Anne-Sophie Beignon

Immunization onto bare skin with heat-labile enterotoxin of Escherichia coli enhances immune responses to coadministered protein and peptide antigens and protects mice against lethal toxin challenge

In this study, the potential of the bare skin as a non‐invasive route for vaccination was examined. Following application of heat‐labile enterotoxin (LT) of Escherichia coli onto bare skin of BALB/c mice, strong serum anti‐LT antibody responses were observed, and mucosal immunoglobulin A (IgA) and IgG antibodies were measured in vagina washes. In addition, LT enhanced the serum and mucosal antibody and proliferative T‐cell responses to the model protein antigen β‐galactosidase (β‐gal) when coadministered onto bare skin, highlighting its potential to exert an adjuvant effect. When a peptide representing a T‐helper epitope (aa 307–319) from the haemagglutinin of influenza virus was applied onto bare skin with LT or cholera toxin (CT), it primed effectively peptide‐ and virus‐specific T cells, as measured in vitro by the interleukin‐2 (IL‐2) secretion assay. LT was shown to be as immunogenic as CT. Binding activity to GM1 gangliosides was essential for effective induction of anti‐CT serum and mucosal antibody responses. Finally, mice immunized onto bare skin with LT were protected against intraperitoneal challenge with a lethal dose of the homologous toxin. These findings give further support to a growing body of evidence on the potential of skin as a non‐invasive route for vaccine delivery. This immunization strategy might be advantageous for vaccination programmes in Third World countries, because administration by this route is simple, painless and economical.

Transcutaneous Immunization with Tetanus Toxoid and Mutants of Escherichia coli Heat-Labile Enterotoxin as Adjuvants Elicits Strong Protective Antibody Responses

In this study, the adjuvanticity of 2 nontoxic derivatives (LTK63 and LTR72) of heat-labile enterotoxin of Escherichia coli (LT) was evaluated and was compared with that of a cytosine phosphodiester-guanine (CpG) motif, after transcutaneous immunization with tetanus toxoid (TT). TT plus LTR72 elicited the strongest antibody responses, compared with those elicited by the other vaccines (TT, TT plus LTK63, TT plus CpG, and TT plus LTK63 plus CpG); it neutralized the toxin and conferred full protection after passive transfer in mice. Preexisting immunity to LT mutants did not adversely affect their adjuvant potency. Both LTK63 and LTR72 promoted the induction of IgG1 antibodies. In contrast, mice receiving either CpG motif alone or CpG motif plus LTK63 produced strong IgG2a anti-TT antibody responses. Overall, these findings demonstrate that mutants of enterotoxins with reduced toxicity are effective adjuvants for transcutaneous immunization.

Immunization onto bare skin with synthetic peptides: immunomodulation with a CpG-containing oligodeoxynucleotide and effective priming of influenza virus-specific CD4+ T cells

Exploiting the immune system of the skin for vaccine administration offers an attractive alternative to the currently used invasive immunization procedures. In this study we report that a synthetic peptide representing a T‐helper (Th) epitope from influenza virus haemagglutinin (aa 307–319) can be an effective immunogen when coapplied with cholera toxin (CT) onto bare skin. Proliferation of both peptide‐ and influenza virus‐specific CD4+ T cells was measured in lymphocyte cultures from spleens and regional lymph nodes. The presence of the CpG oligodeoxynucleotide 1826 in the peptide/CT formulation, enhanced the proliferation of peptide‐ and virus‐specific T cells as measured by the conventional [3H]thymidine uptake and interleukin (IL)‐2 assays. Furthermore, the bias towards Th2‐type of responses stimulated by CT was shifted towards Th1 as demonstrated (i) by the increase of interferon‐γ and decrease of IL‐4 cytokine levels measured in culture supernatants, (ii) by the predominance of IG2a anti‐CT antibodies in the serum, and (iii) by the down‐regulation of total serum IgE antibody levels. These findings demonstrate the potential of the bare skin as a non‐invasive route for administration of small molecular size peptide antigens. Furthermore, with the selection and combination of the appropriate type of adjuvants, immune responses can be modulated towards the desired type of Th phenotype.

Immunity under the skin: potential application for topical delivery of vaccines

With the technological advances in biomedical sciences and the better understanding of how the immune system works, new immunisation strategies and vaccine delivery options, such sprays, patches, and edible formulations have been developed. This has opened up the possibility of administering vaccines without the use of needles and syringes. Already topical immunisation is a reality and it has the potential to make vaccine delivery more equitable, safer, and efficient. Furthermore, it would increase the rate of vaccine compliance and greatly facilitate the successful implementation of worldwide mass vaccination campaigns against infectious diseases. This review gives a brief account of the latest developments of application of candidate vaccine antigens onto bare skin and describes some of our recent observations using peptide and glycoconjugate vaccines as immunogens.

