Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe.

BACKGROUND: The replication-competent recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing a Zaire ebolavirus (ZEBOV) glycoprotein was selected for rapid safety and immunogenicity testing before its use in West Africa. METHODS: We performed three open-label, dose-escalation phase 1 trials and one randomized, double-blind, controlled phase 1 trial to assess the safety, side-effect profile, and immunogenicity of rVSV-ZEBOV at various doses in 158 healthy adults in Europe and Africa. All participants were injected with doses of vaccine ranging from 300,000 to 50 million plaque-forming units (PFU) or placebo. RESULTS: No serious vaccine-related adverse events were reported. Mild-to-moderate early-onset reactogenicity was frequent but transient (median, 1 day). Fever was observed in up to 30% of vaccinees. Vaccine viremia was detected within 3 days in 123 of the 130 participants (95%) receiving 3 million PFU or more; rVSV was not detected in saliva or urine. In the second week after injection, arthritis affecting one to four joints developed in 11 of 51 participants (22%) in Geneva, with pain lasting a median of 8 days (interquartile range, 4 to 87); 2 self-limited cases occurred in 60 participants (3%) in Hamburg, Germany, and Kilifi, Kenya. The virus was identified in one synovial-fluid aspirate and in skin vesicles of 2 other vaccinees, showing peripheral viral replication in the second week after immunization. ZEBOV-glycoprotein-specific antibody responses were detected in all the participants, with similar glycoprotein-binding antibody titers but significantly higher neutralizing antibody titers at higher doses. Glycoprotein-binding antibody titers were sustained through 180 days in all participants. CONCLUSIONS: In these studies, rVSV-ZEBOV was reactogenic but immunogenic after a single dose and warrants further evaluation for safety and efficacy. (Funded by the Wellcome Trust and others; ClinicalTrials.gov numbers, NCT02283099, NCT02287480, and NCT02296983; Pan African Clinical Trials Registry number, PACTR201411000919191.).

MF59 Mediates Its B Cell Adjuvanticity by Promoting T Follicular Helper Cells and Thus Germinal Center Responses in Adult and Early Life.

The early life influenza disease burden calls for more effective vaccines to protect this vulnerable population. Influenza vaccines including the MF59 oil-in-water adjuvant induce higher, broader, and more persistent Ab responses in adults and particularly in young, through yet undefined mechanisms. In this study, we show that MF59 enhances adult murine IgG responses to influenza hemagglutinin (HA) by promoting a potent T follicular helper cells (TFH) response, which directly controls the magnitude of the germinal center (GC) B cell response. Remarkably, this enhancement of TFH and GC B cells is already fully functional in 3-wk-old infant mice, which were fully protected by HA/MF59 but not HA/PBS immunization against intranasal challenge with the homologous H1N1 (A/California/7/2009) strain. In 1-wk-old neonatal mice, MF59 recruits and activates APCs, efficiently induces CD4(+) effector T cells and primes for enhanced infant responses but induces few fully functional TFH cells, which are mostly follicular regulatory T cells, and poor GC and anti-HA responses. The B cell adjuvanticity of MF59 appears to be mediated by the potent induction of TFH cells which directly controls GC responses both in adult and early life, calling for studies assessing its capacity to enhance the efficacy of influenza immunization in young infants.

Notch signaling regulates follicular helper T cell differentiation.

Follicular helper T (TFH) cells are specialized in providing help for B cell differentiation and Ab secretion. Several positive and negative regulators of TFH cell differentiation have been described but their control is not fully understood. In this study, we show that Notch signaling in T cells is a major player in the development and function of TFH cells. T cell-specific gene ablation of Notch1 and Notch2 impaired differentiation of TFH cells in draining lymph nodes of mice immunized with T-dependent Ags or infected with parasites. Impaired TFH cell differentiation correlated with deficient germinal center development and the absence of high-affinity Abs. The impact of loss of Notch on TFH cell differentiation was largely independent of its effect on IL-4. These results show a previously unknown role for Notch in the regulation of TFH cell differentiation and function with implications for the control of this T cell population.

Redundant Notch1 and Notch2 signaling is necessary for IFNγ secretion by T helper 1 cells during infection with Leishmania major.

The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+) T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+) T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4(+) T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre)) were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre) mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+) T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+) T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.

Environmental and T cell-intrinsic factors limit the expansion of neonatal follicular T helper cells but may be circumvented by specific adjuvants.

