Else Marie Agger

The majority of vaccine candidates in clinical development are highly purified proteins and peptides relying on adjuvants to enhance and/or direct immune responses. Despite the acknowledged need for novel adjuvants, there are still very few adjuvants in licensed human vaccines. A vast number of adjuvants have been tested pre-clinically using different experimental conditions, rendering it impossible to directly compare their activity. We performed a head-to-head comparison of five different adjuvants Alum, MF59®, GLA-SE, IC31® and CAF01 in mice and combined these with antigens from M. tuberculosis, influenza and chlamydia to test immune-profiles and efficacy in infection models using standardized protocols. Regardless of antigen, each adjuvant had a unique immunological signature suggesting that the adjuvants have potential for different disease targets. Alum increased antibody titers; MF59® induced strong antibody and IL-5 responses; GLA-SE induced antibodies and Th1; CAF01 showed a mixed Th1/Th17 profile and IC31® induced strong Th1 responses. MF59® and GLA-SE were strong inducers of influenza HI titers while CAF01, GLA-SE and IC31® enhanced protection to TB and chlamydia. Importantly, this is the first extensive attempt to categorize clinical-grade adjuvants based on their immune profiles and protective efficacy to inform a rational development of next generation vaccines for human use.

The Autotransporter pathway, ubiquitous in Gram-negative bacteria, allows the efficient secretion of large passenger proteins via a relatively simple mechanism. Capitalizing on its crystal structure, we have engineered the Escherichia coli autotransporter Hemoglobin protease (Hbp) into a versatile platform for secretion and surface display of multiple heterologous proteins in one carrier molecule. As proof-of-concept, we demonstrate efficient secretion and high-density display of the sizeable Mycobacterium tuberculosis antigens ESAT6, Ag85B and Rv2660c in E. coli simultaneously. Furthermore, we show stable multivalent display of these antigens in an attenuated Salmonella Typhimurium strain upon chromosomal integration. To emphasize the versatility of the Hbp platform, we also demonstrate efficient expression of multiple sizeable antigenic fragments from Chlamydia trachomatis and the influenza A virus at the Salmonella cell surface. The successful efficient cell surface display of multiple antigens from various pathogenic organisms highlights the potential of Hbp as a universal platform for the development of multivalent recombinant bacterial vector vaccines.

Significance: Despite the availability of a number of antibiotics and extensive use of the live vaccine bacillus Calmette–Guérin, tuberculosis remains a major global health problem. Antigen 85 is by far the most popular antigen in current clinical trials vaccines. However, a recent failure of a virus-based vaccine expressing antigen 85 emphasizes the continued need for identifying antigens, testing their protective efficacy, and learning from this process to develop a protective vaccine. The H65 vaccine demonstrates that it is possible to develop antigen 85-free vaccines that does not interfere with current immune-based diagnostic assays.

Else Marie Agger speaks to Katie Lockwood, Assistant Commissioning Editor Else Marie Agger was appointed Director of the Department of Infectious Disease Immunology, Statens Serum Institut in Denmark in 2011. Prior to this, she had been heading two different research groups focusing on tuberculosis (TB) vaccine research and adjuvant development, respectively. She holds a postdoctoral degree within immunology from the University of Copenhagen (Denmark). Agger has extensive experience within adjuvant research, TB vaccine research and preclinical development of vaccines. In addition, she has previously been involved in antigen discovery, primarily with a focus on developing novel diagnostic tests within TB. She has been involved in the coordination of vaccine delivery optimization within the EU-funded TBVAC and NEWTBVAC consortia and also has previous experience as a work-package leader within an EU-funded project, focusing on adjuvantation of influenza vaccines. Agger is the coinventor of several adjuvant patents and has published more than 60 papers within TB, vaccines and adjuvant research.

It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed. Herein, we describe a cationic adjuvant formulation (CAF01) consisting of DDA as a delivery vehicle and synthetic mycobacterial cordfactor as immunomodulator. CAF01 primes strong and complex immune responses and using ovalbumin as a model vaccine antigen in mice, antigen specific cell-mediated- and humoral responses were obtained at a level clearly above a range of currently used adjuvants (Aluminium, monophosphoryl lipid A, CFA/IFA, Montanide). This response occurs through Toll-like receptor 2, 3, 4 and 7-independent pathways whereas the response is partly reduced in MyD88-deficient mice. In three animal models of diseases with markedly different immunological requirement; Mycobacterium tuberculosis (cell-mediated), Chlamydia trachomatis (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge. CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection.

Summary: It is known that protection against tuberculosis is mediated primarily by T helper type 1 (Th1) cells but the influence of the Th1/Th2 balance of a vaccination response on the subsequent protection and pathology during infection has not been studied in detail. We designed a panel of Ag85B‐ESAT‐6 subunit vaccines based on adjuvants with different Th1/Th2‐promoting activities and studied cellular responses, bacterial replication and pathology in the lungs of mice infected with Mycobacterium tuberculosis. All vaccines induced cell‐mediated and humoral responses but with markedly different interferon‐γ : interleukin‐5 (IFN‐γ : IL‐5) and immunoglobulin G1 (IgG1) : IgG2 ratios. The vaccines promoted different levels of control of bacterial replication with the most efficient protection being exerted by cationic liposomes containing monophosphoryl lipid A and low to completely absent immunity with conventional aluminium. The level of protection correlated with the amount of IFN‐γ produced in response to the vaccine whereas there was no inverse correlation with the level of IL‐5. Characterizing a protective response was an accelerated recruitment of IL‐17 and IFN‐γ‐producing lymphocytes resulting in the early formation of granulomas containing clustered inducible nitric oxide synthase‐activated macrophages. In comparison, non‐protected mice exhibited a different inflammatory infiltrate rich in neutrophil granulocytes. This study indicates that the adjuvant component of a tuberculosis vaccine may be crucial in determining the kinetics by which effective granulomas, pivotal in controlling bacterial growth, are formed.

Summary: Cationic liposomes are being used increasingly as efficient adjuvants for subunit vaccines but their precise mechanism of action is still unknown. Here, we investigated the adjuvant mechanism of cationic liposomes based on the synthetic amphiphile dimethyldioctadecylammonium (DDA). The liposomes did not have an effect on the maturation of murine bone‐marrow‐derived dendritic cells (BM‐DCs) related to the surface expression of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86. We found that ovalbumin (OVA) readily associated with the liposomes (> 90%) when mixed in equal concentrations. This efficient adsorption onto the liposomes led to an enhanced uptake of OVA by BM‐DCs as assessed by flow cytometry and confocal fluorescence laser‐scanning microscopy. This was an active process, which was arrested at 4° and by an inhibitor of actin‐dependent endocytosis, cytochalasin D. In vivo studies confirmed the observed effect because adsorption of OVA onto DDA liposomes enhanced the uptake of the antigen by peritoneal exudate cells after intraperitoneal injection. The liposomes targeted antigen preferentially to antigen‐presenting cells because we only observed a minimal uptake by T cells in mixed splenocyte cultures. The adsorption of antigen onto the liposomes increased the efficiency of antigen presentation more than 100 times in a responder assay with MHC class II‐restricted OVA‐specific T‐cell receptor transgenic DO11.10 T cells. Our data therefore suggest that the primary adjuvant mechanism of cationic DDA liposomes is to target the cell membrane of antigen‐presenting cells, which subsequently leads to enhanced uptake and presentation of antigen.