Sublingual vaccination and delivery systems.

Most infectious agents use mucosal tissues as entry portals, thus, mucosae are frequently defined as a first line of defense against pathogens. Mucosal protection generally operates through antibody-mediated and cytotoxic T-cell responses which can be triggered by mucosal vaccines. Sublingual vaccination provides many advantages such as systemic and mucosal responses (both locally and at remote mucosal sites), besides being a needle-free administration route with high patient compliance and limited adverse effects. Buccal mucosa complexity nonetheless represents a challenge for vaccine administration, hence, many efforts were recently deployed to improve vaccine components, mucoadhesion and/or penetration. Several innovative approaches indeed confirmed that a robust and protective immunity can be achieved by sublingual vaccines. This review will then specify the most recent delivery systems and improvements developed to increase sublingual vaccines efficiency. We will focus our description on the immune mechanisms involved and the requirements for optimal sublingual immunization and mucosal protection.

Development of an antibacterial nanocomposite hydrogel for human dental pulp engineering.

Hydrogel-based regenerative endodontic procedures (REPs) are considered to be very promising therapeutic strategies to reconstruct the dental pulp (DP) tissue in devitalized human teeth. However, the success of the regeneration process is limited by residual bacteria that may persist in the endodontic space after the disinfection step and contaminate the biomaterial. The aim of this work was to develop an innovative fibrin hydrogel incorporating clindamycin (CLIN)-loaded Poly (d,l) Lactic Acid (PLA) nanoparticles (NPs) to provide the hydrogel with antibacterial properties. CLIN-PLA-NPs were synthesized by a surfactant-free nanoprecipitation method and their microphysical properties were assessed by dynamic light scattering, electrophoretic mobility and scanning electron microscopy. Their antimicrobial efficacy was evaluated on Enteroccocus fæcalis by the determination of the minimal inhibitory concentration (MIC) and the minimal biofilm inhibition and eradication concentrations (MBIC and MBEC). Antibacterial properties of the nanocomposite hydrogel were verified by agar diffusion assays. NP distribution into the hydrogel and release from it were evaluated using fluorescent PLA-NPs. NP cytotoxicity was assessed on DP mesenchymal stem cells (DP-MSCs) incorporated into the hydrogel. Type I collagen synthesis was investigated after 7 days of culture by immunohistochemistry. We found that CLIN-PLA-NPs displayed a drug loading of 10 ± 2 µg per mg of PLA polymer and an entrapment efficiency of 43 ± 7%. Antibiotic loading did not affect NP size, polydispersity index and zeta potential. The MIC for Enterococcus fæcalis was 32 µg mL-1. MBIC50 and MBEC50 were 4 and 16 µg mL-1, respectively. CLIN-PLA-NPs appeared homogenously distributed throughout the hydrogel. CLIN-PLA-NP-loaded hydrogels clearly inhibited E. faecalis growth. DP-MSC viability and type I collagen synthesis within the fibrin hydrogel were not affected by CLIN-PLA-NPs. In conclusion, CLIN-PLA-NP incorporation into the fibrin hydrogel gave the latter antibacterial and antibiofilm properties without affecting cell viability and function. This formulation could help establish an aseptic environment supporting DP reconstruction and, accordingly, might be a valuable tool for REPs.

Innovative drug vehicle for local treatment of inflammatory skin diseases: Ex vivo and in vivo screening of five topical formulations containing poly(lactic acid) (PLA) nanoparticles.

