Immune responses and protection induced by mucosal and systemic immunisation with recombinant measles nucleoprotein in a mouse model of measles virus-induced encephalitis.

In this study the immunogenicity of recombinant nucleoprotein (Np) administered intranasally or intraperitoneally, and its ability to support a systemic protective anti-virus antibody response was examined, in a mouse model of measles virus (MV)-induced encephalitis. Although both intranasal and intraperitoneal routes of immunisation resulted in priming Np- and MV-specific T-cell responses, the intraperitoneal route was shown to prime for a predominantly IgG2a serum anti-MV antibody response of high avidity, which confered complete protection following intracranial challenge with a neuroadapted strain of MV. On the other hand, intranasal priming resulted in a mixed IgG1, IgG2a serum anti-MV antibody response of low avidity, and only 43% of immunised mice survived following intracranial challenge with the neuroadapted strain of MV. These findings suggest that the route of immunisation in combination with an appropriate adjuvant could influence the induction of a quality antibody response with protective capacity

HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨm) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor

Immune Modulation by Adjuvants Combined with Diphtheria Toxoid Administered Topically in BALB/c Mice After Microneedle Array Pretreatment

Purpose. In this study, modulation of the immune response against diphtheria toxoid (DT) by various adjuvants in transcutaneous immunization (TCI) with microneedle array pretreatment was investigated. Methods. TCI was performed on BALB/c mice with or without microneedle array pretreatment using DT as a model antigen co-administrated with lipopolysaccharide (LPS), Quil A, CpG oligo deoxynucleotide (CpG) or cholera toxin (CT) as adjuvant. The immunogenicity was evaluated by measuring serum IgG subtype titers and neutralizing antibody titers. Results. TCI with microneedle array pretreatment resulted in a 1,000-fold increase of DT-specific serum IgG levels as compared to TCI. The immune response was further improved by co-administration of adjuvants, showing a progressive increase in serum IgG titers when adjuvanted with LPS, Quil A, CpG and CT. IgG titers of the CT-adjuvanted group reached levels comparable to those obtained after DTalum subcutaneous injection. The IgG1/IgG2a ratio of DT-specific antibodies decreased in the following sequence: plain DT, Quil A, CT and CpG, suggesting that the immune response was skewed towards the Th1 direction. Conclusions. The potency and the quality of the immune response against DT administered by microneedle array mediated TCI can be modulated by co-administration of adjuvants. KEY WORDS: cholera toxin; CpG; diphtheria toxoid; microneedle array; transcutaneous immunization

Synthetic Double-Stranded RNAs Are Adjuvants for the Induction of T Helper 1 and Humoral Immune Responses to Human Papillomavirus in Rhesus Macaques

Toll-like receptor (TLR) ligands are being considered as adjuvants for the induction of antigen-specific immune responses, as in the design of vaccines. Polyriboinosinic-polyribocytoidylic acid (poly I:C), a synthetic double-stranded RNA (dsRNA), is recognized by TLR3 and other intracellular receptors. Poly ICLC is a poly I:C analogue, which has been stabilized against the serum nucleases that are present in the plasma of primates. Poly I:C12U, another analogue, is less toxic but also less stable in vivo than poly I:C, and TLR3 is essential for its recognition. To study the effects of these compounds on the induction of protein-specific immune responses in an animal model relevant to humans, rhesus macaques were immunized subcutaneously (s.c.) with keyhole limpet hemocyanin (KLH) or human papillomavirus (HPV)16 capsomeres with or without dsRNA or a control adjuvant, the TLR9 ligand CpG-C. All dsRNA compounds served as adjuvants for KLH-specific cellular immune responses, with the highest proliferative responses being observed with 2 mg/animal poly ICLC (p = 0.002) or 6 mg/animal poly I:C12U (p = 0.001) when compared with immunization with KLH alone. Notably, poly ICLC—but not CpG-C given at the same dose—also helped to induce HPV16-specific Th1 immune responses while both adjuvants supported the induction of strong anti-HPV16 L1 antibody responses as determined by ELISA and neutralization assay. In contrast, control animals injected with HPV16 capsomeres alone did not develop substantial HPV16-specific immune responses. Injection of dsRNA led to increased numbers of cells producing the T cell–activating chemokines CXCL9 and CXCL10 as detected by in situ hybridization in draining lymph nodes 18 hours after injections, and to increased serum levels of CXCL10 (p = 0.01). This was paralleled by the reduced production of the homeostatic T cell–attracting chemokine CCL21. Thus, synthetic dsRNAs induce an innate chemokine response and act as adjuvants for virus-specific Th1 and humoral immune responses in nonhuman primates

Immunoprotectivity of HLA-A2 CTL Peptides Derived from Respiratory Syncytial Virus Fusion Protein in HLA-A2 Transgenic Mouse

