Molecular Methods and Bioinformatic Tools for Adjuvant Characterization by High-Throughput Sequencing.

Adjuvants in vaccine formulations are designed to enhance immune responses against a target antigen or pathogen. The ability of these vaccines to induce activation and differentiation of mature naïve B cells to produce pathogen-specific antibodies (immunoglobulins; Ig) helps guarantee long-lived humoral immunity. This process involves clonal expansion of antigen-specific B cells, genomic rearrangement of Ig heavy (IgH) and light (IgL) loci, somatic hypermutation (SHM), and clonal selection for affinity-matured antibody, resulting in a vast but directed repertoire of B cells expressing highly specific antibody proteins.

From mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE.

Key antigens of Leishmania species identified in the context of host responses in Leishmania-exposed individuals from disease-endemic areas were prioritized for the development of a subunit vaccine against visceral leishmaniasis (VL), the most deadly form of leishmaniasis. Two Leishmania proteins-nucleoside hydrolase and a sterol 24-c-methyltransferase, each of which are protective in animal models of VL when properly adjuvanted- were produced as a single recombinant fusion protein NS (LEISH-F3) for ease of antigen production and broad coverage of a heterogeneous major histocompatibility complex population. When formulated with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE), a Toll-like receptor 4 TH1 (T helper 1) promoting nanoemulsion adjuvant, the LEISH-F3 polyprotein induced potent protection against both L.

CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen.

Chemokine-dependent localization of specific B cell subsets within the immune microarchitecture is essential to ensure successful cognate interactions. Although cognate interactions between T cells and memory B cells (B(mem)) are essential for the secondary humoral immune responses, the chemokine response patterns of B(mem) cells are largely unknown. In contrast to naive B cells, this study shows that Ag-specific B(mem) cells have heightened expression of CCR6 and a selective chemotactic response to the CCR6 ligand, CCL20.

Regulatory actions of Toll-like receptor 2 (TLR2) and TLR4 in Leishmania donovani infection in the liver.

In livers of susceptible but self-curing C57BL/6 mice, intracellular Leishmania donovani infection enhanced Toll-like receptor 4 (TLR4) and TLR2 gene expression. In the liver, infected TLR4(-/-) mice showed reduced gamma interferon (IFN-γ), tumor necrosis factor (TNF), and inducible nitric oxide synthase (iNOS) mRNA expression, higher-level and slowly resolving infection, delayed granuloma formation, and little response to low-dose chemotherapy; in serum, the ratio of IFN-γ to interleukin 10 (IL-10) activity was decreased by 50%. In contrast, in TLR2(-/-) mice, control of liver infection, parasite killing, and granuloma assembly were accelerated and chemotherapy's efficacy enhanced.

Development and pre-clinical assessment of a 73 kD chimeric fusion protein as a defined sub-unit vaccine for leprosy.

Despite the advances toward the elimination of leprosy through widespread provision of multi-drug therapy to registered patients over the last 2 decades, new case detection rates have stabilized and leprosy remains endemic in a number of localized regions. A vaccine could overcome the inherent limitations of the drug treatment program by providing protection in individuals who are not already harboring the Mycobacterium leprae bacilli at the time of administration and effectively interrupt the transmission cycle over a wider timespan. In this report we present data validating the production of 73f, a chimeric fusion protein incorporating the M.

Adjuvants for Leishmania vaccines: from models to clinical application.

Two million new cases of leishmaniasis occur every year, with the cutaneous leishmaniasis (CL) presentation accounting for approximately two-thirds of all cases. Despite the high incidence rates and geographic expansion of the disease, CL remains a neglected tropical disease without effective intervention strategies. Efforts to address this deficit have given rise to the experimental murine model of CL.

The development and clinical evaluation of second-generation leishmaniasis vaccines.

Infection with Leishmania parasites results in a range of clinical manifestations and outcomes. Control of Leishmania parasite transmission is extremely difficult due to the large number of vectors and potential reservoirs, and none of the current treatments are ideal. Vaccination could be an effective strategy to provide sustained control.

KSAC, the first defined polyprotein vaccine candidate for visceral leishmaniasis.

A subunit vaccine using a defined antigen(s) may be one effective solution for controlling leishmaniasis. Because of genetic diversity in target populations, including both dogs and humans, a multiple-antigen vaccine will likely be essential. However, the cost of a vaccine to be used in developing countries must be considered.

A synthetic TLR4 agonist formulated in an emulsion enhances humoral and Type 1 cellular immune responses against GMZ2--a GLURP-MSP3 fusion protein malaria vaccine candidate.

GMZ2 adjuvanted by aluminum hydroxide is a candidate malaria vaccine that has successfully passed phase 1 clinical testing in adult German and Gabonese volunteers and Gabonese children under five. Here we report a preclinical study screening a series of adjuvant vehicles and Toll-like receptor (TLR) agonists in CB6F1 mice to identify an improved formulation of GMZ2 suitable for further human clinical studies. GMZ2 formulated in an oil-in-water emulsion plus the synthetic TLR4 agonist GLA elicits the highest (a) vaccine-specific IgG2a and total IgG titers, (b) parasite-specific IFA titers, (c) levels of Type 1 cytokine responses (IFN-γ), and (d) number of long-lived-plasma cells (LLPC) secreting antibodies against both the GMZ2 fusion and its two components.

A synthetic adjuvant to enhance and expand immune responses to influenza vaccines.

