In the past 25 years, the development of an effective malaria vaccine has become one of the biggest riddles in the biomedical sciences. Experimental data using animal infection models demonstrated that it is possible to induce protective immunity against different stages of malaria parasites. Nonetheless, the vast body of knowledge has generated disappointments when submitted to clinical conditions and presently a single antigen formulation has progressed to the point where it may be translated into a human vaccine. In parallel, new means to increase the protective effects of antigens in general have been pursued and depicted, such as the use of bacterial flagellins as carriers/adjuvants. Flagellins activate pathways in the innate immune system of both mice and humans. The recent report of the first Phase I clinical trial of a vaccine containing a
The seminal work using radiation-attenuated sporozoites has been reproduced in multiple experimental systems, demonstrating that attenuated infective forms of
Likewise, genetically attenuated blood-stage forms of rodent malaria parasites have been successfully generated in the past few years and have been proposed as an antigen source for human vaccination trials [
The difficulties associated with large-scale generation of whole parasite antigens for mass vaccination led to the search for recombinant subunit vaccines based on immunodominant malarial antigens. Again, the mouse malaria model was very instructive for narrowing the search for the most promising antigens and testing different recombinant formulations, which provided the proof of principle for these subunit recombinant vaccines. Based on many years of detailed studies, some leading candidate antigens and formulations for vaccines were selected and exhaustively tested, but these tests have mainly occurred in the mouse model of infection as well as nonhuman primates.
Immunity generated by radiation-attenuated sporozoites is targeted to a dominant protective antigen, the circumsporozoite (CS) protein [
High antibody titres can be achieved, for example, using multiple synthetic peptides (MAPs) formulated in the presence of strong adjuvants [
The most reliable and reproducible results of vaccination against pre-erythrocytic stages in rodent malaria models were obtained by vaccination with two recombinant viral vectors. Using attenuated viruses containing the entire CS protein or its immunodominant epitopes, a solid and long-lasting protective immunity against sporozoite challenge was elicited [
These exciting results obtained in the mouse model led to a number of clinical trials. Unfortunately, these trials provided only limited protective immunity in Phase II trials performed in the laboratory, and none in the field [
Despite the great progress in malaria vaccine research against pre-erythrocytic stages in the past 20 years, a single type of formulation has shown promising results when tested in humans. This vaccine formulation consists of a large C-terminal fragment of the CS protein sequence fused to the Hepatitis B antigen S (conventional hepatitis B vaccine, Engerix B) and expressed as a recombinant protein in
In recent Phase II trials performed in naïve human volunteers challenged with
The erythrocytic forms of malaria grow and multiply within the host erythrocytes, and the rupture of the host cells is responsible for the clinical symptoms, including fever, of the noncomplicated forms of the disease. Nevertheless, infected erythrocytes (iEs) can also cytoadhere by means of variant surface antigens (VSA) to endothelial receptors on the microvasculature of certain organs or to the placental trophoblast, thereby disappearing from peripheral blood circulation. This phenomenon, named sequestration, is responsible for the majority of the deaths caused by malaria because it leads to the severe forms of malaria, including cerebral malaria, severe anaemia, acute respiratory distress symptoms or pregnancy-associated malaria (PAM) [
In the case of PAM, the scenario is complex because cytoadherence is mediated by VSA. Given that
Concerning blood-stage antigens of
In different species of
During invasion of the host erythrocytes, MSP-1 is a target of proteolytic processes. Various studies have demonstrated that MSP142 is found as a dimer, while MSP119 remains monomeric, suggesting a secondary process that would cause the dissociation of the protein before the erythrocytes are invaded (reviewed in [
The precise biological role of MSP-1 is still unknown. However, several studies have demonstrated that antibodies specific for the MSP119 of
Based on these positive attributes, the C-terminal region of MSP-1 (MSP119 or MSP142) has been heavily pursued as a vaccine candidate. Initial studies in mouse models demonstrated solid levels of protective immunity after vaccination with strong adjuvants, such as Complete Freund’s Adjuvant [
Based on the results obtained from the experimental models of malaria infection, a recent Phase IIb vaccine trial was performed in Africa. In this clinical trial, children were vaccinated with a formulation containing a recombinant His-tagged fusion protein encompassing the MSP142 of the
As thoroughly discussed above, vaccination of mice with recombinant proteins or viral vectors may confer significant protection against an infectious challenge with malaria parasites. The main problem is that some of these formulations, such as the ones containing strong adjuvants, simply cannot be used in humans. Others, such as the recombinant viruses, failed to reproduce in humans the immunogenicity observed in mice. These successive failures have led to the search for new alternatives that could eventually bridge the gap between mice and humans.
