Nicolas Rochereau

Traffic of poly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to sub-epithelial immune competent cells

Mucosal immunization is designed to induce strong immune responses at portal of pathogen entry. Unfortunately, mechanisms underlying the fate of the vaccine vector co-administered with antigens are still partially uncovered and limit further development of mucosal vaccines. Hence, poly(lactic acid) (PLA) nanoparticles being a versatile vaccine vehicle, we have analyzed the fate of these PLA nanoparticles during their uptake at intestinal mucosal sites, both in vivo and ex vivo, to decipher the mechanisms involved during this process. We first designed specific fluorescent PLA nanoparticles exhibiting strong colloidal stability after encapsulation of either 6-coumarin or CellTrace BODIPY before monitoring their transport through mucosa in the mouse ligated ileal loop model. The journey of the particles appears to follow a three-step process. Most particles are first entrapped in the mucus. Then, crossing of the epithelial barrier takes place exclusively through M-cells, leading to an accumulation in Peyers patches (PP). Lastly, we noticed specific interaction of these PLA nanoparticles with underlying B cells and dendritic cells (DCs) of PP. Furthermore, we could document that DCs engulfing some nanoparticles could exhibit a TLR8+ specific expression. Specific targeting of these two cell types strongly supports the use of PLA nanoparticles as a vaccine delivery system for oral use. Indeed, following oral gavage of mice with PLA nanoparticles, we were able to observe the same biodistribution patterns, indicating that these nanoparticles specifically reach immune target required for oral immunization.

Dectin-1 Is Essential for Reverse Transcytosis of Glycosylated SIgA-Antigen Complexes by Intestinal M Cells

This work reports the long-awaited identification of Dectin-1 and Siglec-5 as the M cell co-receptors that mediate the reverse transcytosis of secretory IgA molecules to mount a gut immune response.

Cutting edge: New chimeric NOD2/TLR2 adjuvant drastically increases vaccine immunogenicity.

TLR ligands are critical activators of innate immunity and are being developed as vaccine adjuvants. However, their usefulness in conjunction with NOD-like receptor agonists remains poorly studied. In this study, we evaluated a new ligand that targets both TLR2 and NOD2 receptors. We assessed its ability to enhance dendritic cell maturation in vitro in addition to improving systemic and mucosal immune responses in mice. The chimeric NOD2/TLR2 ligand induced synergistic upregulation of dendritic cell maturation markers, costimulatory molecules, and secretion of proinflammatory cytokines compared with combinations of separate ligands. Furthermore, when coadministered with biodegradable nanoparticles carrying a model Ag, the ligand was able to induce high Ag-specific IgA and IgG titers at both systemic and mucosal sites after parenteral immunizations. These findings point out the potential utility of chimeric molecules TLR/NOD as adjuvants for vaccines to induce systemic and mucosal immune responses.

Delivery of antigen to nasal-associated lymphoid tissue microfold cells through secretory IgA targeting local dendritic cells confers protective immunity.

BACKGROUND Transmission of mucosal pathogens relies on their ability to bind to the surfaces of epithelial cells, to cross this thin barrier, and to gain access to target cells and tissues, leading to systemic infection. This implies that pathogen-specific immunity at mucosal sites is critical for the control of infectious agents using these routes to enter the body. Although mucosal delivery would ensure the best onset of protective immunity, most of the candidate vaccines are administered through the parenteral route. OBJECTIVE The present study evaluates the feasibility of delivering the chemically bound p24gag (referred to as p24 in the text) HIV antigen through secretory IgA (SIgA) in nasal mucosae in mice. RESULTS We show that SIgA interacts specifically with mucosal microfold cells present in the nasal-associated lymphoid tissue. p24-SIgA complexes are quickly taken up in the nasal cavity and selectively engulfed by mucosal dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-positive dendritic cells. Nasal immunization with p24-SIgA elicits both a strong humoral and cellular immune response against p24 at the systemic and mucosal levels. This ensures effective protection against intranasal challenge with recombinant vaccinia virus encoding p24. CONCLUSION This study represents the first example that underscores the remarkable potential of SIgA to serve as a carrier for a protein antigen in a mucosal vaccine approach targeting the nasal environment.

Phenotypic localization of distinct DC subsets in mouse Peyer Patch.