Safe, effective adjuvants that enhance vaccine potency, including induction of neutralizing Abs against a broad range of variant strains, is an important strategy for the development of seasonal influenza vaccines which can provide optimal protection, even during seasons when available vaccines are not well matched to circulating viruses. We investigated the safety and ability of Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE), a synthetic Toll-like receptor (TLR)4 agonist formulation, to adjuvant Fluzone® in mice and non-human primates. The GLA-SE adjuvanted Fluzone vaccine caused no adverse reactions, increased the induction of T helper type 1 (T(H)1)-biased cytokines such as IFNγ, TNF and IL-2, and broadened serological responses against drifted A/H1N1 and A/H3N2 influenza variants.

Applying TLR synergy in immunotherapy: implications in cutaneous leishmaniasis.

Therapy of intracellular pathogens can be complicated by drug toxicity, drug resistance, and the need for prolonged treatment regimens. One approach that has shown promise is immunotherapy. Leishmaniasis, a vector-borne disease ranked among the six most important tropical infectious diseases by the World Health Organization, has been treated clinically with crude or defined vaccine preparations or cytokines, such as IFN-gamma and GM-CSF, in combination with chemotherapy.

Vaccination with the ML0276 antigen reduces local inflammation but not bacterial burden during experimental Mycobacterium leprae infection.

Leprosy elimination has been a goal of the WHO for the past 15 years. Widespread BCG vaccination and multidrug therapy have dramatically reduced worldwide leprosy prevalence, but new case detection rates have remained relatively constant. These data suggest that additional control strategies, such as a subunit vaccine, are required to block transmission and to improve leprosy control.

Enhanced humoral and Type 1 cellular immune responses with Fluzone adjuvanted with a synthetic TLR4 agonist formulated in an emulsion.

Impairments in anti-influenza T helper 1 (Th1) responses are associated with greater risk of influenza-related mortality in the elderly. Addition of adjuvants to existing influenza vaccines could improve immune responses in the elderly. In this study, the activity of three adjuvants, an oil-in-water emulsion and a synthetic lipid A adjuvant formulated with or without the emulsion, is compared.

Leishmania infantum sterol 24-c-methyltransferase formulated with MPL-SE induces cross-protection against L. major infection.

The enzyme sterol 24-c-methyltranferase (SMT) is required for the biosynthesis of ergosterol, the major membrane sterol in Leishmania parasites. SMT and ergosterol are not found in mammals, so this protein may be an attractive target for anti-leishmanial vaccines and drugs. We have previously demonstrated that SMT from L.

Antigen-specific cellular and humoral responses are induced by intradermal Mycobacterium leprae infection of the mouse ear.

Leprosy is caused by infection with Mycobacterium leprae. The immune response of leprosy patients can be highly diverse, ranging from strong cellular responses accompanied by an apparent deficit of M. leprae-specific antibodies to strong humoral responses with a deficit of cell-mediated responses.

Leish-111f, a recombinant polyprotein vaccine that protects against visceral Leishmaniasis by elicitation of CD4+ T cells.

The Leishmania-derived recombinant polyprotein Leish-111f or its three component proteins, thiol-specific antioxidant (TSA), Leishmania major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF), have previously been demonstrated to be efficacious against cutaneous or mucosal leishmaniasis in mice, nonhuman primates, and humans. In this study we demonstrate that Leish-111f is also a vaccine antigen candidate against visceral leishmaniasis (VL) caused by Leishmania infantum. We evaluated the immune response and protection induced by Leish-111f formulated with monophosphoryl lipid A in a stable emulsion (Leish-111f+MPL-SE) and demonstrated that mice developed strong humoral and T-cell responses to the vaccine antigen.

Strategies for selective priming of memory B cells.

Long-term humoral immunity elicited by pathogens and vaccines alike relies upon the generation of both memory B cells (B(mem)) and long-lived plasma cells (PCs). Virtually all vaccine formulations induce the concomitant emergence of both B(mem) and PCs, suggesting that the emergence of these two differentiated B cells subsets is commonly controlled. Evidence presented shows specific Toll-like receptor (TLR) agonists coupled with soluble protein antigen (sAg) can selectively induce the expansion of antigen specific B(mem) in the absence of PC generation.

The in vivo function of a noncanonical TRAF2-binding domain in the C-terminus of CD40 in driving B-cell growth and differentiation.

The recruitment of tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 to the CD40 cytoplasmic tail upon CD40 trimerization results in downstream signaling events that ultimately lead to CD40-dependent, thymus-dependent (TD) humoral immune responses. Previously, we have shown signaling through the C-terminal tail of CD40 in the absence of canonical TRAF-binding sites is capable of signaling through an alternative TRAF2-binding site. Here, we demonstrate that B cells from mice harboring CD40 with only the C-terminal tail can activate both canonical and noncanonical NFkappaB signaling pathways.

BCMA is essential for the survival of long-lived bone marrow plasma cells.

Long-lived humoral immunity is manifested by the ability of bone marrow plasma cells (PCs) to survive for extended periods of time. Recent studies have underscored the importance of BLyS and APRIL as factors that can support the survival of B lineage lymphocytes. We show that BLyS can sustain PC survival in vitro, and this survival can be further enhanced by interleukin 6.

Ligation of CD27 on B cells in vivo during primary immunization enhances commitment to memory B cell responses.

Ligation of CD27 on B cells has been shown to inhibit terminal differentiation of activated murine B cells into plasma cells. We show in this study that this inhibition is accompanied by an enhanced movement of activated B cells toward differentiation into memory cells. Treatment of mice with anti-CD27 during immunization leads to the generation of greater numbers of Ag-binding B cells in draining lymph nodes that persist for longer periods of time, and they contain a greater proportion of cells of a postgerminal center phenotype.