Flagella represent a complex locomotion device expressed by different bacterial species. They encompass at least 50 different proteins. Flagella may also behave as virulence-associated factors by helping pathogenic bacteria to bind to epithelial cells and to overcome non-immune defence mechanisms such as liquid flow in the urinary tract. Flagellin, the structural protein of the flagellar filament, is the most abundant protein of the flagellar apparatus, which can assemble by the polymerisation of thousands of flagellin monomers. The molecular masses of flagellins can vary from 28 kDa to 80 kDa. The N- and C-terminal regions of these molecules are highly conserved among different bacterial species and are folded together in parallel as a packed
Flagellin is a Pathogen-Associated Molecular Pattern (PAMP) recognised by Pattern Recognition Receptors (PRRs) in the innate immune system that triggers inflammatory responses and activates the adaptative immune system. At least two classes of PRRs, Toll-like Receptors (TLRs) and NOD-like Receptors (NLRs), respond to flagellin.TLR5 is a transmembrane receptor with a conserved cytoplasmic signalling domain—the Toll/interleukin (IL)-1 receptor homology domain (TIR), a common signature for TLRs—that specifically recognises a conserved site in the N- and C-terminal regions of flagellin that is formed after the correct assembly of the molecule; in fact, this site is essential for the correct flagella assembly [
In the case of the NLRs, flagellin that is expressed by intracellularly replicating pathogens or specifically injected by a secretory apparatus displayed by some bacterial pathogens, such as
Clearly, mammalian cells evolved the ability to very efficiently sense the presence of extra- and intracellular flagellins and respond with strong inflammatory signals that may ultimately enhance the induction of adaptive immune defences. Based on these natural properties, bacterial flagellins have been intensively investigated as vaccine adjuvants that can either be co-administered with purified recombinant antigens or as a carrier/adjuvant molecule genetically fused to the target antigen.
In the late 80s, a seminal work envisaged the use of bacterial flagellins as carriers/adjuvants for vaccine antigens by cloning synthetic oligonucleotides encoding specific B or T-cell epitopes at the central hypervariable region of the
More recently, the description of innate immune receptors that recognise flagellin and their capacity to activate innate and adaptive immune responses intensified the studies testing bacterial flagellins as vaccine adjuvants for the induction of both antibody and cellular immune responses. Different systems that used flagellins as carriers/adjuvants for recombinant fusion proteins or with coadministered recombinant antigens were tested in the last few years; for a detailed and updated compilation of these data, we recommend a very recently published paper [
For brevity and the purpose of our review, we selected the vaccine field that is highly advanced: the development of an anti-influenza vaccine. Two formulations of fusion proteins of flagellin and influenza antigen were generated. First, four tandem copies of the ectodomain of the conserved matrix protein M2 (M2e) of human influenza A virus were fused to the C-terminal domain of the flagellin of
Based on these successful pre-clinical studies, the influenza vaccine candidate VAX125 underwent a complete Phase I clinical trial that tested safety, reactogenicity, immunogenicity, tolerogenicity, and escalating dose-range. The results from this trial showed that recombinant flagellin was generally well tolerated by vaccinated individuals. Importantly, 91% of the individuals who received any dose of recombinant protein developed titres of neutralising antibodies compatible with protective status against influenza infection (clinicaltrials.gov/NCT00921947) [
Based on the great capacity of flagellin to induce humoral and cellular immune responses against foreign antigens, we explored the use of this molecule for the generation of fusion proteins containing malarial antigens. We selected for our initial studies the 19-kDa C-terminal region of MSP-1 from
PvMSP119 was fused to the C-terminal end of the FliC flagellin from
We immunised mice and rabbits with the recombinant fusion protein in the absence of any other adjuvant. Immunised animals developed strong, specific, and long-lasting antibody-mediated responses. The antibody titres after the second dose were similar to the ones obtained following immunisation with a recombinant protein containing only the PvMSP119 emulsified in Complete/Incomplete Freund’s Adjuvant. Additionally, the antibodies raised after these immunisations recognised
In the absence of any other adjuvant, the pattern of the immune response was biased toward a type 2 response, with a high IgG1/2 ratio and limited amounts of IFN-
Because long-term
Using another approach, we inserted the CS protein CD8+ T-cell epitope CS280–288) from the murine malaria parasite
To our knowledge, these were the first reports of an induction of specific humoral and cellular immune responses in experimental immunisations with recombinant proteins based on malaria vaccine antigens without the addition of any conventional adjuvant. As mentioned above, the magnitude of an immune response induced against recombinant proteins is highly dependent on the potency of the adjuvant formulation.
The mechanisms by which flagellin acts as an adjuvant are not fully understood, but it is likely to be dependent on direct APC activation, especially DC activation, to enhance antigen presentation. Flagellin up-regulates the expression of costimulatory molecules such as B7-1, B7-2, and CD40 in murine bone marrow-derived DCs [
The requirement for the physical linking of flagellin to the antigen is still a matter of debate. The fusion requirement may be dependent on the amount of antigen injected. One possibility is that when flagellin is bound to the antigen, there is concomitant APC activation and antigen uptake, as opposed to coadministered soluble antigen and flagellin when an activated APC may not uptake the antigen at the same time of activation. The fact that activated DCs down-regulate endocytic activity supports this hypothesis, indicating that if activation does not happen in the presence of the antigen, uptake may be impaired, and more antigen would be required to increase the chance of concomitant antigen uptake and APC activation. In fact, when we tested equimolar amounts of flagellin and antigen that were fused or co-administered, we found that the responses are comparable in the range of 10
RTS,S is arguably the most advanced vaccine against
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (Proc. no. 09/15132-4), The National Institute for Vaccine Technology (INCTV-CNPq), and The Millennium Institute for Vaccine Development and Technology (CNPq-420067/2005-1). Daniel Y. Bargieri is supported by a Manlio Cantarini fellowship. Catarina J. Braga is supported by a fellowship from FAPESP. Irene S. Soares, Fabio T. M. Costa, Luis C. S. Ferreira and Mauricio M. Rodrigues are recipients of fellowships from CNPq. The authors thank Dr. Bruna C. de Alencar for the suggestions.