Persistence of Protective Immunity to Malaria Induced by DNA Priming and Poxvirus Boosting: Characterization of Effector and Memory CD8+-T-Cell Populations

The persistence of immunity to malaria induced in mice by a heterologous DNA priming and poxvirus boosting regimen was characterized. Mice were immunized by priming with DNA vaccine plasmids encoding the Plasmodium yoelii circumsporozoite protein (PyCSP) and murine granulocyte-macrophage colony-stimulating factor and boosting with recombinant vaccinia encoding PyCSP. BALB/c mice immunized with either high-dose (100 µg of p PyCSP plus 30 µg of pGM-CSF) or low-dose (1 µg of p PyCSP plus 1 µg of pGM-CSF DNA) priming were protected against challenge with 50 P. yoelii sporozoites. Protection 2 weeks after immunization was 70 to 100%, persisted at this level for at least 20 weeks, and declined to 30 to 40% by 28 weeks. Eight of eight mice protected at 20 weeks were still protected when rechallenged at 40 weeks. The antigen (Ag)-specific effector CD8+-T-cell population present 2 weeks after boosting had ex vivo Ag-specific cytolytic activity, expressed both gamma interferon (IFN-{gamma}) and tumor necrosis factor alpha, and constituted 12 to 20% of splenic CD8+ T cells. In contrast, the memory CD8+-Ag-specific-cell population at 28 weeks lacked cytolytic activity and constituted only 6% of splenic CD8+ T cells, but at the single-cell level it produced significantly higher levels of IFN-{gamma} than the effectors. High levels of Ag- or parasite-specific antibodies present 2 weeks after boosting had declined three- to sevenfold by 28 weeks. Low-dose priming was similarly immunogenic and as protective as high-dose priming against a 50-, but not a 250-, sporozoite challenge. These results demonstrate that a heterologous priming and boosting vaccination can provide lasting protection against malaria in this model system

Vaxfectin® enhances both antibody and in vitro T cell responses to each component of a 5-gene Plasmodium falciparum plasmid DNA vaccine mixture administered at low doses

We previously reported the capacity of the cationic lipid-based formulation, Vaxfectin®, to enhance the immunogenicity and protective efficacy of a low dose plasmid DNA vaccine against Plasmodium yoelii malaria in mice. Here, we have extended this finding to human Plasmodium falciparum genes, evaluating the immune enhancing effect of Vaxfectin® formulation on a mixture, designated CSLAM, of five plasmid DNA vaccines encoding antigens from the sporozoite (PfCSP, PfSSP2/TRAP), intrahepatic (PfLSA1), and erythrocytic (PfAMA1, PfMSP1) life cycle stages of P. falciparum administered at 2, 10 or 50 μg doses. Vaxfectin® formulation enhanced both antibody and cellular immune responses to each component of the multi-antigen vaccine mixture, as assessed by ELISA, IFAT, and IFN-γ ELIspot, respectively. There was no apparent antigenic competition, as indicated by comparison of responses induced in mice immunized with PfCSP vs. CSLAM. These data showing that Vaxfectin® can enhance the immunogenicity of plasmid DNA vaccines administered at low doses per body weight, and in combinations, has important clinical implications for the development of a vaccine against malaria, as well as against other public health threats

Vaxfectin™ enhances immunogenicity and protective efficacy of P. yoelii circumsporozoite DNA vaccines

We evaluated the capacity of the cationic lipid based formulation, Vaxfectin™, to enhance the immunogenicity and protective efficacy of DNA-based vaccine regimens in the Plasmodium yoelii murine malaria model. We immunized Balb/c mice with varying doses (0.4–50 μg) of plasmid DNA (pDNA) encoding the P. yoelii circumsporozoite protein (PyCSP), either in a homologous DNA/DNA regimen (D-D) or a heterologous prime-boost DNA-poxvirus regimen (D-V). At the lowest pDNA doses, Vaxfectin™ substantially enhanced IFA titers, ELISPOT frequencies, and protective efficacy. Clinical trials of pDNA vaccines have often used low pDNA doses based on a per kilogram weight basis. Formulation of pDNA vaccines in Vaxfectin™ may improve their potency in human clinical trials

Correction: Vaccine Strain-Specificity of Protective HLA-Restricted Class 1 P. falciparum Epitopes.

[This corrects the article DOI: 10.1371/journal.pone.0163026.]

ELISpot and CD8+ T cell IFN-γ responses of DNA/HuAd5 and HuAd5 immunized subjects to <i>P</i>. <i>falciparum</i> strains 3D7 and 7G8 AMA1 A*03 protective epitopes.

<p>ELISpot and CD8+ T cell IFN-γ activities are shown in Panels A–D. <b>Panel A:</b> ELISpot IFN-γ response of the A*03 protected subject (v11) are positive with Ap8 and the 3D7 A*03 epitope but not the 7G8 epitope (arrow). <b>Panel B:</b> ELISpot activity of v11 is not affected by CD4+-depletion but is abolished after CD8+ depletion (arrow). <b>Panel C:</b> CD8+ T cell IFN-γ responses of v11 are much higher (p = 0.001) to the 3D7 epitope than to the 7G8 epitope (arrow). <b>Panel D:</b> ELISpot IFN-γ responses of two of four non-protected subjects from the HuAd5 trial were weakly positive with the 3D7 epitope but all four subjects were negative with the 7G8 epitope (arrows).</p

Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein.

Malaria remains a major global public health problem with an estimated 200 million cases detected in 2012. Although the most advanced candidate malaria vaccine (RTS,S) has shown promise in clinical trials, its modest efficacy and durability have created uncertainty about the impact of RTS,S immunization (when used alone) on global malaria transmission. Here we describe the development and characterization of a novel modified vaccinia virus Ankara (MVA)-based malaria vaccine which co-expresses the Plasmodium yoelii circumsporozoite protein (CSP) and IL-15. Vaccination/challenge studies showed that C57BL/6 mice immunized with the MVA-CSP/IL15 vaccine were protected significantly better against a P. yoelii 17XNL sporozoite challenge than either mice immunized with an MVA vaccine expressing only CSP or naïve controls. Importantly, the levels of total anti-CSP IgG were elevated about 100-fold for the MVA-CSP/IL15 immunized group compared to mice immunized with the MVA-CSP construct that does not express IL-15. Among the IgG subtypes, the IL-15 expressing MVA-CSP vaccine induced levels of IgG1 (8 fold) and IgG2b (80 fold) higher than the MVA-CSP construct. The significantly enhanced humoral responses and protection detected after immunization with the MVA-CSP/IL15 vaccine suggest that this IL-15 expressing MVA construct could be considered in the development of future malaria immunization strategies

Human adenovirus 5-vectored Plasmodium falciparum NMRC-M3V-Ad-PfCA vaccine encoding CSP and AMA1 is safe, well-tolerated and immunogenic but does not protect against controlled human malaria infection.

BACKGROUND: In a prior study, a DNA prime / adenovirus boost vaccine (DNA/Ad) expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) (NMRC-M3V-D/Ad-PfCA Vaccine) induced 27% protection against controlled human malaria infection (CHMI). To investigate the contribution of DNA priming, we tested the efficacy of adenovirus vaccine alone (NMRC-M3V-Ad-PfCA ) in a Phase 1 clinical trial. METHODOLOGY/PRINCIPAL FINDINGS: The regimen was a single intramuscular injection with two non-replicating human serotype 5 adenovectors encoding CSP and AMA1, respectively. One x 10 (10) particle units of each construct were combined prior to administration. The regimen was safe and well-tolerated. Four weeks later, 18 study subjects received P. falciparum CHMI administered by mosquito bite. None were fully protected although one showed delayed onset of parasitemia. Antibody responses were low, with geometric mean CSP ELISA titer of 381 (range SIGNIFICANCE: In contrast to DNA/Ad, Ad alone did not protect against CHMI despite inducing broad, cell-mediated immunity, indicating that DNA priming is required for protection by the adenovirus-vectored vaccine. ClinicalTrials.gov Identifier: NCT00392015

IMRAS-A clinical trial of mosquito-bite immunization with live, radiation-attenuated P. falciparum sporozoites: Impact of immunization parameters on protective efficacy and generation of a repository of immunologic reagents.

BackgroundImmunization with radiation-attenuated sporozoites (RAS) by mosquito bite provides >90% sterile protection against Plasmodium falciparum (Pf) malaria in humans. RAS invade hepatocytes but do not replicate. CD8+ T cells recognizing parasite-derived peptides on the surface of infected hepatocytes are likely the primary protective mechanism. We conducted a randomized clinical trial of RAS immunization to assess safety, to achieve 50% vaccine efficacy (VE) against controlled human malaria infection (CHMI), and to generate reagents from protected and non-protected subjects for future identification of protective immune mechanisms and antigens.MethodsTwo cohorts (Cohort 1 and Cohort 2) of healthy, malaria-naïve, non-pregnant adults age 18-50 received five monthly immunizations with infected (true-immunized, n = 21) or non-infected (mock-immunized, n = 5) mosquito bites and underwent homologous CHMI at 3 weeks. Immunization parameters were selected for 50% protection based on prior clinical data. Leukapheresis was done to collect plasma and peripheral blood mononuclear cells.ResultsAdverse event rates were similar in true- and mock-immunized subjects. Two true- and two mock-immunized subjects developed large local reactions likely caused by mosquito salivary gland antigens. In Cohort 1, 11 subjects received 810-1235 infected bites; 6/11 (55%) were protected against CHMI vs. 0/3 mock-immunized and 0/6 infectivity controls (VE 55%). In Cohort 2, 10 subjects received 839-1131 infected bites with a higher first dose and a reduced fifth dose; 9/10 (90%) were protected vs. 0/2 mock-immunized and 0/6 controls (VE 90%). Three/3 (100%) protected subjects administered three booster immunizations were protected against repeat CHMI vs. 0/6 controls (VE 100%). Cohort 2 uniquely showed a significant rise in IFN-γ responses after the third and fifth immunizations and higher antibody responses to CSP.ConclusionsPfRAS were generally safe and well tolerated. Cohort 2 had a higher first dose, reduced final dose, higher antibody responses to CSP and significant rise of IFN-γ responses after the third and fifth immunizations. Whether any of these factors contributed to increased protection in Cohort 2 requires further investigation. A cryobank of sera and cells from protected and non-protected individuals was generated for future immunological studies and antigen discovery.Trial registrationClinicalTrials.gov NCT01994525