One of the main goals in the galenic development of innovative topical treatment options for inflammatory skin diseases such as psoriasis and atopic dermatitis is to selectively deliver the drug at the inflammation site. Recent studies have highlighted the beneficial use of polymeric nanoparticles for anti-inflammatory therapy and topical anti-inflammatory drug delivery due to their ability to form a drug reservoir retaining the drug locally at the site of action. Our approach consisted in designing innovative topical semi-solid formulations of poly(lactic acid) (PLA) nanoparticles as anti-inflammatory drug vehicles for local treatment of inflammatory skin diseases. In the course of this work, five topical formulations containing fluorescent PLA nanoparticles were initially developed, and then screened depending on their physico-chemical properties, toxicity and delivery efficacy. The penetration and permeation of a fluorophore vectorized by PLA nanoparticles into healthy and inflammatory skin were assessed using an alternative device to classical Franz cells: VitroPharma. All these investigations led to the selection of two satisfactory formulations out of five initial candidates.

Role of human immunodeficiency virus type 1 envelope structure in the induction of broadly neutralizing antibodies.

Very soon after the discovery of neutralizing antibodies (NAbs) toward human immunodeficiency virus type 1 (HIV-1) infection, it became apparent that characterization of these NAbs would be an important step in finding a cure for or a vaccine to eradicate HIV-1. Since the initial description of broadly cross-clade NAbs naturally produced in HIV-1 patients, numerous studies have described new viral targets for these antibodies. More recently, studies concerning new groups of patients able to control their viremia, such as long-term nonprogressors (LTNPs) or elite controllers, have described the generation of numerous envelope-targeted NAbs. Recent studies have marked a new stage in research on NAbs with the description of antibodies obtained from a worldwide screening of HIV-positive patients. These studies have permitted the discovery of NAb families with great potential for both neutralization and neutralization breadth, such as PG, PGT, CH, and highly active agonistic anti-CD4 binding site antibodies (HAADs), of which VRC01 and its variants are members. These antibodies are able to neutralize more than 80% of circulating strains without any autoreactivity and can be rapidly integrated into clinical trials in order to test their protective potential. In this review, we will focus on new insights into HIV-1 envelope structure and their implications for the generation of potent NAbs.

Síndrome de Cushing iatrogénico por corticoides tópicos en dos adultos / Iatrogenic Cushing’s syndrome caused by topical steroid therapy in two adults

Los corticoides tópicos representan el escalón principal en el tratamiento de muchas enfermedades dermatológicas no infecciosas. Si se usan de forma apropiada son seguros y efectivos, pero sin una supervisión médica adecuada pueden producir efectos secundarios graves, tanto locales como sistémicos. Presentamos dos pacientes adultos diagnosticados de síndrome de Cushing iatrogénico en nuestro hospital. Aunque es una complicación poco frecuente, se debe tener en cuenta en pacientes con dermatosis inflamatorias de larga evolución, ya que puede ser de difícil diagnóstico si no se sospecha (AU)

Topical corticosteroids are the mainstay of treatment for many non- infectious dermatoses. If used appropriately they are a safe and effective therapy, but without medical supervision severe local and systemic adverse effects may occur. We report two patients followed at our Dermatology unit that developed iatrogenic Cushing's syndrome after using topical corticosteroids. Although it's an extremely rare complication in adults, we must be aware of this adverse effect in patients with chronic inflammatory dermatoses, as diagnosis requires a high grade of suspicion (AU)

Pivotal advance: The promotion of soluble DC-SIGN release by inflammatory signals and its enhancement of cytomegalovirus-mediated cis-infection of myeloid dendritic cells.

DC-SIGN is a member of the C-type lectin family. Mainly expressed by myeloid DCs, it is involved in the capture and internalization of pathogens, including human CMV. Several transcripts have been identified, some of which code for putative soluble proteins. However, little is known about the regulation and the functional properties of such putative sDC-SIGN variants. To better understand how sDC-SIGN could be involved in CMV infection, we set out to characterize biochemical and functional properties of rDC-SIGN as well as naturally occurring sDC-SIGN. We first developed a specific, quantitative ELISA and then used it to detect the presence sDC-SIGN in in vitro-generated DC culture supernatants as cell-free secreted tetramers. Next, in correlation with their inflammatory status, we demonstrated the presence of sDC-SIGN in several human body fluids, including serum, joint fluids, and BALs. CMV infection of human tissues was also shown to promote sDC-SIGN release. Based on the analysis of the cytokine/chemokine content of sDC-SIGN culture supernatants, we identified IFN-γ and CXCL8/IL-8 as inducers of sDC-SIGN production by MoDC. Finally, we demonstrated that sDC-SIGN was able to interact with CMV gB under native conditions, leading to a significant increase in MoDC CMV infection. Overall, our results confirm that sDC-SIGN, like its well-known, counterpart mDC-SIGN, may play a pivotal role in CMV-mediated pathogenesis.

Characterization of the antibody response elicited by HIV-1 Env glycomutants in rabbits.

HIV-1 N-glycans are known to shield underlying epitopes towards the protective antibody repertoire. We previously described HIV-1 acute infection Env glycomutants designed from 3D-model in which the removal of clustered N-glycans did not disturb the envelope antigenicity, but increased the neutralization sensitivity. The potential of such immunogens to elicit neutralizing responses was estimated after rabbit immunizations with a DNA/protein protocol. Maturation of the Env-specific antibody response was confirmed by a change in avidity and conformational dependence. For one immunogen, the neutralizing response was increased with a higher breadth compared to the Wild-Type. Our data suggest that Env selective deglycosylation based on 3D data may represent a valuable strategy to improve elicitation of neutralizing antibodies.

Evidence for CTL-mediated selection of Tat and Rev mutants after the onset of the asymptomatic period during HIV type 1 infection.

The evolution of HIV-1 sequences over time is the result of the selection of mutant variants that have escaped from host immune responses or the outgrowth of mutants with increased viral replication, or both. We investigated the contribution of both selection processes to the overall evolution of the Tat and Rev regulatory gene sequences from four individuals, ranging in time from just prior to seroconversion to stable asymptomatic infection. After sequencing at least 15 clones per sample per gene, we analyzed the sequence evolution of the MHC-I motifs that were predicted from the MHC-I haplotypes of these patients. For each identified Tat sequence, we tested the activity of the corresponding encoded protein in a transactivation assay in vitro. Our results suggest that the evolution of the Tat and Rev sequences from these individuals can be explained by mutational escape of the MHC-I epitopes and that no mutations that replaced the original sequences in the viral population are associated with either an increase or decrease in Tat activity. CTL-mediated selection appears to be an important determinant of HIV-1 regulatory gene sequence evolution during the early stages of infection.

[HIV1 Env N-glycans and their interactions with the immune system]. / Les N-glycanes de l'enveloppe du VIH1 et leurs interactions avec le système immunitaire.

N-glycans determine both the conformational and functional characteristics of the HIV1 envelope glycoprotein (Env). In addition, the significant glycosylation modulates the recognition of Env by the different immune system effectors. N-glycans confer to Env the capacity to interact with the type C lectins. Thus, MBLs (mannose binding lectins) recognise the gp120 in a specific manner and exert an antiviral action. Conversely, the interaction between gp120 and DC-SIGN at the dendritic cells surface interferes with the induction of the adaptive immune response. N-glycans also modulate the presentation of the T helper epitopes and thus indirectly influence the induction and the maturation of both the Env cytotoxic cellular and humoral response. Moreover, the N-glycans form a shield which limits the accessibility of the proteic backbone to the antibodies. The constant evolving glycan shield enables neutralising response escape. However, in some cases, N-glycans can constitute clusters which represent a target for the antibodies. The understanding of the multiple roles of N-glycans could significantly contribute to the optimisation of an Env immunogen potentially usable in a vaccine preparation.

[Vaccines against HIV/AIDS]. / Vaccins préventifs anti-VIH/sida.