The bare skin and the nose as non-invasive routes for administering peptide vaccines

Among the different technologies currently tested for the development of novel vaccines, synthetic peptides represent a promising option, since they are chemically pure and induce immune responses of predetermined specificity. Furthermore, they can be replaced with pseudopeptides or peptide mimetics that contain changes in the amide bond, resulting in more stable and immunogenic molecules. Administration of peptide vaccines via non-invasive routes, such as the nose or the bare skin, allows the efficient uptake of antigen by antigen-presenting cells, which are abundant in the associated lymphoid tissues, ensuring the induction of effective systemic and mucosal immune responses. Using non-invasive routes could be advantageous for vaccination programs in third-world countries, since vaccine administration is simple, painless and economical. In this review, we discuss and present some preliminary data on the advantages of synthetic peptides and peptidomimetics as candidate vaccines, and their potential for administration via the skin and the nose.

A retro-inverso peptide analogue of influenza virus hemagglutinin B-cell epitope 91-108 induces a strong mucosal and systemic immune response and confers protection in mice after intranasal immunization.

In this study, a novel approach for the development of a peptide-based vaccine has been tested. We investigated the possibility of replacing an all-L amino acid peptide sequence corresponding to the protective B-cell epitope hemagglutinin (HA) 91-108 from influenza HA with a retro-inverso analogue encompassing this sequence. Retro-inverso peptides are composed of D-amino acids assembled in a reverse order from that of the parent L-sequence, thus maintaining the overall topology of the native sequence. This explains the observed antigenic cross-reactivity with anti-influenza virus antibodies. Mice immunized intranasally with the ovalbumin-conjugated retro-inverso analogue and cholera toxin as an adjuvant, produced strong systemic (serum IgG) and mucosal (lung IgA) antibody responses, and were protected against intranasal challenge with a lethal dose of influenza virus. The weight loss pattern in the protected group indicated that the vaccinated animals developed a disease of low severity resulting in a quick recovery. Furthermore, splenocytes of the immunized mice cultured in the presence of inactivated influenza virus, secreted high levels of IFN-gamma. The half-life of the retro-inverso analogue in the presence of lung homogenate proteases was at least 700 times greater than that of the parent L-peptide. These results demonstrate that peptidomimetic analogues with high resistance to proteolytic degradation are very effective immunogens when administered via the intranasal route, inducing protective immunity against a viral infection. This approach might be advantageous for vaccine development.

Successful Induction of Protective Antibody Responses against Haemophilus influenzae Type b and Diphtheria after Transcutaneous Immunization with the Glycoconjugate Polyribosyl Ribitol Phosphate—Cross-Reacting Material197 Vaccine

We examined the antibody responses elicited in rats after transcutaneous immunization (TCI) with the Haemophilus influenzae type b (Hib)-cross-reacting material (CRM(197)) glycoconjugate vaccine coadministered with cholera toxin or mutants of heat-labile enterotoxin of Escherichia coli (LTK63 and LTR72) as adjuvants. The glycoconjugate vaccine was immunogenic, eliciting high antibody responses to the capsular polysaccharide of Hib and to diphtheria toxin. Passively transferred immune serum protected infant rats against challenge with the Hib Eagan strain and exhibited strong neutralizing activity against diphtheria toxin both in vitro and in vivo. The finding that TCI of rats can elicit antibody responses surpassing the minimum levels required for protective immunity against Hib and diphtheria suggests that this immunization strategy holds a lot of promise for future pediatric use. However, further studies are required to confirm the potential of TCI with glycoconjugate vaccines in humans.

Applying peptide antigens onto bare skin: induction of humoral and cellular immune responses and potential for vaccination

The development of non-invasive immunisation procedures is a top priority for public health agencies when it is realised that the current immunisation practices are unsafe, particularly in developing countries due to the widespread reuse of non-sterile syringes. There is a risk of abscess formation resulting in impairment of meat quality or the value of the hide, and the risk of transmission of infectious diseases when vaccines are administered to food animals by injection. Recently, the skin has emerged as an alternative route for non-invasive delivery of vaccines. Topical application of various types of antigens (mainly proteins and toxoids) with an adjuvant resulted in the induction of systemic and mucosal immune responses. However, due to skin barrier constraints and the physicochemical properties of large molecular weight proteins, the immune responses are variable and require further optimisation. Small molecular size synthetic peptides when applied onto bare skin with an adjuvant are effective immunogens, inducing both humoral and cellular immune responses. Their use as vaccines offers considerable advantages over conventional preparations in terms of safety, purity, stability, availability and cost. Therefore, they could be the most suitable candidate immunogens for skin immunisation. This review describes our recent observations on the immunogenicity of synthetic peptides applied onto bare skin in relation to vaccination.