USP39 Regulates NF-κB-Mediated Inflammatory Responses through Deubiquitinating K48-Linked IκBα.

IκBα is a critical protein that inhibits NF-κB nuclear translocation and impairs NF-κB-mediated signaling. The abundance of IκBα determines the activation and restoration of the inflammatory response. However, posttranslational regulation of IκBα remains to be fully understood. In this study, we identified ubiquitin-specific protease 39 (USP39) as a negative regulator in the NF-κB inflammatory response by stabilizing basal IκBα. The expression of USP39 in macrophages was reduced under LPS-induced inflammation. Knockdown or knockout of USP39 in macrophages significantly increased the expression and secretion of proinflammatory cytokines upon exposure to LPS or Escherichia coli, whereas reexpression of exogenous USP39 in USP39-deficient macrophages rescued the effect. Moreover, USP39-defective mice were more sensitive to LPS or E. coli-induced systemic sepsis. Mechanistically, USP39 interacted with and stabilized IκBα by reducing K48-linked polyubiquination of IκBα. Taken together, to our knowledge, our study for the first time revealed the inhibitory function of USP39 in the NF-κB inflammatory response, providing a previously unknown mechanism for control of inflammatory cytokine induction in the cellular anti-inflammatory response.

A Phase 2 Clinical Trial to Evaluate the Safety, Reactogenicity, and Immunogenicity of Different Prime-Boost Vaccination Schedules of 2013 and 2017 A(H7N9) Inactivated Influenza Virus Vaccines Administered With and Without AS03 Adjuvant in Healthy US Adults.

INTRODUCTION: A surge of human influenza A(H7N9) cases began in 2016 in China from an antigenically distinct lineage. Data are needed about the safety and immunogenicity of 2013 and 2017 A(H7N9) inactivated influenza vaccines (IIVs) and the effects of AS03 adjuvant, prime-boost interval, and priming effects of 2013 and 2017 A(H7N9) IIVs. METHODS: Healthy adults (n = 180), ages 19-50 years, were enrolled into this partially blinded, randomized, multicenter phase 2 clinical trial. Participants were randomly assigned to 1 of 6 vaccination groups evaluating homologous versus heterologous prime-boost strategies with 2 different boost intervals (21 vs 120 days) and 2 dosages (3.75 or 15 µg of hemagglutinin) administered with or without AS03 adjuvant. Reactogenicity, safety, and immunogenicity measured by hemagglutination inhibition and neutralizing antibody titers were assessed. RESULTS: Two doses of A(H7N9) IIV were well tolerated, and no safety issues were identified. Although most participants had injection site and systemic reactogenicity, these symptoms were mostly mild to moderate in severity; injection site reactogenicity was greater in vaccination groups receiving adjuvant. Immune responses were greater after an adjuvanted second dose, and with a longer interval between prime and boost. The highest hemagglutination inhibition geometric mean titer (95% confidence interval) observed against the 2017 A(H7N9) strain was 133.4 (83.6-212.6) among participants who received homologous, adjuvanted 3.75 µg + AS03/2017 doses with delayed boost interval. CONCLUSIONS: Administering AS03 adjuvant with the second H7N9 IIV dose and extending the boost interval to 4 months resulted in higher peak antibody responses. These observations can broadly inform strategic approaches for pandemic preparedness. Clinical Trials Registration. NCT03589807.

Identification of Beclin-1 from orange-spotted grouper (Epinephelus coioides) involved in viral infection.

Beclin-1 is an essential autophagic regulator that plays diverse roles in physiology and disease. However, reports about the function of fish Beclin-1 during pathogen infection are still very limited. In this study, a Beclin-1 homolog (EcBeclin-1) from orange-spotted grouper (Epinephelus coioides) was identified and its roles in viral infection were investigated. EcBeclin-1 encoded 447amino acids protein with a BH3 domain, a CCD domain and an ECD domain, which shared high identities (97%-82%) with reported Beclin-1 proteins from mammal to fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBeclin-1 was predominantly expressed in brain and muscle of healthy grouper. Using fluorescence microscopy, we found that EcBeclin-1 was co-localized with endoplasmic reticulum (ER) in grouper spleen cells (EAGS). After red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro, EcBeclin-1 transcript was significantly up-regulated, implying that EcBeclin-1 might be involved in viral infection. Furthermore, the in vitro studies of EcBeclin-1 overexpression promoted RGNNV induced autophagy, as well as the expression of coat protein (CP) and RNA-dependent RNA polymerase (RdRp). The overexpression of EcBeclin-1 suppressed the expressions of interferon pathway-related factors, inflammatory-related factors and activities of NF-κB and ISRE. Additionally, EcBeclin-1 could interact with EcBcl-xL in vitro. These data suggest that EcBeclin-1 affect viral replication through modulating IFN and inflammatory responses, as well as virus-induced cell death, which will help us to further explore the immune response of fish during viral infection.