Some 50 phase I clinical trials of candidate vaccines against HIV/AIDS, 2 phase II trials and 2 phase III trials have been completed since the 1980s, altogether involving more than 16,000 volunteers. Although several neutralization epitopes have been identified on the surface of the virus glycoprotein spikes, the design of an envelope-based HIV vaccine capable of eliciting broadly reactive neutralizing antibodies remains as an elusive goal. A gp120- based vaccine, which was tested in two phase III trials, one in the USA and the other in Thailand, was found to be devoid of protective efficacy. The observation was made in the monkey model, using the simian immunodeficiency virus (SIV), that both virus loads and the clinical evolution of the disease were controlled by the CD8+ T-cell response (CTL) of the animals. This has prompted the development of vaccine candidates capable of inducing HIV-specific T-cell responses. A series of HIV vaccines based on live virus vectors already are in clinical studies, including a live recombinant canarypox virus vaccine (ALVAC), which is in phase III in Thailand, a non-replicative adenovirus type 5 (Ad5) vaccine, which has entered phase II clinical trials in the USA and The Caribbeans, and live recombinant vaccines based on the attenuated vaccinia virus MVA vector, which already have been through several phase I/II studies. These live recombinant vaccines have been evaluated either alone or as booster immunizations after priming with DNA vaccines. A whole array of other vaccines based on live vector vaccines, pseudoviral particles, peptides and other designs, have been tested in nonhuman primate models. So far, using the macaque/SIV model, none of the available vaccine candidates has been able to prevent infection following experimental challenge of the animals, but the vaccinated animals showed significant reduction of viral loads as compared to controls and were able to maintain their CD4+ T-cell count. T-cell stimulating vaccines thus illustrate a new paradigm in vaccinology, that of vaccines which are unable to prevent infection, but can prevent the occurrence of disease or at least slow down its evolution through continuous control of virus replication in the vaccinated host. The efficacy of these vaccines in humans now remains to be established.

[Mechanisms of resistance to sexual transmission of HIV-1]. / Les mécanismes de résistance à la transmission sexuelle du VIH-1.

Sexual transmission is the most common pathway for HIV-1; nevertheless some individuals remain seronegative despite repeated high risk sexual exposure. These were grouped in cohorts of "highly exposed but persistently seronegative" individuals, mostly prostitutes and flailing couples. Three lines of defence were observed in these cohorts. The first one is the mucosal barrier, the determining factors of which are the type of epithelium (monolayer or multilayer), epithelial integrity, and the pre-existing microflora. The second one is linked to innate immunity directly related to the genetic and/or immune predispositions of the individual: mutations affecting the CCR5 chemokine receptor, secretion of protective soluble factors, and particular HLA alleles. The third one is acquired immunity via the mechanisms of humoral and/or specific cellular immunity. These studies suggest anti HIV-1 vaccinal strategies aiming at a local immunization combining the different types of responses observed in these individuals.

Synthesis, assembly, and processing of the Env ERVWE1/syncytin human endogenous retroviral envelope.

Syncytin is a fusogenic protein involved in the formation of the placental syncytiotrophoblast layer. This protein is encoded by the envelope gene of the ERVWE1 proviral locus belonging to the human endogenous retrovirus W (HERV-W) family. The HERV-W infectious ancestor entered the primate lineage 25 to 40 million years ago. Although the syncytin fusion property has been clearly demonstrated, little is known about this cellular protein maturation process with respect to classical infectious retrovirus envelope proteins. Here we show that the cellular syncytin protein is synthesized as a glycosylated gPr73 precursor cleaved into two mature proteins, a gp50 surface subunit (SU) and a gp24 transmembrane subunit (TM). These SU and TM subunits are found associated as homotrimers. The intracytoplasmic tail is critical to the fusogenic phenotype, although its cleavage requirements seem to have diverged from those of classical retroviral maturation.

Therapeutic vaccination for chronic infectious diseases: lessons from HIV-1.

Most therapeutic vaccines are usually divided into two groups according to their mode of action on immunity, based on the induction of either a Immoral response (antibodies) or a cellular response, mostly cytotoxic T lymphocytes (CTLs). The latter ones are in fact the most promising candidates for the treatment of cancer and chronic viral infections. However, we must admit that the design of such vaccines is far from being simple as the biology of the chronic infectious diseases involves complex issues such as viral latency, the existence of reservoirs or immune escape mechanisms. Furthermore, the concept of therapeutic vaccination implies that the host immune system is still competent for eliciting an immune response after vaccination, but patients suffering from chronic infectious diseases usually exhibit impaired immune defenses. To overcome this challenge, the actual tendency is to combine chemotherapy and therapeutic vaccination, playing around with schedules of vaccine administration and standard chemotherapy. To illustrate the different steps in the design and testing of a therapeutic vaccine, the human immunodeficiency virus (HIV-1) for which the efficacy of therapeutic vaccines is currently being evaluated, could serve as a model. Specific points like the rationale of using HIV-1 regulatory genes instead of structural genes, the possibility of using multiple injections of vaccine candidates or the importance of pre-existing immunity will be emphasized, together with the risks of inducing the emergence of new HIV-1 variants upon vaccine treatment of chronically infected HIV-1 patients. The current expertise in the field of therapeutic vaccines in chronically infected HIV-1 patients could be of interest for the design of a therapeutic vaccine for HBV infection.

Negative regulation of CFTR activity by extracellular ATP involves P2Y2 receptors in CFTR-expressing CHO cells.

Extracellular nucleotides exert autocrine/ paracrine effects on ion transport by activating P2 receptors. We studied the effects of extracellular ATP and UTP on the cystic fibrosis transmembrane conductance regulator (CFTR) channel stably expressed in Chinese Hamster Ovary cells (CHO-BQI cells). CFTR activity was measured using the (125I) iodide efflux technique and whole-cell patch-clamp recording in response to either forskolin or xanthine derivatives. Using RT-PCR and intracellular calcium concentration ([Ca2+]i) measurement, we showed that CHO-BQI cells express P2Y2 but not P2Y4 receptors. While ATP and UTP induced similar increases in [Ca2+]i, pre-addition by one of these two agonists desensitized the response for the other, suggesting that ATP- and UTP-induced [Ca2+]i increases were mediated by a common receptor, which was identified as the P2Y2 subtype. CFTR activity was reduced by ATP and UTP but not by ADP or adenosine applications. This inhibitory effect of ATP on CFTR activity was not due to a change in cAMP level. Furthermore, CFTR activation by forskolin or IBMX failed to promote [Ca2+]i increase, suggesting that CFTR activation did not generate an ATP release large enough to stimulate P2Y2 receptors. Taken together, our results show that endogenous P2Y2 receptor activation downregulates CFTR activity in a cAMP-independent manner in CHO cells.

Immunogenicity of recombinant envelope glycoproteins derived from T-cell line-adapted isolates or primary HIV isolates: a comparative study using multivalent vaccine approaches.

We investigated immunogenic properties of native envelope glycoproteins derived from HIV-1 (subtype B). Our main objective was to assess whether the design of multivalent vaccines affects generation of neutralizing antibodies against primary viruses. Recombinant Semliki Forest virus (SFV) particles producing various HIV-1 envelope glycoproteins were used as vaccine vectors. The following multivalent vaccination approaches were compared: 1) immunization with a mixture of recombinant SFV expressing envelope glycoproteins derived from three HIV-1 primary isolates and two T-cell laboratory-adapted (TCLA) viruses; 2) immunization with a mixture of recombinant SFV expressing only the envelope glycoproteins derived from three HIV-1 primary isolates; 3) sequential immunizations with the recombinant SFV expressing the envelope glycoproteins derived from three HIV-1 primary isolates and two TCLA viruses, respectively. Two monovalent vaccine approaches using SFV expressing envelope glycoproteins derived from a single primary isolate or TCLA virus were also included in the study. The multivalent vaccination strategies based on SFV vaccine vectors did not induce more neutralizing antibodies than the previously tested TCLA envelope immunogens, which gave disappointing results against primary isolates.