Peyers patch have been extensively studied as a major inductive site for mucosal immunity within the small intestine. The intestinal mucosa contains numerous dendritic cells, which induce either protective immunity to infectious agents or tolerance to innocuous antigens, including food and commensal bacteria. Although during the past few years, several subsets of human mucosal dendritic cells have been described, a precise characterization of the different mouse mucosal dendritic cells subpopulations remains to be achieved with regard to their phenotype and localization in Peyers patch. In this report, we have investigated by immunofluorescence on cryosection and by flow cytometry, the phenotype and the localization of dendritic cells into Peyers patch of C57Bl/6 mouse intestine using dendritic cells markers. Positive and double staining for CD11c and BDCA-2, pDC/IPC, DC-LAMP, DC-SIGN, TLR8 and Langerin have been observed revealing new mouse intestinal DC subsets. This study provides new insight in the understanding of mucosal immune responses induced by natural processes as infections but also new perspectives for the evaluation of oral vaccines.

Recent progress in HIV vaccines inducing mucosal immune responses.

In spite of several attempts over many years at developing a HIV vaccine based on classical strategies, none has convincingly succeeded to date. As HIV is transmitted primarily by the mucosal route, particularly through sexual intercourse, understanding antiviral immunity at mucosal sites is of major importance. An ideal vaccine should elicit HIV-specific antibodies and mucosal CD8+ cytotoxic T-lymphocyte (CTL) as a first line of defense at a very early stage of HIV infection, before the virus can disseminate into the secondary lymphoid organs in mucosal and systemic tissues. A primary focus of HIV preventive vaccine research is therefore the induction of protective immune responses in these crucial early stages of HIV infection. Numerous approaches are being studied in the field, including building upon the recent RV144 clinical trial. In this article, we will review current strategies and briefly discuss the use of adjuvants in designing HIV vaccines that induce mucosal immune responses.

Secretory IgA specific for MPER can protect from HIV-1 infection in vitro.

This preliminary study was performed to assess the neutralization activity of anti-MPER-secretory IgA (SIgA) from parotid saliva in 88 HIV+ patients, in five exposed seronegative (ESN) and in five seronegative volunteers. 48.9% of the HIV+ patients and 100% of ESN patients showed antigp41 SIgA. 25.6% of anti-MPER SIgA from HIV+ patients and 100% from ESN patients neutralized efficiently HIV-1 primary isolate SF162 (IC 50). The neutralization activity is maintained for much lower concentrations in ESN than in HIV+ secretions.

Vaccination against Salmonella Infection: the Mucosal Way

SUMMARY Salmonella enterica subspecies enterica includes several serovars infecting both humans and other animals and leading to typhoid fever or gastroenteritis. The high prevalence of associated morbidity and mortality, together with an increased emergence of multidrug-resistant strains, is a current global health issue that has prompted the development of vaccination strategies that confer protection against most serovars. Currently available systemic vaccine approaches have major limitations, including a reduced effectiveness in young children and a lack of cross-protection among different strains. Having studied host-pathogen interactions, microbiologists and immunologists argue in favor of topical gastrointestinal administration for improvement in vaccine efficacy. Here, recent advances in this field are summarized, including mechanisms of bacterial uptake at the intestinal epithelium, the assessment of protective host immunity, and improved animal models that closely mimic infection in humans. The pros and cons of existing vaccines are presented, along with recent progress made with novel formulations. Finally, new candidate antigens and their relevance in the refined design of anti-Salmonella vaccines are discussed, along with antigen vectorization strategies such as nanoparticles or secretory immunoglobulins, with a focus on potentiating mucosal vaccine efficacy.

Brief Report: A High Rate of β7+ Gut-Homing Lymphocytes in HIV-Infected Immunological Nonresponders is Associated With Poor CD4 T-Cell Recovery During Suppressive HAART

Objective:Correlation between GALT homing markers on lymphocytes and the low blood CD4 T-cell reconstitution in immunological nonresponders (INRs) has been studied. Design:Thirty-one INRs, 19 immunological responders (IRs), and 12 noninfected controls were enrolled in this study. INRs were defined by an undetectable plasma viral load RNA less than 40 copies per milliliter and CD4+ T-cell count <500 cells per cubic milliliter in at least 3 years. Methods:A complete peripheral and mucosal lymphocyte immunophenotyping was performed on these patients with a focus on the CCR9, CCR6, and &agr;4&bgr;7 gut-homing markers. Results:A highly significant upregulation of &agr;4&bgr;7 on INRs peripheral lymphocytes compared with that of IRs has been observed. This upregulation impacts different lymphocyte subsets namely CD4+, CD8+, and B lymphocytes. The frequency of &bgr;7+ Th17 and Treg cells are increased compared with IRs and healthy controls. The frequency of &bgr;7+ CD8+ T cells in the blood is negatively correlated with integrated proviral DNA in rectal lymphoid cells in contrast to &bgr;7+ CD4+ T cells associated with HIV integration. Conclusions:Alteration of lymphocyte homing abilities would have deleterious effects on GALT reconstitution and could participate to HIV reservoir constitution. These results emphasize the great interest to consider &agr;4&bgr;7-targeted therapy in INR patients to block homing of lymphocytes and/or to directly impair gp120-&agr;4&bgr;7 interactions.

