcutaneous immunization shapes the immune response toward vaccine-specific CD8 T cells. Others have shown that ID inoculation of a vaccine, which targets dermal APCs, mobilizes both the cellular and humoral arms of immunity. Here, we investigated the participation of epidermal LCs in response to ID immunization. When human or mouse skin was injected ID with a particle-based vaccine, we observed significant modifications in the morphology of epidermal LCs and their mobilization to the dermis. We further established that this LC recruitment after ID administration was essential for the induction of antigen-specific CD8 T cells, but was, however, dispensable for the generation of specific CD4 T cells and neutralizing antibodies. Thus, epidermal and dermal APCs shape the outcome of the immune responses to ID vaccination. Their combined potential provides new avenues for the development of vaccination strategies against infectious diseases.