Innate immune responses to vaccine adjuvants based on lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, are driven by Toll-like receptor (TLR) 4 and adaptor proteins ...including MyD88 and TRIF, leading to the production of inflammatory cytokines, type I interferons, and chemokines. We report here on the characterization of a synthetic hexaacylated lipid A derivative, denoted as glucopyranosyl lipid adjuvant (GLA). We assessed the effects of GLA on murine and human dendritic cells (DC) by combining microarray, mRNA and protein multiplex assays and flow cytometry analyses. We demonstrate that GLA has multifunctional immunomodulatory activity similar to naturally-derived monophosphory lipid A (MPL) on murine DC, including the production of inflammatory cytokines, chemokines, DC maturation and antigen-presenting functions. In contrast, hexaacylated GLA was overall more potent on a molar basis than heterogeneous MPL when tested on human DC and peripheral blood mononuclear cells (PBMC). When administered in vivo, GLA enhanced the immunogenicity of co-administered recombinant antigens, producing strong cell-mediated immunity and a qualitative T(H)1 response. We conclude that the GLA adjuvant stimulates and directs innate and adaptive immune responses by inducing DC maturation and the concomitant release of pro-inflammatory cytokines and chemokines associated with immune cell trafficking, activities which have important implications for the development of future vaccine adjuvants.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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. These results suggest that synthetic TLR4 adjuvants can enhance the magnitude and quality of protective immunity induced by influenza vaccines.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a ...CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recent studies have shown that immune responses against the cell-traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) can inhibit parasite infection. While these studies provide ...important evidence toward the development of vaccines targeting this protein, it remains unknown whether these responses could engage the Plasmodium falciparum CelTOS in vivo Using a newly developed rodent malaria chimeric parasite expressing the P. falciparum CelTOS (PfCelTOS), we evaluated the protective effect of in vivo immune responses elicited by vaccination and assessed the neutralizing capacity of monoclonal antibodies specific against PfCelTOS. Mice immunized with recombinant P. falciparum CelTOS in combination with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) or glucopyranosyl lipid adjuvant-liposome-QS21 (GLA-LSQ) adjuvant system significantly inhibited sporozoite hepatocyte infection. Notably, monoclonal antibodies against PfCelTOS strongly inhibited oocyst development of P. falciparum and Plasmodium berghei expressing PfCelTOS in Anopheles gambiae mosquitoes. Taken together, our results demonstrate that anti-CelTOS responses elicited by vaccination or passive immunization can inhibit sporozoite and ookinete infection and impair vector transmission.
One third of the world is infected with Mycobacterium tuberculosis (Mtb) with eight million new cases of active tuberculosis (TB) each year. Development of a new vaccine to augment or replace the ...only approved TB vaccine, BCG, is needed to control this disease. Mtb infection is primarily controlled by TH1 cells through the production of IFN-γ and TNF which activate infected macrophages to kill the bacterium. Here we examine an array of adjuvant formulations containing the TLR4 agonist GLA to identify candidate adjuvants to pair with ID93, a lead TB vaccine antigen, to elicit protective TH1 responses. We evaluate a variety of adjuvant formulations including alum, liposomes, and oil-in-water emulsions to determine how changes in formulation composition alter adjuvant activity. We find that alum and an aqueous nanosuspension of GLA synergize to enhance generation of ID93-specific TH1 responses, whereas neither on their own are effective adjuvants for generation of ID93-specific TH1 responses. For GLA containing oil-in-water emulsions, the selection of the oil component is critical for adjuvant activity, whereas a variety of lipid components may be used in liposomal formulations of GLA. The composition of the liposome formulation of ID93/GLA does alter the magnitude of the TH1 response. These results demonstrate that there are multiple solutions for an effective formulation of a novel TB vaccine candidate that enhances both TH1 generation and protective efficacy.