Answering the call for educating the new generation of vaccinologists - A new European Erasmus+ Joint Master degree in vaccinology

Vaccines are among the most cost-effective health measures, saving millions of lives each year (WHO). Immunization has led to the eradication of smallpox and poliomyelitis. Immunization also contributes to broader economic benefits and increased cognitive abilities [1,2]. However, the benefits of vaccines are often not fully valued as evidenced by public concerns regarding vaccine safety, quality or efficacy. In addition, the spread of public misinformation via the internet and other media has the potential to impair immunization programs. Elements to improve public confidence in vaccines include evidence-based decision-making procedures and recommendations, controlled processes for licensing and monitoring vaccine safety, effectiveness, disease surveillance, and high quality education programs. Engagement of the community with appropriate communication approaches for each audience is a key factor in building trust in vaccines [3]. Vaccine safety/quality issues should be handled quickly and transparently to be effective. In order to maximize the utilization of vaccines, and thus sustain trust in vaccines, partnerships are needed between public and private health sector stakeholders. Confidence in vaccines can be improved through collaborations that concomitantly ensure (i) efficient highlevel vaccine coverage; (ii) information for the public and other stakeholders concerning the benefits of vaccines; and (iii) ongoing evaluation of vaccine safety monitored by an independent expert body with full transparency. All of the above are best accomplished by insuring adequate numbers of professionals trained in the sciences of vaccinology. Vaccinology is a multidisciplinary discipline that includes vaccine development, education, utilization, policy, and safety. As such, it combines knowledge from basic sciences, medical sciences, public health and social sciences. Vaccinology requires inputs from various basic sciences (mainly immunology, virology and genetics), clinical medicine (clinical trials development, pediatrics, internal medicine), public health (methodology, epidemiology and biostatistics) and social sciences (psychology, communications, public health policy, anthropology). There is clearly an important need to train young scientists as vaccinologists across these areas of content, in order to create a continuous pipeline of vaccinologists for the future [4–7]. There is also a clear need for innovation in vaccine development. Ideally, a vaccinologist grasps a general overview of all disciplines involved, but is also able to focus on a single discipline in depth, as related to vaccinology. A vaccinologist holds a clear overview of how these elements could be assembled to generate new concepts. In our point of view, the three main aspects to train vaccinologists are (1) in-depth exposure of trainees to the multidisciplinary aspects of vaccinology; (2) academic teaching in basic and clinical sciences; (3) practical vaccinology training undertaken in both public sector and industry settings. Such a program prepares vaccinologists to conceive, develop, and assess new vaccines and vaccine policy. A previous editorial in Vaccine called for educating the next generation of vaccinologists, and the clear-cut need for advanced training programs in vaccinology [4]. A new Erasmus Mundus – Joint Master degrees granted by EACEA, the Education, Audiovisual and Culture Executive Agency of the European Commission, entitled “Leading International Vaccinology Education” (LIVE), is scheduled to start in 2016. The general objective of the new LIVE program is to train the next generation of vaccinologists who will have to manage an increasing number of infectious and non-infectious vaccine targets for many important issues: unsolved and still emerging infectious diseases, immunosenescence in an era where there is exponential aging of the population, non-infectious but immune-related diseases (e.g., allergy, cancer and chronic inflammatory diseases such as atherosclerosis, obesity, diabetes, addictions, etc.), and others. Such needs parallel the global requirement to decrease health care expenses while increasing quality and health care outcomes. Meeting these needs starts with providing the funding, teachers, excellent training and career pathways for smart and dedicated students who will devote their professional careers to Vaccinology. LIVE is a novel two-year program for talented and motivated students interested in multidisciplinary studies in Vaccinology. It is a joint project between five European universities (Barcelona, Antwerp, Saint-Etienne and Lyon), each one awarding a Master degree of excellent quality. Academic internationality is enriched by a worldwide network of 12 academic universities from Brazil, Canada, China, Cuba, Europe, and USA. LIVE students will develop a trans-national appreciation for vaccine issues by in-residence participation in educational activities in at least three different countries during the program. Graduates are will also be well prepared for doctoral-level research in Ph.D. programs funded by associated partners. We have identified five specific areas that must be addressed in educating future leaders in vaccinology: