Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display.

Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo-electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach.

Exploiting Natural Cross-reactivity between Human Immunodeficiency Virus (HIV)-1 p17 Protein and Anti-gp41 2F5 Antibody to Induce HIV-1 Neutralizing Responses In Vivo.

Anti-p17 antibodies are able to neutralize human immunodeficiency virus (HIV) entry in a mouse model. In this study, we identified a region of sequence similarity between the epitopes of anti-p17 neutralizing antibodies and anti-gp41 neutralizing 2F5 antibody and verified cross-reactivity between p17 and 2F5 in vitro. The p17 sequence was modified to increase sequence identity between the p17 and 2F5 epitopes, which resulted in enhanced cross-reactivity in vitro. Immunogenicity of wild-type and modified p17 was characterized in a rabbit model. Both wild-type and mutated p17 induced anti-gp41 responses in rabbits; sera from these animals reacted with gp41 from different HIV clades. Moreover, introduction of the 2F5 sequence in p17 resulted in induction of antibodies with partially neutralizing activity. Based upon these data, we suggest that the natural cross-reactivity between HIV-1 p17 protein and 2F5 antibody can be exploited to induce antibodies with neutralizing activity in an animal model.

Improving bioassay sensitivity through immobilization of bio-probes onto reactive micelles.

An antigen probe (HIV-1 p24) immobilized onto N-succinimidyl ester based micelles was used as a solid phase coating in ELISA test, and induced a significant improvement in antibody detection sensitivity as compared to the standard free antigen coating. The relevance of this straightforward approach to improve the bioassay sensitivity was confirmed by using biotin as a generic probe.

Poly(Lactic Acid) Nanoparticles Targeting α5ß1 Integrin as Vaccine Delivery Vehicle, a Prospective Study.

Biodegradable polymeric nanoparticles are vehicles of choice for drug delivery and have the ability to encapsulate and present at their surface different molecules of interest. Among these bio-nanocarriers, poly(lactic acid) (PLA) nanoparticles have been used as adjuvant and vehicle for enhanced vaccine efficacy. In order to develop an approach to efficient vaccine delivery, we developed nanoparticles to target α5ß1 positive cells. We first overproduced, in bacteria, human fibronectin FNIII9/10 recombinant proteins possessing an integrin α5ß1 binding site, the RGDS sequence, or a mutated form of this site. After having confirmed the integrin binding properties of these recombinant proteins in cell culture assays, we were able to formulate PLA nanoparticles with these FNIII9/10 proteins at their surface. We then confirmed, by fluorescence and confocal microscopy, an enhanced cellular uptake by α5ß1+ cells of RGDS-FNIII9/10 coated PLA nanoparticles, in comparison to KGES-FNIII9/10 coated or non-coated controls. As a first vaccination approach, we prepared PLA nanoparticles co-coated with p24 (an HIV antigen), and RGDS- or KGES-FNIII9/10 proteins, followed by subcutaneous vaccine administration, in mice. Although we did not detect improvements in the apparent humoral response to p24 antigen in the serum of RGDS/p24 nanoparticle-treated mice, the presence of the FNIII proteins increased significantly the avidity index of anti-p24 antibodies compared to p24-nanoparticle-injected control mice. Future developments of this innovative targeted vaccine are discussed.

Preparation and in vitro evaluation of imiquimod loaded polylactide-based micelles as potential vaccine adjuvants.

PURPOSE: Activation of immune cells through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) or NOD-like receptors (NLRs), has been identified as a key issue in the development of new efficient vaccine adjuvants. We report here on the elaboration and immunostimulatory potential of polylactide (PLA)-based micelles core-loaded with imiquimod TLR7 ligand and able to be further surface-functionalized with antigenic protein (HIV-1 Gag p24) for antigen delivery purpose. METHODS: Micelles prepared from poly(D,L-lactide)-b-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) amphiphilic copolymer were incubated in the presence of imiquimod, leading to 1.2 wt% loading, and further conjugated to p24 antigen through reaction of p24 lysines and N-terminal amine with the N-succinimidyl pendant groups of the micelle corona. The impact of imiquimod encapsulation in the micelles on its immunostimulatory properties was investigated in vitro, by monitoring: (i) the NF-κB and mitogen-activated protein kinases (MAPK) pathways through experiments with RAW-Blue™ cells, a mouse macrophage cell line encoding an NF-κB/AP-1-inducible reporter construct; (ii) human dendritic cells (DCs) maturation markers by flow cytometry. RESULTS: RAW-Blue™ cells based experiments showed that imiquimod encapsulated in the micelles was much more efficient to activate the NF-κB and MAPK pathways than free imiquimod. Furthermore, encapsulated imiquimod was found to induce much higher maturation of DCs than the free analog. Finally, these immunostimulatory properties of the loaded imiquimod were shown to be conserved when the p24 antigen was coupled at the micelle surface. CONCLUSIONS: Taken together, these data regarding improved immunostimulatory efficiency suggest the strong potential of our micelle-based nano-system for vaccine delivery.

Formulation of HIV-1 Tat and p24 antigens by PLA nanoparticles or MF59 impacts the breadth, but not the magnitude, of serum and faecal antibody responses in rabbits.