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Despite the widespread use of the childhood vaccine against tuberculosis (TB), Mycobacterium bovis bacillus Calmette-Guérin (BCG), the disease remains a serious global health problem. A successful ...vaccine against TB that replaces or boosts BCG would include antigens that induce or recall the appropriate T cell responses. Four Mycobacterium tuberculosis (Mtb) antigens--including members of the virulence factor families PE/PPE and EsX or antigens associated with latency--were produced as a single recombinant fusion protein (ID93). When administered together with the adjuvant GLA-SE, a stable oil-in-water nanoemulsion, the fusion protein was immunogenic in mice, guinea pigs, and cynomolgus monkeys. In mice, this fusion protein-adjuvant combination induced polyfunctional CD4 T helper 1 cell responses characterized by antigen-specific interferon-γ, tumor necrosis factor, and interleukin-2, as well as a reduction in the number of bacteria in the lungs of animals after they were subsequently infected with virulent or multidrug-resistant Mtb strains. Furthermore, boosting BCG-vaccinated guinea pigs with fusion peptide-adjuvant resulted in reduced pathology and fewer bacilli, and prevented the death of animals challenged with virulent Mtb. Finally, the fusion protein elicited polyfunctional effector CD4 and CD8 T cell responses in BCG-vaccinated or Mtb-exposed human peripheral blood mononuclear cells. This study establishes that the protein subunit vaccine consisting of the fusion protein and adjuvant protects against TB and drug-resistant TB in animals and is a candidate for boosting the protective efficacy of the childhood BCG vaccine in humans.
Greater than 99% of the Norwalk virus (NV) capsid consists of 180 copies of a single 58-kDa protein. Recombinantly expressed monomers self-assemble into virus-like particles (VLPs) with a well ...defined icosahedral structure. NV-VLPs are an appropriate vaccine antigen since the antigenic determinants of the parent virion are preserved. They also constitute very simple models to study the mechanisms of assembly and disassembly of viral capsids. This work examines the inherent stability of NV-VLPs over a range of pH and temperature values and provides detailed insight into structural perturbations that accompany disassembly. The NV-VLP structure was monitored using a variety of biophysical techniques including intrinsic and extrinsic fluorescence, high resolution second-derivative UV absorption spectroscopy, circular dichroism (CD), dynamic light scattering, differential scanning calorimetry, and direct observation employing transmission electron microscopy. The data demonstrate that NV-VLPs are highly stable over a pH range of 3-7 and up to 55 degrees C. At pH 8, however, reversible capsid dissociation was correlated with increased solvent exposure of tyrosine residues and subtle changes in secondary structure. Above 60 degrees C NV-VLPs undergo distinct phase transitions arising from secondary-, tertiary-, and quaternary-level protein structural perturbations. By combining the spectroscopic data employing a multidimensional eigenvector phase space approach, an empirical phase diagram for NV-VLP was constructed. This strategy of visualization provides a comprehensive description of the physical stability of NV-VLP over a broad range of pH and temperature. Complementary, differential scanning calorimetric analyses suggest that the two domains of VP1 unfold independently in a pH-dependent manner.
Adjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase ...the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against
(
), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE).
In this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations.
We identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies.
These efforts will aid in the development of a platform formulation for use with other similar vaccines.
Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of Ags that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 ...Mtb genes based on criteria that included growth in macrophages, up- or down-regulation under hypoxic conditions, secretion, membrane association, or because they were members of the PE/PPE or EsX families. Recombinant proteins encoded by these genes were evaluated for IFN-gamma recall responses using PBMCs from healthy subjects previously exposed to Mtb. From this screen, dominant human T cell Ags were identified and 49 of these proteins, formulated in CpG, were evaluated as vaccine candidates in a mouse model of tuberculosis. Eighteen of the individual Ags conferred partial protection against challenge with virulent Mtb. A combination of three of these Ags further increased protection against Mtb to levels comparable to those achieved with bacillus Calmette-Guérin vaccination. Vaccine candidates that led to reduction in lung bacterial burden following challenge-induced pluripotent CD4 and CD8 T cells, including Th1 cell responses characterized by elevated levels of Ag-specific IgG2c, IFN-gamma, and TNF. Priority vaccine Ags elicited pluripotent CD4 and CD8 T responses in purified protein derivative-positive donor PBMCs. This study identified numerous novel human T cell Ags suitable to be included in subunit vaccines against tuberculosis.
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