The transmembrane protein of HIV-1 primary isolates modulates cell surface expression of their envelope glycoproteins.

We have recently shown that the level of cell surface expression of envelope glycoproteins derived from various human immunodeficiency virus type 1 (HIV-1) primary isolates (PI) was lower than those of envelope glycoproteins derived from T-cell laboratory-adapted (TCLA) HIV-1 (D. Brand et al., 2000, Virology 271, 350-362). We investigated this phenomenon by comparing the cell surface expression of chimeric envelope glycoproteins constructed by swapping the gp120 surface and gp41 transmembrane glycoproteins of the TCLA HIV-1MN and the PI HIV-1(133), HIV-1G365, or HIV-1EFRA. We found that each chimeric envelope construct had a cell surface-specific pattern of expression similar to that of the parental envelope glycoproteins corresponding to the gp41. Thus, the difference in cell surface expression observed between TCLA viruses and various PI is probably due to a signal located in gp41. Identification of this signal may be important for the design of PI envelope-derived immunogens and may increase our understanding of the mechanisms by which HIV-1 escapes from the immune system.

Long-term safety and antiretroviral activity of hydroxyurea and didanosine in HIV-infected patients.

Long-term safety, immunologic effects, and antiretroviral activity of hydroxyurea and didanosine were evaluated in this retrospective study. Some 65 HIV-1-infected patients (39 of whom were antiretroviral naive) were studied (mean baseline CD4 count, 362 cells/mm3; mean plasma HIV-1 RNA viral load, 4.8 log10 copies/ml). The mean treatment duration was 20 months. Overall tolerance was good: 15 patients interrupted treatment because of clinical or biologic side effects. Four patients experienced a category B event. Patients had a mean increase of 27 CD4 cell counts after 12 months, of 112 after 24 months and of 59 after 36 months. They had a mean 1. 03 log10 fall in HIV-1 RNA after 12 months, 1.59 log10 after 24 months, and 1.27 log10 after 36 months. After 12 months, 35% developed an HIV-1 RNA viral load <200 copies/ml, 53% after 24 months, and 36% after 36 months. Those whose viral load became undetectable after 12 months have significantly lower baseline RNA values (p =.03). Fourteen patients had a viral load <3.4 log10 copies/ml after 24 months of the double therapy. A prolonged viral load suppression can be achieved using a simple combination of two drugs that are inexpensive and well tolerated.

Antigenic properties of recombinant envelope glycoproteins derived from T-cell-line-adapted isolates or primary human immunodeficiency virus isolates and their relationship to immunogenicity.

The native envelope glycoproteins of primary HIV-1 virions have weaker antigenicity than do T-cell laboratory-adapted (TCLA) viruses. These antigenic properties require further evaluation if recombinant envelope glycoproteins are produced as part of a vaccine strategy. In this study, we compared the antigenicity of recombinant envelope glycoproteins derived from three primary isolates (PI) (HIV-1(BX08), HIV-1(CHA), and HIV-1(133)) and two TCLA viruses (HIV-1(HXB2) and HIV-1(MN)) produced using the Semliki Forest virus (SFV) system. This analysis was performed by radioimmunoprecipitation assays and flow cytometry. The results suggest that the SFV produces envelope glycoproteins with features in common with the envelopes found in naturally occurring virions. In particular, the PI envelopes had weak heterogeneous antigenic properties. However, the cytometric analysis also showed that there was less envelope glycoprotein on the cell surface for the PI envelopes than for those of TCLA viruses, suggesting differences in their intracellular trafficking. The immunogenic properties of the various envelope glycoproteins were evaluated in mice using recombinant SFV particles as vaccine vectors. The PI envelopes were less immunogenic than the TCLA envelopes, probably due to both their low antigenicity and cell surface expression level. Thus, it may be difficult to design an effective vaccine based on native recombinant PI envelopes.