Follicular Th (T(FH)) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5(high)PD-1(high) CD4(+) T(FH) cells that exhibit T(FH) features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal T(FH) cells fail to expand and to acquire a full-blown GC T(FH) phenotype, as reflected by a higher ratio of GC T(FH)/non-GC CD4(+) T cells in immunized adults than neonates (3.8 × 10(-3) versus 2.2 × 10(-3), p = 0.01). Following the adoptive transfer of naive adult OT-II CD4(+) T cells, OT-II T(FH) cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4(+) OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell-intrinsic factors. Postponing immunization to later in life increases the number of T(FH) cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC T(FH) and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the T(FH) cell development limits early life GC responses and that adjuvants/delivery systems supporting T(FH) differentiation may restore adultlike early life GC B cell responses.

Role of Toll-like receptor 9 signaling in experimental Leishmania braziliensis infection.

Infection with Leishmania braziliensis causes cutaneous or mucocutaneous leishmaniasis in humans. Toll-like receptor 9 (TLR9) expression has been found in granulomas of lesions in L. braziliensis-infected individuals. L. braziliensis inoculation in mice induces very small lesions that are self-healing, whereas deficiency in the TLR adaptor molecule, MyD88, renders mice susceptible to infection. The TLR involved has not been identified, prompting us to investigate if TLR9 triggering by the parasite contributes to the strong resistance to infection observed in L. braziliensis-inoculated mice. The parasites activated wild-type (WT) dendritic cells (DCs) in vitro but not DCs derived from TLR9(-/-) mice. TLR9(-/-) mice inoculated with L. braziliensis exhibited a transient susceptibility characterized by increased lesion size and parasite burden compared to those of WT mice. Surprisingly, elevated levels of gamma interferon (IFN-γ) were measured at the site of infection and in draining lymph node T cells of TLR9(-/-) mice at the peak of susceptibility, suggesting that unlike observations in vitro, the parasite could induce DC activation leading to the development of Th1 cells in the absence of TLR9 expression. Taken together, these data show that TLR9 signaling is important for the early control of lesion development and parasite burden but is dispensable for the differentiation of Th1 cells secreting IFN-γ, and the high levels of this cytokine are not sufficient to control early parasite replication following L. braziliensis infection.

The role of Notch in the differentiation of CD4⁺ T helper cells.

CD4⁺ T helper cells are playing critical roles in host defense to pathogens and in the maintenance of immune homeostasis. Naïve CD4⁺T cells, upon antigen-specific recognition, receive signals to differentiate into distinct effector T helper cell subsets characterized by their pattern of cytokine production and specific immune functions. A tight balance between these different subsets ensures proper control of the immune response. There is increasing evidence revealing an important role for Notch signaling in the regulation of CD4⁺T helper cell differentiation or function in the periphery. However, the exact mechanisms involved remain unclear and appear contradictory. In this review, we summarize current knowledge and discuss recent advances in the field to reconcile different views on the role of Notch signaling in the differentiation of functional T helper subsets.

Human in vitro modeling of adjuvant formulations demonstrates enhancement of immune responses to SARS-CoV-2 antigen.

Adjuvants can enhance vaccine immunogenicity, but their mechanism of action is often incompletely understood, hampering rapid applicability for pandemic vaccines. Herein, we characterized the cellular and molecular activity of adjuvant formulations available for pre-clinical evaluation, including several developed for global open access. We applied four complementary human in vitro platforms to assess individual and combined adjuvants in unformulated, oil-in-water, and liposomal delivery platforms. Liposomal co-formulation of MPLA and QS-21 was most potent in promoting dendritic cell maturation, selective production of Th1-polarizing cytokines, and activation of SARS-CoV-2 Spike-specific CD4+ and CD8+ T cells in a co-culture assay. Select formulations also significantly enhanced Spike antigen-specific humoral immunity in vivo. This study confirms the utility of the cumulative use of human in vitro tools to predict adjuvanticity potential. Thus, human in vitro modeling may advance public health by accelerating the development of affordable and scalable adjuvants for vaccines tailored to vulnerable populations.

Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms.

Adjuvanted protein vaccines offer high efficacy, yet most potent adjuvants remain proprietary. Several adjuvant compounds are being developed by the Vaccine Formulation Institute in Switzerland for global open access clinical use. In the context of the R21 malaria vaccine, in a mouse challenge model, we characterize the efficacy and mechanism of action of four Vaccine Formulation Institute adjuvants: two liposomal (LQ and LMQ) and two squalene emulsion-based adjuvants (SQ and SMQ), containing QS-21 saponin (Q) and optionally a synthetic TLR4 agonist (M). Two R21 vaccine formulations, R21/LMQ and R21/SQ, offer the highest protection (81%-100%), yet they trigger different innate sensing mechanisms in macrophages with LMQ, but not SQ, activating the NLRP3 inflammasome. The resulting in vivo adaptive responses have a different TH1/TH2 balance and engage divergent innate pathways while retaining high protective efficacy. We describe how modular changes in vaccine formulation allow for the dissection of the underlying immune pathways, enabling future mechanistically informed vaccine design.