Innate, T-, and B-Cell Responses in Acute Human Zika Patients.

Background: There is an urgent need for studies of viral persistence and immunity during human Zika infections to inform planning and conduct of vaccine clinical trials. Methods: In 5 returned US travelers with acute symptomatic Zika infection, clinical features, viral RNA levels, and immune responses were characterized. Results: Two pregnant, flavivirus-experienced patients had viral RNA persist in plasma for >44 and >26 days. Three days after symptom onset, transient increases in proinflammatory monocytes began followed at 5 days by transient decreases in myeloid dendritic cells. Anti-Zika virus immunoglobulin M was detected at day 7 after symptom onset, persisted beyond 103 days, and remained equivocal through day 172. Zika virus-specific plasmablasts and neutralizing antibodies developed quickly; dengue virus-specific plasmablasts and neutralizing antibodies at high titers developed only in flavivirus-experienced patients. Zika virus- and dengue virus-specific memory B cells developed in both flavivirus-naive and -experienced patients. CD4+ T cells were moderately activated and produced antiviral cytokines after stimulation with Zika virus C, prM, E, and NS5 peptides in 4/4 patients. In contrast, CD8+ T cells were massively activated, but virus-specific cells that produced cytokines were present in only 2/4 patients assessed. Conclusions: Acute infections with Zika virus modulated antigen-presenting cell populations early. Flavivirus-experienced patients quickly recalled cross-reactive MBCs to secrete antibodies. Dengue virus-naive patients made little dengue-specific antibody but developed MBCs that cross-reacted against dengue virus. Zika virus-specific functional CD4+ T cells were readily detected, but few CD8+ T cells specific for the tested peptides were found.

Hybrid Indicators for Fast and Sensitive Voltage Imaging.

Membrane voltage is an important biophysical signal that underlies intercellular electrical communications. A fluorescent voltage indicator is presented that enables the investigation of electrical signaling at high spatial resolution. The method is built upon the site-specific modification of microbial rhodopsin proteins with organic fluorophores, resulting in a hybrid indicator scaffold that is one of the most sensitive and fastest orange-colored voltage indicators developed to date. We applied this technique to optically map electrical connectivity in cultured cells, which revealed gap junction-mediated long-range coupling that spanned over hundreds of micrometers.

Zinc finger protein 451 is a novel Smad corepressor in transforming growth factor-ß signaling.

ZNF451 is a transcriptional cofactor localized to promyelocytic leukemia bodies. Here, we present evidence demonstrating that ZNF451 physically interacts with Smad3/4 and functionally inhibits TGF-ß signaling. Increased expression of ZNF451 attenuates TGF-ß-induced growth inhibitory and gene transcriptional responses, whereas depletion of ZNF451 enhances TGF-ß responses. Mechanistically, ZNF451 blocks the ability of Smad3/4 to recruit p300 in response to TGF-ß, which causes reduction of histone H3K9 acetylation on the promoters of TGF-ß target genes. Taken together, ZNF451 acts as a transcriptional corepressor for Smad3/4 and negatively regulates TGF-ß signaling.

Emergency postexposure vaccination with vesicular stomatitis virus-vectored Ebola vaccine after needlestick.