Biodegradable nanoparticles coated with proteins represent a promising method for in vivo delivery of vaccines. Here we used a rabbit model to compare quantitatively and qualitatively the antibody responses induced by poly(D,L-lactide) nanoparticles (PLA) and by emulsion adjuvant MF59 using three HIV-1 antigens: p24gag, WT Tat and a mutated, detoxified form of Tat. We could show that all antigens and adjuvants lead to the induction of similar level of IgG titres in serum when injected subcutaneously. p24, but not Tat, could also induce faecal IgG in rabbits when formulated with PLA or MF59. The nature of the adjuvant had consequences on the spectrum of specificity induced, depending on the antigen: PLA adjuvant focussed the anti-p24 response to an immunodominant domain when compared to MF59. With wild-type Tat, no difference between adjuvants was observed in the spectrum of specificity induced. On the opposite, detoxified Tat coated on PLA increased the number of epitopes recognized by serum IgG compared to MF59 adjuvantation. The impact of these qualitative differences depending on the antigen/adjuvant associations will be important to take into account for further designs of vaccinal formulation using particulate adjuvants.

Surfactant-free anionic PLA nanoparticles coated with HIV-1 p24 protein induced enhanced cellular and humoral immune responses in various animal models.

Microparticles and nanoparticles prepared with poly(D,L-lactide-co-glycolide) (PLGA) or poly(D,L-lactide) (PLA) polymers represent a promising method for in vivo delivery of encapsulated peptide, protein or DNA antigens. However, one major issue that limits the potential of these delivery systems is the instability or the degradation of the entrapped antigen. Charged microparticles carrying surface adsorbed antigen were developed to resolve this problem and appear more suitable for vaccine applications. We describe here new anionic PLA nanoparticles obtained by the dialysis method that are absolutely surfactant-free, which makes them more appropriate for use in humans. The potency of this delivery system as a vaccine carrier was tested in various animal models using HIV-1 p24 protein. p24-coated PLA nanoparticles (p24/PLA) induced high antibody titres (>10(6)) in mice, rabbits and macaques. Moreover, p24/PLA nanoparticles elicited strong CTL responses and a Th1-biased cytokine release (IFNgamma, IL-2) in mice. p24 protein seemed to generate a more Th1-oriented response when administered coated onto the surface of PLA nanoparticles than adjuvanted with Freund's adjuvant. Most importantly, the ability of p24/PLA particles to induce Th1 responses was also confirmed in the macaque model, since high levels of IFNgamma-producing CD4+ T cells and CD8+ T cells could be detected by the ELISPOT assay. This protein delivery system confirms the potential of charged nanoparticles in the field of vaccine development.

Short communication: retrospective study to time the introduction of HIV type 1 non-B subtypes in Lyon, France, using env genes obtained from primary infection samples.

Using blood samples from primary HIV-1 infection (PHI) patients obtained in Lyon, France, we characterized the newly transmitted HIV-1 variants in this area during the 1992-1996 period. As PHI samples allowed the precise timing of the transmission event, we were able to date the introduction of non-B subtypes or recombinant forms of the virus in Lyon. Genomic DNA from 18 HIV-1-positive patients at primary infection was used to amplify the full-length env gene by nested PCR; after cloning, the gene was sequenced for subsequent phylogenetic analysis. Several non-B subtypes and recombinant forms of HIV-1 were identified among the 18 patients studied (1 subtype F1, 1 CRF01-AE, 2 subtype G and 2 CRF02-AG). We also found a new J/K recombinant form transmitted in 1995 and never described until now. The introduction of CRF02-AG in Lyon, France, occurred prior to 1992 and six transmission events including non-B subtypes were documented in the following 4 years. Heterosexual contacts appeared as the main introduction pathway for non-B subtypes or recombinant forms. Nevertheless, as transmission of these viruses occurred not only during travel to endemic regions, but also in France or Germany, we conclude that non-B strains entered Europe before the studied period. This retrospective study showed that even if subtype B remained prevalent in the spreading HIV-1 infection in Lyon between 1992 and 1996, non-B subtypes and circulating recombinant forms represented a significantly growing part.

Evaluation in rhesus macaques of Tat and rev-targeted immunization as a preventive vaccine against mucosal challenge with SHIV-BX08.

Recent evidence suggests that a CD8-mediated cytotoxic T-cell response against the regulatory proteins of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) may control infection after pathogenic virus challenge. Here, we evaluated whether vaccination with Tat or Tat and Rev could significantly reduce viral load in nonhuman primates. Rhesus macaques were primed with Semliki forest Virus (SFV) expressing HIV-1 tat (SFV-tat) and HIV-1 rev (SFV-rev) and boosted with modified vaccinia virus Ankara (MVA) expressing tat and rev. A second group of monkey was primed with SFV-tat only and boosted with MVA-tat. A third group received a tat and rev DNA/MVA prime-boost vaccine regimen. Monitoring of anti-Tat and anti-Rev antibody responses or antigen-specific IFN-gamma production, as measured by enzyme-linked immunospot assays revealed no clear differences between the three groups. These results suggest that priming with either DNA or SFV seemed to be equivalent, but the additive or synergistic effect of a rev vaccine could not be clearly established. The animals were challenged by the rectal route 9 weeks after the last booster immunization, using 10 MID(50) of a SHIV-BX08 stock. Postchallenge follow-up of the monkeys included testing seroconversion to Gag and Env antigens, measuring virus infectivity in PBMC by cocultivation with noninfected human cells, and monitoring of plasma viral load. None of the animals was protected from infection as assessed by PCR, but peak viremia was reduced more than 200-fold compared to sham controls in one third (6/18) of vaccinated macaques, whatever the vaccine regimen they received. Interestingly, among these six protected animals four did not seroconvert. Altogether, these results clearly indicated that the addition of early HIV proteins like Tat and Rev in a multicomponent preventive vaccine including structural proteins like Env or Gag may be beneficial in preventive vaccinal strategies.