Neutrophil-derived CCL3 is essential for the rapid recruitment of dendritic cells to the site of Leishmania major inoculation in resistant mice.

Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3(-/-) mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.

Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies.

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

The prominent role of neutrophils during the initial phase of infection by Leishmania parasites.

Neutrophils are rapidly and massively recruited to the site of Leishmania inoculation, where they phagocytose the parasites, some of which are able to survive within these first host cells. Neutrophils can thus provide a transient safe shelter for the parasites, prior to their entry into macrophages where they will replicate. In addition, neutrophils release and synthesize rapidly several factors including cytokines and chemokines. The mechanism involved in their rapid recruitment to the site of parasite inoculation, as well as the putative consequences of their massive presence on the microenvironment of the focus of infection will be discussed in the context of the development of the Leishmania-specific immune response.

A TLR7/8 Agonist-Including DOEPC-Based Cationic Liposome Formulation Mediates Its Adjuvanticity Through the Sustained Recruitment of Highly Activated Monocytes in a Type I IFN-Independent but NF-κB-Dependent Manner.

Novel adjuvants, such as Toll-like receptors (TLRs) agonists, are needed for the development of new formulations able to circumvent limitations of current vaccines. Among TLRs, TLR7/8 agonists represent promising candidates, as they are well described to enhance antigen-specific antibody responses and skew immunity toward T helper (TH) 1 responses. We find here that the incorporation of the synthetic TLR7/8 ligand 3M-052 in a cationic DOEPC-based liposome formulation shifts immunity toward TH1 responses and elicits strong and long-lasting germinal center and follicular T helper cell responses in adult mice. This reflects the prolonged recruitment of innate cells toward the site of immunization and homing of activated antigen-loaded monocytes and monocyte-derived dendritic cells toward draining lymph nodes. We further show that this adjuvanticity is independent of type I IFN but NF-κB-dependent. Overall, our data identify TLR7/8 agonists incorporated in liposomes as promising and effective adjuvants to enhance TH1 and germinal center responses.

Erratum: Author Correction: Rapid dose-dependent Natural Killer (NK) cell modulation and cytokine responses following human rVSV-ZEBOV Ebolavirus vaccination.

[This corrects the article DOI: 10.1038/s41541-020-0179-4.].

Rapid dose-dependent Natural Killer (NK) cell modulation and cytokine responses following human rVSV-ZEBOV Ebolavirus vaccination.

The rVSV-ZEBOV Ebolavirus vaccine confers protection within days after immunization, suggesting the contribution of innate immune responses. We report modulation of rVSV-ZEBOV vaccinee blood CD56+ NK cell numbers, NKG2D or NKp30 surface receptor expression, Killer Immunoglobulin-like Receptor (KIR)+ cell percentages and NK-cell-related genes on day 1 post immunization. Inverse correlations existed between the concentration of several plasma cytokines and inhibitory KIR+ CD56dim or cytokine-responsive CD56bright NK cells. Thus, NK cells may contribute to the early protective efficacy of rVSV-ZEBOV in humans.

Site-Specific DC Surface Signatures Influence CD4+ T Cell Co-stimulation and Lung-Homing.

Dendritic cells (DCs) that drain the gut and skin are known to favor the establishment of T cell populations that home to the original site of DC-antigen (Ag) encounter by providing soluble "imprinting" signals to T cells in the lymph node (LN). To study the induction of lung T cell-trafficking, we used a protein-adjuvant murine intranasal and intramuscular immunization model to compare in vivo-activated Ag+ DCs in the lung and muscle-draining LNs. Higher frequencies of Ag+ CD11b+ DCs were observed in lung-draining mediastinal LNs (MedLN) compared to muscle-draining inguinal LNs (ILN). Ag+ CD11b+ MedLN DCs were qualitatively superior at priming CD4+ T cells, which then expressed CD49a and CXCR3, and preferentially trafficked into the lung parenchyma. CD11b+ DCs from the MedLN expressed higher levels of surface podoplanin, Trem4, GL7, and the known co-stimulatory molecules CD80, CD86, and CD24. Blockade of specific MedLN DC molecules or the use of sorted DC and T cell co-cultures demonstrated that DC surface phenotype influences the ability to prime T cells that then home to the lung. Thus, the density of dLN Ag+ DCs, and DC surface molecule signatures are factors that can influence the output and differentiation of lung-homing CD4+ T cells.

Corrigendum: Site-Specific DC Surface Signatures Influence CD4+ T Cell Co-stimulation and Lung-Homing.

[This corrects the article DOI: 10.3389/fimmu.2019.01650.].

Maternal Antibodies Inhibit Neonatal and Infant Responses to Vaccination by Shaping the Early-Life B Cell Repertoire within Germinal Centers.