IMPORTANCE: Safe and effective vaccines and drugs are needed for the prevention and treatment of Ebola virus disease, including following a potentially high-risk exposure such as a needlestick. OBJECTIVE: To assess response to postexposure vaccination in a health care worker who was exposed to the Ebola virus. DESIGN AND SETTING: Case report of a physician who experienced a needlestick while working in an Ebola treatment unit in Sierra Leone on September 26, 2014. Medical evacuation to the United States was rapidly initiated. Given the concern about potentially lethal Ebola virus disease, the patient was offered, and provided his consent for, postexposure vaccination with an experimental vaccine available through an emergency Investigational New Drug application. He was vaccinated on September 28, 2014. INTERVENTIONS: The vaccine used was VSVΔG-ZEBOV, a replicating, attenuated, recombinant vesicular stomatitis virus (serotype Indiana) whose surface glycoprotein gene was replaced by the Zaire Ebola virus glycoprotein gene. This vaccine has entered a clinical trial for the prevention of Ebola in West Africa. RESULTS: The vaccine was administered 43 hours after the needlestick occurred. Fever and moderate to severe symptoms developed 12 hours after vaccination and diminished over 3 to 4 days. The real-time reverse transcription polymerase chain reaction results were transiently positive for vesicular stomatitis virus nucleoprotein gene and Ebola virus glycoprotein gene (both included in the vaccine) but consistently negative for Ebola virus nucleoprotein gene (not in the vaccine). Early postvaccination cytokine secretion and T lymphocyte and plasmablast activation were detected. Subsequently, Ebola virus glycoprotein-specific antibodies and T cells became detectable, but antibodies against Ebola viral matrix protein 40 (not in the vaccine) were not detected. CONCLUSIONS AND RELEVANCE: It is unknown if VSVΔG-ZEBOV is safe or effective for postexposure vaccination in humans who have experienced a high-risk occupational exposure to the Ebola virus, such as a needlestick. In this patient, postexposure vaccination with VSVΔG-ZEBOV induced a self-limited febrile syndrome that was associated with transient detection of the recombinant vesicular stomatitis vaccine virus in blood. Strong innate and Ebola-specific adaptive immune responses were detected after vaccination. The clinical syndrome and laboratory evidence were consistent with vaccination response, and no evidence of Ebola virus infection was detected.

Biomass ash as soil fertilizers: Supercharging biomass accumulation by shifting auxin distribution.

Growing quantities of biomass ashes (phyto-ashs) are currently produced worldwide due to the increasing biomass consumption in energy applications. Utilization of phyto-ash in agriculture is environmentally friendly solution. However, mechanisms involving the coordination of carbon metabolism and distribution in plants and soil amendment are not well known. In the present study, tobacco plants were chemically-fertilized with or without 2‰ phyto-ash addition. The control had sole chemical fertilizer; for two phyto-ash treatments, the one (T1) received comparable levels of nitrogen, phophorus, and potassium from phyto-ash and fertilizers as the control and another (T2) had 2‰ of phyto-ash and the same rates of fertilizers as the control. Compared with the control, phyto-ash addition improved the soil pH from 5.94 to about 6.35; T2 treatment enhanced soil available potassium by 30% but no difference of other elements was recorded among three treatments. Importantly, bacterial (but not fungal) communities were significantly enriched by phyto-ash addition, with the rank of richness as: T2 > T1 > control. Consistent with amelioration of soil properties, phyto-ash promoted plant growth through enlarged leaf area and photosynthesis and induced outgrowth of lateral roots (LRs). Interestingly, increased auxin content was recorded in 2nd and 3rd leaves and roots under phyto-ash application, also with the rank level as T2 > T1 > control, paralleling with higher transcripts of auxin synthetic genes in the topmost leaf and stronger [3H]IAA activity under phyto-ash addition. Furthermore, exogenous application of analog exogenous auxin (NAA) restored leaf area, photosynthesis and LR outgrowth to the similar level as T2 treatment; conversely, application of auxin transport inhibitor (NPA) under T2 treatment retarded leaf and root development. We demonstrated that phyto-ash addition improved soil properties and thus facilitated carbon balance within plants and biomass accumulation in which shifting auxin distribution plays an important role.

Cellular and humoral immunity to Ebola Zaire glycoprotein and viral vector proteins following immunization with recombinant vesicular stomatitis virus-based Ebola vaccine (rVSVΔG-ZEBOV-GP).

