Investigation of liposomal self-adjuvanting peptide epitopes derived from conserved blood-stage Plasmodium antigens.

Malaria is a vector born parasitic disease causing millions of deaths every year. Despite the high mortality rate, an effective vaccine against this mosquito-borne infectious disease is yet to be developed. Up to date, RTS,S/AS01 is the only vaccine available for malaria prevention; however, its efficacy is low. Among a variety of malaria antigens, merozoite surface protein-1(MSP-1) and ring-infected erythrocyte surface antigen (RESA) have been proposed as promising candidates for malaria vaccine development. We developed peptide-based Plasmodium falciparum vaccine candidates that incorporated three previously reported conserved epitopes from MSP-1 and RESA into highly effective liposomal polyleucine delivery system. Indeed, MSP-1 and RESA-derived epitopes conjugated to polyleucine and formulated into liposomes induced higher epitope specific antibody titres. However, immunized mice failed to demonstrate protection in a rodent malaria challenge study with Plasmodium yoelii. In addition, we found that the three reported P. falciparum epitopes did not to share conformational properties and high sequence similarity with P. yoelii MSP-1 and RESA proteins, despite the epitopes were reported to protect mice against P. yoelii challenge.

Development and Evaluation of a Cryopreserved Whole-Parasite Vaccine in a Rodent Model of Blood-Stage Malaria.

Infection with malaria parasites continues to be a major global public health issue. While current control measures have enabled a significant decrease in morbidity and mortality over the last 20 years, additional tools will be required if we are to progress toward malaria parasite eradication. Malaria vaccine research has focused on the development of subunit vaccines; however, more recently, interest in whole-parasite vaccines has reignited. Whole-parasite vaccines enable the presentation of a broad repertoire of antigens to the immune system, which limits the impact of antigenic polymorphism and genetic restriction of the immune response. We previously reported that whole-parasite vaccines can be prepared using chemically attenuated parasites within intact red blood cells or using killed parasites in liposomes, although liposomes were less immunogenic than attenuated parasites. If they could be frozen or freeze-dried and be made more immunogenic, liposomal vaccines would be ideal for vaccine deployment in areas where malaria is endemic. Here, we develop and evaluate a Plasmodium yoelii liposomal vaccine with enhanced immunogenicity and efficacy due to incorporation of TLR4 agonist, 3D(6-acyl) PHAD, and mannose to target the liposome to antigen-presenting cells. Following vaccination, mice were protected, and strong cellular immune responses were induced, characterized by parasite-specific splenocyte proliferation and a mixed Th1/Th2/Th17 cytokine response. Parasite-specific antibodies were induced, predominantly of the IgG1 subclass. CD4+ T cells and gamma interferon were critical components of the protective immune response. This study represents an important development toward evaluation of this whole-parasite blood-stage vaccine in a phase I clinical trial. IMPORTANCE Malaria is a mosquito-borne infectious disease that is caused by parasites of the genus, Plasmodium. There are seven different Plasmodium spp. that can cause malaria in humans, with P. falciparum causing the majority of the morbidity and mortality. Malaria parasites are endemic in 87 countries and continue to result in >200 million cases of malaria and >400,000 deaths/year, mostly children <5 years of age. Malaria infection initially presents as a flu-like illness but can rapidly progress to severe disease in nonimmune individuals if treatment is not initiated promptly. Existing control strategies for the mosquito vector (insecticides) and parasite (antimalarial drugs) are becoming increasingly less effective due to the development of resistance. While artemisinin combination therapies are frontline treatment for P. falciparum malaria, resistance has been documented in numerous countries. A highly effective malaria vaccine is urgently required to reduce malaria-attributable clinical disease and death and enable progression toward the ultimate goal of eradication.

Pre-clinical evaluation of a whole-parasite vaccine to control human babesiosis.

Babesia spp. are tick-transmitted intra-erythrocytic protozoan parasites that infect humans and animals, causing a flu-like illness and hemolytic anemia. There is currently no human vaccine available. People most at risk of severe disease are the elderly, immunosuppressed, and asplenic individuals. B. microti and B. divergens are the predominant species affecting humans. Here, we present a whole-parasite Babesia vaccine. To establish proof-of-principle, we employed chemically attenuated B. microti parasitized red blood cells from infected mice. To aid clinical translation, we produced liposomes containing killed parasite material. Vaccination significantly reduces peak parasitemia following challenge. B cells and anti-parasite antibodies do not significantly contribute to vaccine efficacy. Protection is abrogated by the removal of CD4+ T cells or macrophages prior to challenge. Importantly, splenectomized mice are protected by vaccination. To further facilitate translation, we prepared a culture-based liposomal vaccine and demonstrate that this performs as a universal vaccine inducing immunity against different human Babesia species.

Mannosylated liposomes formulated with whole parasite P. falciparum blood-stage antigens are highly immunogenic in mice.

The development of a blood-stage malaria vaccine has largely focused on the subunit approach. However, the limited success of this strategy, mainly due to antigenic polymorphism and the failure to maintain potent parasite-specific immune responses, indicates that other approaches must be considered. Whole parasite (WP) vaccines offer many advantages over sub-units; they represent every antigen on the organism, thus limiting the effects of antigenic polymorphism, and similarly they compensate for individual Immune-Response (Ir) gene-regulated non-responsiveness to any particular antigen. From a development perspective, they negate the need to identify and compare the relative efficacies of individual candidate antigens. WP vaccines induce protective immunity that is largely cell-mediated. However, WP blood-stage vaccines present a number of challenges for the development pathway. Key issues are cryopreservation and storage and the possible induction of antibodies against red blood cell surface antigens, even if the parasites are grown in blood group O, Rh negative blood. Here, we used a novel adaptation of an immunomagnetic method from STEMCELL™ Technologies to remove the red cell membranes from human red blood cells parasitized with P. falciparum. We then used these antigens to construct liposomes which were modified to present mannose on their membrane to target the liposome to antigen presenting cells. We then compared the immunogenicity of freshly prepared and lyophilized liposome vaccines. Following vaccination of mice, liposomes induced significantly lower antibody responses to human red cells but potent strain- and species-transcending cell-mediated immune responses to parasite antigens. These data support transitioning the P. falciparum liposomal vaccine into clinical studies.

Novel platform technology for modular mucosal vaccine that protects against streptococcus.

The upper respiratory tract (URT) is the major entry site for human pathogens and strategies to activate this network could lead to new vaccines capable of preventing infection with many pathogens. Group A streptococcus (GAS) infections, causing rheumatic fever, rheumatic heart disease, and invasive disease, are responsible for substantial morbidity and mortality. We describe an innovative vaccine strategy to induce mucosal antibodies of significant magnitude against peptide antigens of GAS using a novel biocompatible liposomal platform technology. The approach is to encapsulate free diphtheria toxoid (DT), a standard vaccine antigen, within liposomes as a source of helper T-cell stimulation while lipidated peptide targets for B-cells are separately displayed on the liposome surface. As DT is not physically conjugated to the peptide, it is possible to develop modular epitopic constructs that simultaneously activate IgA-producing B-cells of different and complementary specificity and function that together neutralize distinct virulence factors. An inflammatory cellular immune response is also induced. The immune response provides profound protection against streptococcal infection in the URT. The study describes a new vaccine platform for humoral and cellular immunity applicable to the development of vaccines against multiple mucosal pathogens.