Identification of HLA-restricted CD8+ T cell epitopes is important to study RSV-induced immunity and illness. We algorithmically analyzed the sequence of the fusion protein (F) of respiratory syncytial virus (RSV) and generated synthetic peptides that can potentially bind to HLA-A*0201. Four out of the twenty-five 9-mer peptides tested: peptides 3 (F33–41), 13 (F214–222), 14 (F273–281), and 23 (F559–567), were found to bind to HLA-A*0201 with moderate to high affinity and were capable of inducing IFN-γ and IL-2 secretion in lymphocytes from HLA-A*0201 transgenic (HLA-Tg) mice pre-immunized with RSV or recombinant adenovirus expressing RSV F. HLA-Tg mice were immunized with these four peptides and were found to induce both Th1 and CD8+ T cell responses in in vitro secondary recall. Effector responses induced by these peptides were observed to confer differential protection against live RSV challenge. These peptides also caused better recovery of body weight loss induced by RSV. A significant reduction of lung viral load was observed in mice immunized with peptide 23, which appeared to enhance the levels of inflammatory chemokines (CCL17, CCL22, and IL-18) but did not increase eosinophil infiltration in the lungs. Whereas, significant reduction of infiltrated eosinophils induced by RSV infection was found in mice pre-immunized with peptide 13. Our results suggest that HLA-A2-restricted epitopes of RSV F protein could be useful for the development of epitope-based RSV vaccine

Recombinant Modified Vaccinia Virus Ankara Expressing Glycoprotein E2 of Chikungunya Virus Protects AG129 Mice against Lethal Challenge

Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections

Mimotopes of viral antigens and biologically important molecules as candidate vaccines and potential immunotherapeutics.

Antigen recognition by antibodies or ligand-receptor interactions involve small areas of the molecule named epitopes that are normally conformational in nature. The availability of combinatorial peptide libraries has provided a powerful tool for selecting novel sequences which mimic conformational epitopes (mimotopes) either structurally and/or immunologically. These mimotopes can be particularly useful in a number of situations, including: the development of vaccines against tumors, infectious diseases or allergic conditions; the design of molecules which can act as agonists or antagonists of various biologically-important molecules; and for the development of diagnostic assays. This article reviews the authors work on the application of combinatorial peptide libraries to identify mimotopes of protective B-cell epitopes from various pathogens, and the search for molecules able to block the biological activities of TNF-alpha, a cytokine which plays a key role in inflammation

Evaluation of the LTK63 adjuvant effect on cellular immune responses to measles virus nucleoprotein

Background: A lot of pathogens enter the body via the nasal route. The construction of non-toxic mutants of heat labile Escherichia coli enterotoxin (LT), which is a potent mucosal adjuvant, represents a major breakthrough for the development of mucosal vaccines. Objective: This study was undertaken to critically evaluate the adjuvanticity of the mutant of LT (LTK63) on the cellular immune responses to intranasally co-administered recombinant measles virus nucleoprotein (rMVNP). Methods: Groups of CBA mice were immunized intranasally with rMVNP with or without LT or LTK63 as adjuvants. Another group was immunized subcutaneously with rMVNP in Freund&apos;s adjuvant. rMVNP and measles virus (MV) were used in a proliferation assay to test the LTK63 potentiating ability to induce T cell responses. Subsequently MVNP synthetic peptides spanning the length of the N protein were used with a proliferation assay to identify the T cell epitopes. Results: Splenocytes from mice immunized intranasally with rMVNP plus LT or LTK63, showed strong dose dependent proliferative responses to both the MVNP and MV. However, proliferative responses from the latter group were significantly lower than the former group (P < 0.05). Splenocytes tested recognized peptides 20, 21, 28, 31, 39, 40 and 50, suggesting these to be among important epitopes. Subcutaneous route was not effective in priming for T cell responses to rMVNP. Conclusion: These data further demonstrate the great potential of LTK63 as a safe mucosal vaccine adjuvant

Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: towards the construction of nanotube-based gene delivery vectors

10In the present report, we study the interactions of three types of f-CNTs, ammonium-functionalized single-walled and multiwalled carbon nanotubes (SWNT-NH3 +; MWNT-NH3 +), and lysine-functionalized single-walled carbon nanotubes (SWNTLys- NH3 +), with plasmid DNA. Nanotube-DNA complexes were analyzed by scanning electron microscopy, surface plasmon resonance, PicoGreen dye exclusion, and agarose gel shift assay. The results indicate that all three types of cationic carbon nanotubes are able to condense DNA to varying degrees, indicating that both nanotube surface area and charge density are critical parameters that determine the interaction and electrostatic complex formation between f-CNTs with DNA.This is a highly interdisciplinary paper, describing the use of carbon nanotubes in gene delivery. The article has received, so far, 283 citations.nonemixedR. SINGH; D. PANTAROTTO; D. MCCARTHY; O. CHALOIN; J. HOEBEKE; C. D. PARTIDOS; J.-P. BRIAND; PRATO M.; A. BIANCO; K. KOSTARELOSR., Singh; Pantarotto, Davide; D., Mccarthy; O., Chaloin; J., Hoebeke; C. D., Partidos; J. P., Briand; Prato, Maurizio; A., Bianco; K., Kostarelo