While effective at preventing Zaire ebolavirus (ZEBOV) disease, cellular immunity to ZEBOV and vector-directed immunity elicited by the recombinant vesicular stomatitis virus expressing ZEBOV glycoprotein (rVSVΔG-ZEBOV-GP) vaccine remain poorly understood. Sera and peripheral blood mononuclear cells were collected from 32 participants enrolled in a prospective multicenter study [ClinicalTrials.gov NCT02788227] before vaccination and up to six months post-vaccination. IgM and IgG antibodies, IgG-producing memory B cells (MBCs), and T cell reactivity to ZEBOV glycoprotein (ZEBOV-GP), vesicular stomatitis virus-Indiana strain (VSV-I) matrix (M) protein, and VSV-I nucleoprotein (NP) were measured using ELISA, ELISpot, and flow cytometry, respectively. 11/32 (34.4%) participants previously received a different investigational ZEBOV vaccine prior to enrollment and 21/32 (65.6%) participants were ZEBOV vaccine naïve. Both ZEBOV vaccine naïve and experienced participants had increased ZEBOV-GP IgG optical densities (ODs) post-rVSVΔG-ZEBOV-GP vaccination while only ZEBOV vaccine naïve participants had increased ZEBOV-GP IgM ODs. Transient IgM and IgG antibody responses to VSV-I M protein and NP were observed in a minority of participants. All participants had detectable ZEBOV-GP specific IgG-producing MBCs by 6 months post-vaccination while no changes were observed in the median IgG-producing MBCs to VSV-I proteins. T cell responses to ZEBOV-GP differed between ZEBOV vaccine experienced and ZEBOV vaccine naïve participants. T cell responses to both VSV-I M protein and VSV-I NP were observed, but were of a low magnitude. The rVSVΔG-ZEBOV-GP vaccine elicits robust humoral and memory B cell responses to ZEBOV glycoprotein in both ZEBOV vaccine naïve and experienced individuals and can generate vector-directed T cell immunity. Further research is needed to understand the significance of pre-existing vector and target antigen immunity on responses to booster doses of rVSVΔG-ZEBOV-GP and other rVSV-vectored vaccines.

Contextual Fear Learning and Extinction in the Primary Visual Cortex of Mice.

Fear memory contextualization is critical for selecting adaptive behavior to survive. Contextual fear conditioning (CFC) is a classical model for elucidating related underlying neuronal circuits. The primary visual cortex (V1) is the primary cortical region for contextual visual inputs, but its role in CFC is poorly understood. Here, our experiments demonstrated that bilateral inactivation of V1 in mice impaired CFC retrieval, and both CFC learning and extinction increased the turnover rate of axonal boutons in V1. The frequency of neuronal Ca2+ activity decreased after CFC learning, while CFC extinction reversed the decrease and raised it to the naïve level. Contrary to control mice, the frequency of neuronal Ca2+ activity increased after CFC learning in microglia-depleted mice and was maintained after CFC extinction, indicating that microglial depletion alters CFC learning and the frequency response pattern of extinction-induced Ca2+ activity. These findings reveal a critical role of microglia in neocortical information processing in V1, and suggest potential approaches for cellular-based manipulation of acquired fear memory.

Robust and adjustable dynamic scattering compensation for high-precision deep tissue optogenetics.

The development of high-precision optogenetics in deep tissue is limited due to the strong optical scattering induced by biological tissue. Although various wavefront shaping techniques have been developed to compensate the scattering, it is still a challenge to non-invasively characterize the dynamic scattered optical wavefront inside the living tissue. Here, we present a non-invasive scattering compensation system with fast multidither coherent optical adaptive technique (fCOAT), which allows the rapid wavefront correction and stable focusing in dynamic scattering medium. We achieve subcellular-resolution focusing through 500-µm-thickness brain slices, or even three pieces overlapped mouse skulls after just one iteration with a 589 nm CW laser. Further, focusing through dynamic scattering medium such as live rat ear is also successfully achieved. The formed focus can maintain longer than 60 s, which satisfies the requirements of stable optogenetics manipulation. Moreover, the focus size is adjustable from subcellular level to tens of microns to freely match the various manipulation targets. With the specially designed fCOAT system, we successfully achieve single-cellular optogenetic manipulation through the brain tissue, with a stimulation efficiency enhancement up to 300% compared with that of the speckle.

Phase 1 safety and immunogenicity testing of DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles.

BACKGROUND: Recombinant DNA and modified vaccinia virus Ankara (rMVA) vaccines represent a promising approach to an HIV/AIDS vaccine. This Phase 1 clinical trial compared the safety and immunogenicity of a rMVA vaccine administered with and without DNA vaccine priming METHODS: GeoVax pGA2/JS7 DNA (D) and MVA/HIV62 (M) vaccines encode noninfectious virus-like particles. Intramuscular needle injections were used to deliver placebo, 2 doses of DNA followed by 2 doses of rMVA (DDMM), one dose of DNA followed by 2 doses of rMVA (DMM), or 3 doses of rMVA (MMM) to HIV-seronegative participants. RESULTS: Local and systemic symptoms were mild or moderate. Immune response rates for CD4 + and CD8 + T cells were highest in the DDMM group and lowest in the MMM group (77% vs 43% CD4 + and 42% vs 17% CD8 +). In contrast, response rates for Env binding and neutralizing Ab were highest in the MMM group. The DMM group had intermediate response rates. A 1/10th-dose DDMM regimen induced similar T cell but reduced Ab response rates compared with the full-dose DDMM. CONCLUSIONS: MVA62 was well tolerated and elicited different patterns of T cell and Ab responses when administered alone or in combination with the JS7 DNA vaccine.