Maternal antibodies (MatAbs) protect offspring from infections but limit their responses to vaccination. The mechanisms of this inhibition are still debated. Using murine early-life immunization models mimicking the condition prevailing in humans, we observed the induction of CD4-T, T follicular helper, and germinal center (GC) B cell responses even when early-life antibody responses were abrogated by MatAbs. GC B cells induced in the presence of MatAbs form GC structures and exhibit canonical GC changes in gene expression but fail to differentiate into plasma cells and/or memory B cells in a MatAb titer-dependent manner. Furthermore, GC B cells elicited in the presence or absence of MatAbs use different VH and Vk genes and show differences in genes associated with B cell differentiation and isotype switching. Thus, MatAbs do not prevent B cell activation but control the output of the GC reaction both quantitatively and qualitatively, shaping the antigen-specific B cell repertoire.

Reactivating Immunity Primed by Acellular Pertussis Vaccines in the Absence of Circulating Antibodies: Enhanced Bacterial Control by TLR9 Rather Than TLR4 Agonist-Including Formulation.

Pertussis is still observed in many countries despite of high vaccine coverage. Acellular pertussis (aP) vaccination is widely implemented in many countries as primary series in infants and as boosters in school-entry/adolescents/adults (including pregnant women in some). One novel strategy to improve the reactivation of aP-vaccine primed immunity could be to include genetically- detoxified pertussis toxin and novel adjuvants in aP vaccine boosters. Their preclinical evaluation is not straightforward, as it requires mimicking the human situation where T and B memory cells may persist longer than vaccine-induced circulating antibodies. Toward this objective, we developed a novel murine model including two consecutive adoptive transfers of the memory cells induced by priming and boosting, respectively. Using this model, we assessed the capacity of three novel aP vaccine candidates including genetically-detoxified pertussis toxin, pertactin, filamentous hemagglutinin, and fimbriae adsorbed to aluminum hydroxide, supplemented-or not-with Toll-Like-Receptor 4 or 9 agonists (TLR4A, TLR9A), to reactivate aP vaccine-induced immune memory and protection, reflected by bacterial clearance. In the conventional murine immunization model, TLR4A- and TLR9A-containing aP formulations induced similar aP-specific IgG antibody responses and protection against bacterial lung colonization as current aP vaccines, despite IL-5 down-modulation by both TLR4A and TLR9A and IL-17 up-modulation by TLR4A. In the absence of serum antibodies at time of boosting or exposure, TLR4A- and TLR9A-containing formulations both enhanced vaccine antibody recall compared to current aP formulations. Unexpectedly, however, protection was only increased by the TLR9A-containing vaccine, through both earlier bacterial control and accelerated clearance. This suggests that TLR9A-containing aP vaccines may better reactivate aP vaccine-primed pertussis memory and enhance protection than current or TLR4A-adjuvanted aP vaccines.

Overcoming the Neonatal Limitations of Inducing Germinal Centers through Liposome-Based Adjuvants Including C-Type Lectin Agonists Trehalose Dibehenate or Curdlan.

Neonates and infants are more vulnerable to infections and show reduced responses to vaccination. Consequently, repeated immunizations are required to induce protection and early life vaccines against major pathogens such as influenza are yet unavailable. Formulating antigens with potent adjuvants, including immunostimulators and delivery systems, is a demonstrated approach to enhance vaccine efficacy. Yet, adjuvants effective in adults may not meet the specific requirements for activating the early life immune system. Here, we assessed the neonatal adjuvanticity of three novel adjuvants including TLR4 (glucopyranosyl lipid adjuvant-squalene emulsion), TLR9 (IC31®), and Mincle (CAF01) agonists, which all induce germinal centers (GCs) and potent antibody responses to influenza hemagglutinin (HA) in adult mice. In neonates, a single dose of HA formulated into each adjuvant induced T follicular helper (TFH) cells. However, only HA/CAF01 elicited significantly higher and sustained antibody responses, engaging neonatal B cells to differentiate into GCs already after a single dose. Although antibody titers remained lower than in adults, HA-specific responses induced by a single neonatal dose of HA/CAF01 were sufficient to confer protection against influenza viral challenge. Postulating that the neonatal adjuvanticity of CAF01 may result from the functionality of the C-type lectin receptor (CLR) Mincle in early life we asked whether other C-type lectin agonists would show a similar neonatal adjuvanticity. Replacing the Mincle agonist trehalose 6,6'-dibehenate by Curdlan, which binds to Dectin-1, enhanced antibody responses through the induction of similar levels of TFH, GCs and bone marrow high-affinity plasma cells. Thus, specific requirements of early life B cells may already be met after a single vaccine dose using CLR-activating agonists, identified here as promising B cell immunostimulators for early life vaccines when included into cationic liposomes.