Development of an In Vitro Sensitisation Test Using a Coculture System of Human Bronchial Epithelium and Immune Cells.

Chemical respiratory sensitisation is a serious health problem. However, to date, there are no validated test methods available for identifying respiratory sensitisers. The aim of this study was to develop an in vitro sensitisation test by modifying the human cell line activation test (h-CLAT) to detect respiratory sensitisers and distinguish them from skin sensitisers. THP-1 cells were exposed to the test chemicals (two skin sensitisers and six respiratory sensitisers), either as monocultures or as cocultures with air-liquid interface-cultured reconstructed human bronchial epithelium. The responses were analysed by measuring the expression levels of surface markers on THP-1 cells (CD86, CD54 and OX40L) and the concentrations of cytokines in the culture media (interleukin (IL)-8, IL-33 and thymic stromal lymphopoietin (TSLP)). The cocultures exhibited increased CD54 expression on THP-1 cells; moreover, in the cocultures but not in the monocultures, exposure to two uronium salts (i.e. respiratory sensitisers) increased CD54 expression on THP-1 cells to levels above the criteria for a positive h-CLAT result. Additionally, exposure to the respiratory sensitiser abietic acid, significantly increased IL-8 concentration in the culture medium, but only in the cocultures. Although further optimisation of the method is needed to distinguish respiratory from skin sensitisers by using these potential markers (OX40L, IL-33 and TSLP), the coculture of THP-1 cells with bronchial epithelial cells offers a potentially useful approach for the detection of respiratory sensitisers.

Sterile protection and transmission blockade by a multistage anti-malarial vaccine in the pre-clinical study.

The Malaria Vaccine Technology Roadmap 2013 (World Health Organization) aims to develop safe and effective vaccines by 2030 that will offer at least 75% protective efficacy against clinical malaria and reduce parasite transmission. Here, we demonstrate a highly effective multistage vaccine against both the pre-erythrocytic and sexual stages of Plasmodium falciparum that protects and reduces transmission in a murine model. The vaccine is based on a viral-vectored vaccine platform, comprising a highly-attenuated vaccinia virus strain, LC16m8Δ (m8Δ), a genetically stable variant of a licensed and highly effective Japanese smallpox vaccine LC16m8, and an adeno-associated virus (AAV), a viral vector for human gene therapy. The genes encoding P. falciparum circumsporozoite protein (PfCSP) and the ookinete protein P25 (Pfs25) are expressed as a Pfs25-PfCSP fusion protein, and the heterologous m8Δ-prime/AAV-boost immunization regimen in mice provided both 100% protection against PfCSP-transgenic P. berghei sporozoites and up to 100% transmission blocking efficacy, as determined by a direct membrane feeding assay using parasites from P. falciparum-positive, naturally-infected donors from endemic settings. Remarkably, the persistence of vaccine-induced immune responses were over 7 months and additionally provided complete protection against repeated parasite challenge in a murine model. We propose that application of the m8Δ/AAV malaria multistage vaccine platform has the potential to contribute to the landmark goals of the malaria vaccine technology roadmap, to achieve life-long sterile protection and high-level transmission blocking efficacy.

Adeno-Associated Virus as an Effective Malaria Booster Vaccine Following Adenovirus Priming.

An ideal malaria vaccine platform should potently induce protective immune responses and block parasite transmission from mosquito to human, and it should maintain these effects for an extended period. Here, we have focused on vaccine development based on adeno-associated virus serotype 1 (AAV1), a viral vector widely studied in the field of clinical gene therapy that is able to induce long-term transgene expression without causing toxicity in vivo. Our results show the potential utility of AAV1 vectors as an extremely potent booster vaccine to induce durable immunity when combined with an adenovirus-priming vaccine in a rodent malaria model. We generated a series of recombinant AAV1s and human adenovirus type 5 (AdHu5) expressing either Plasmodium falciparum circumsporozoite protein (PfCSP) or P25 (Pfs25) protein. Heterologous two-dose immunization with an AdHu5-prime and AAV1-boost (AdHu5-AAV1) elicited robust and durable PfCSP- or Pfs25-specific functional antibodies over 280 days. Regarding protective efficacy, AdHu5-AAV1 PfCSP achieved high sterile protection (up to 80% protection rate) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. When examining transmission-blocking (TB) efficacy, we found that immunization with AdHu5-AAV1 Pfs25 maintained TB activity in vivo against transgenic P. berghei expressing Pfs25 for 287 days (99% reduction in oocyst intensity, 85% reduction in oocyst prevalence). Our data indicate that AAV1-based malaria vaccines can confer potent and durable protection as well as TB efficacy when administered following an AdHu5 priming vaccine, supporting the further evaluation of this regimen in clinical trials as a next-generation malaria vaccine platform.

Adenovirus-prime and baculovirus-boost heterologous immunization achieves sterile protection against malaria sporozoite challenge in a murine model.

With the increasing prevalence of artemisinin-resistant malaria parasites, a highly efficacious and durable vaccine for malaria is urgently required. We have developed an experimental virus-vectored vaccine platform based on an envelope-modified baculovirus dual-expression system (emBDES). Here, we show a conceptually new vaccine platform based on an adenovirus-prime/emBDES-boost heterologous immunization regimen expressing the Plasmodium falciparum circumsporozoite protein (PfCSP). A human adenovirus 5-prime/emBDES-boost heterologous immunization regimen consistently achieved higher sterile protection against transgenic P. berghei sporozoites expressing PfCSP after a mosquito-bite challenge than reverse-ordered or homologous immunization. This high protective efficacy was also achieved with a chimpanzee adenovirus 63-prime/emBDES-boost heterologous immunization regimen against an intravenous sporozoite challenge. Thus, we show that the adenovirus-prime/emBDES-boost heterologous immunization regimen confers sterile protection against sporozoite challenge by two individual routes, providing a promising new malaria vaccine platform for future clinical use.