An Oil-in-Water adjuvant significantly increased influenza A/H7N9 split virus Vaccine-Induced circulating follicular helper T (cTFH) cells and antibody responses.

BACKGROUND: Unadjuvanted A/H7N9 vaccines are poorly immunogenic. The immune response is improved with the addition of MF59, an oil-in-water adjuvant. However, the cellular immunologic responses of MF59-adjuvanted A/H7N9 vaccine are not fully understood. METHODS: 37 participants were vaccinated with 2 doses of 2013 influenza A/H7N9 vaccine (at Days 1 and 21) with or without MF59 and enrolled in an immunology substudy. Responses were assessed at multiple timepoints (Days 0, 8, 21, 29, and 42) for hemagglutination inhibition (HAI) and neutralizing antibody (Neut) assays, memory B cell responses by enzyme-linked ImmunoSpot; circulating follicular helper T cells (cTFH) and CD4 + T cells by intracellular cytokine staining. RESULTS: MF59-adjuvanted influenza A/H7N9 vaccine induced significantly higher hemagglutination inhibition (HAI) and neutralizing antibody (Neut) responses when compared to unadjuvanted vaccine. The adjuvanted vaccine elicited significantly higher levels of Inducible T-cell Co-Stimulator (ICOS) expression by CXCR3+CXCR5+CD4+ cTFH cells, compared to unadjuvanted vaccine. The magnitude of increase in cTFH cells (from baseline to Day 8) and in IL-21 expressing CD154+CD4+ T cells (from baseline to Days 8 and 21) correlated with HAI (at Day 29) and Neut antibody (at Days 8 and 29) titers. The increase in frequency of IL-21 expressing CD154+CD4+T cells (from baseline to Day 21) correlated with memory B cell frequency (at Day 42). CONCLUSION: cTFH activation is associated with HAI and Neut responses in recipients of MF59-adjuvanted influenza A/H7N9 vaccine relative to unadjuvanted vaccine. Future studies should focus on optimizing the cTFH response and use cTFH as an early biomarker of serological response to vaccination. This trial was registered at clinicaltrials.gov, trial number NCT01938742.

Long-term microglial phase-specific dynamics during single vessel occlusion and recanalization.

Vascular occlusion leading to brain dysfunctions is usually considered evoking microglia-induced inflammation response. However, it remains unclear how microglia interact with blood vessels in the development of vascular occlusion-related brain disorders. Here, we illuminate long-term spatiotemporal dynamics of microglia during single vessel occlusion and recanalization. Microglia display remarkable response characteristics in different phases, including acute reaction, rapid diffusion, transition and chronic effect. Fibrinogen-induced microglial cluster promotes major histocompatibility complex II (MHCII) expression. Microglial soma represents a unique filament-shape migration and has slower motility compared to the immediate reaction of processes to occlusion. We capture proliferative microglia redistribute territory. Microglial cluster resolves gradually and microglia recover to resting state both in the morphology and function in the chronic effect phase. Therefore, our study offers a comprehensive analysis of spatiotemporal dynamics of microglia and potential mechanisms to both vessel occlusion and recanalization. Microglial phase-specific response suggests the morphological feature-oriented phased intervention would be an attractive option for vascular occlusion-related diseases treatments.

A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons.

Membrane potential is a key aspect of cellular signalling and is dynamically regulated by an array of ion-selective pumps and channels. Fluorescent voltage indicators enable non-invasive optical recording of the cellular membrane potential with high spatial resolution. Here, we report a palette of bright and sensitive hybrid voltage indicators (HVIs) with fluorescence intensities sensitive to changes in membrane potential via electrochromic Förster resonance energy transfer. Enzyme-mediated site-specific incorporation of a probe, followed by an inverse-electron-demand Diels-Alder cycloaddition, was used to create enhanced voltage-sensing rhodopsins with hybrid dye-protein architectures. The most sensitive indicator, HVI-Cy3, displays high voltage sensitivity (-39% ΔF/F0 per 100 mV) and millisecond response kinetics, enabling optical recording of action potentials at a sampling rate of 400 Hz over 10 min across a large neuronal population. The far-red indicator HVI-Cy5 could be paired with optogenetic actuators and green/red-emitting fluorescent indicators, allowing an all-optical investigation of neuronal electrophysiology.

Immunologic mechanisms of seasonal influenza vaccination administered by microneedle patch from a randomized phase I trial.

In a phase 1 randomized, single-center clinical trial, inactivated influenza virus vaccine delivered through dissolvable microneedle patches (MNPs) was found to be safe and immunogenic. Here, we compare the humoral and cellular immunologic responses in a subset of participants receiving influenza vaccination by MNP to the intramuscular (IM) route of administration. We collected serum, plasma, and peripheral blood mononuclear cells in 22 participants up to 180 days post-vaccination. Hemagglutination inhibition (HAI) titers and antibody avidity were similar after MNP and IM vaccination, even though MNP vaccination used a lower antigen dose. MNPs generated higher neuraminidase inhibition (NAI) titers for all three influenza virus vaccine strains tested and triggered a larger percentage of circulating T follicular helper cells (CD4 + CXCR5 + CXCR3 + ICOS + PD-1+) compared to the IM route. Our study indicates that inactivated influenza virus vaccination by MNP produces humoral and cellular immune response that are similar or greater than IM vaccination.

The Effects of Imprinting and Repeated Seasonal Influenza Vaccination on Adaptive Immunity after Influenza Vaccination.

(1) Background: The influenza virus continues to cause significant annual morbidity and mortality. The overall efficacy of seasonal influenza vaccination is suboptimal, which is partly due to host immune factors. The effects of imprinting and repeated seasonal influenza vaccination were investigated to assess for immune factors and mechanisms that impact influenza vaccine responses. (2) Methods: Twenty participants were enrolled into a prospective pilot study based on birth cohort and seasonal influenza immunization history. Immunologic parameters were assessed over a six-month period after the seasonal influenza vaccine was administered. (3) Results: There was no significant imprinting effect, as measured by hemagglutination inhibition (HAI) fold change, HAI geometric mean titer (GMT) for Day 29 or Day 180 post-vaccination and antigen- specific antibody-secreting cells (ASC) for Day 8 post-vaccination. Individuals who had minimal prior seasonal influenza vaccination had a higher magnitude ASC response and a higher HAI fold change post-vaccination than individuals who were repeatedly vaccinated. (4) Conclusions: Repeated seasonal influenza vaccination resulted in a decreased fold change of the immune response, although individuals in this cohort tended to have high HAI titers at baseline that persisted after vaccination. Imprinting effects were not observed in this cohort. These host immune factors should be considered in the development of universal influenza vaccines. ClinicalTrials.gov Identifier: NCT03686514.

Plasmablast, Memory B Cell, CD4+ T Cell, and Circulating Follicular Helper T Cell Responses to a Non-Replicating Modified Vaccinia Ankara Vaccine.

Background: Vaccinia is known to induce antibody and cellular responses. Plasmablast, circulating follicular helper T (cTFH) cells, cytokine-expressing CD4 T cells, and memory B cells were compared between subcutaneous (SC) and needle-free jet injection (JI) recipients of non-replicating modified vaccinia Ankara (MVA) vaccine. Methods: Vaccinia-naïve adults received MVA SC or by JI on Days 1 and 29. Vaccinia-specific antibodies were quantified by plaque reduction neutralization test (PRNT) and enzyme-linked immunosorbent assay. Plasmablast, cTFH, and cytokine-expressing CD4 T cells were assessed on Days 1, 8, 15, 29, 36, 43 (cTFH and CD4+ only) and 57. Memory B cells were measured on Days 1 and 57. Results: Of the 36 enrolled subjects, only 22 received both vaccinations and had evaluable specimens after the second vaccine. Plasmablasts peaked one week after each vaccine. Day 15 plasmablasts correlated with peak PRNT titers. cTFH peaked on Days 8 and 36 and correlated with Day 36 plasmablasts. CD4+ peaked at Day 29 and one-third produced ≥2 cytokines. Day 57 memory B cells ranged from 0.1% to 0.17% of IgG-secreting B cells. Conclusions: This study provides insights into the cellular responses to non-replicating MVA, currently used as a vector for a variety of novel vaccines.