Prime-and-Trap Malaria Vaccination To Generate Protective CD8+ Liver-Resident Memory T Cells.

Tissue-resident memory CD8+ T (Trm) cells in the liver are critical for long-term protection against pre-erythrocytic Plasmodium infection. Such protection can usually be induced with three to five doses of i.v. administered radiation-attenuated sporozoites (RAS). To simplify and accelerate vaccination, we tested a DNA vaccine designed to induce potent T cell responses against the SYVPSAEQI epitope of Plasmodium yoelii circumsporozoite protein. In a heterologous "prime-and-trap" regimen, priming using gene gun-administered DNA and boosting with one dose of RAS attracted expanding Ag-specific CD8+ T cell populations to the liver, where they became Trm cells. Vaccinated in this manner, BALB/c mice were completely protected against challenge, an outcome not reliably achieved following one dose of RAS or following DNA-only vaccination. This study demonstrates that the combination of CD8+ T cell priming by DNA and boosting with liver-homing RAS enhances formation of a completely protective liver Trm cell response and suggests novel approaches for enhancing T cell-based pre-erythrocytic malaria vaccines.

Defining rules of CD8(+) T cell expansion against pre-erythrocytic Plasmodium antigens in sporozoite-immunized mice.

BACKGROUND: Whole Plasmodium sporozoites serve as both experimental tools and potentially as deployable vaccines in the fight against malaria infection. Live sporozoites infect hepatocytes and induce a diverse repertoire of CD8(+) T cell responses, some of which are capable of killing Plasmodium-infected hepatocytes. Previous studies in Plasmodium yoelii-immunized BALB/c mice showed that some CD8(+) T cell responses expanded with repeated parasite exposure, whereas other responses did not. RESULTS: Here, similar outcomes were observed using known Plasmodium berghei epitopes in C57BL/6 mice. With the exception of the response to PbTRAP, IFNγ-producing T cell responses to most studied antigens, such as PbGAP50, failed to re-expand in mice immunized with two doses of irradiated P. berghei sporozoites. In an effort to boost secondary CD8(+) T cell responses, heterologous cross-species immunizations were performed. Alignment of P. yoelii 17XNL and P. berghei ANKA proteins revealed that >60 % of the amino acids in syntenic orthologous proteins are continuously homologous in fragments ≥8-amino acids long, suggesting that cross-species immunization could potentially trigger responses to a large number of common Class I epitopes. Heterologous immunization resulted in a larger liver burden than homologous immunization. Amongst seven tested antigen-specific responses, only CSP- and TRAP-specific CD8(+) T cell responses were expanded by secondary homologous sporozoite immunization and only those to the L3 ribosomal protein and S20 could be re-expanded by heterologous immunization. In general, heterologous late-arresting, genetically attenuated sporozoites were better at secondarily expanding L3-specific responses than were irradiated sporozoites. GAP50 and several other antigens shared between P. berghei and P. yoelii induced a large number of IFNγ-positive T cells during primary immunization, yet these responses could not be re-expanded by either homologous or heterologous secondary immunization. CONCLUSIONS: These studies highlight how responses to different sporozoite antigens can markedly differ in recall following repeated sporozoite vaccinations. Cross-species immunization broadens the secondary response to sporozoites and may represent a novel strategy for candidate antigen discovery.

A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations.

Development of an antimalarial subunit vaccine inducing protective cytotoxic T lymphocyte (CTL)-mediated immunity could pave the way for malaria eradication. Experimental immunization with sporozoites induces this type of protective response, but the extremely large number of proteins expressed by Plasmodium parasites has so far prohibited the identification of sufficient discrete T-cell antigens to develop subunit vaccines that produce sterile immunity. Here, using mice singly immunized with Plasmodium yoelii sporozoites and high-throughput screening, we identified a unique CTL response against the parasite ribosomal L3 protein. Unlike CTL responses to the circumsporozoite protein (CSP), the population of L3-specific CTLs was not expanded by multiple sporozoite immunizations. CSP is abundant in the sporozoite itself, whereas L3 expression does not increase until the liver stage. The response induced by a single immunization with sporozoites reduces the parasite load in the liver so greatly during subsequent immunizations that L3-specific responses are only generated during the primary exposure. Functional L3-specific CTLs can, however, be expanded by heterologous prime-boost regimens. Thus, although repeat sporozoite immunization expands responses to preformed antigens like CSP that are present in the sporozoite itself, this immunization strategy may not expand CTLs targeting parasite proteins that are synthesized later. Heterologous strategies may be needed to increase CTL responses across the entire spectrum of Plasmodium liver-stage proteins.

Preliminary studies on the immunogenicity of a prime-and-trap malaria vaccine in nonhuman primates.

Development of next-generation vaccines against Plasmodium falciparum (Pf) is a priority. Many malaria vaccines target the pre-erythrocytic sporozoite (SPZ) and liver stages. These include subunit vaccines based on the Pf circumsporozoite protein (CSP) and attenuated PfSPZ vaccines. However, these strategies require 3-4 doses and have not achieved optimal efficacy against field-transmitted malaria. Prime-and-trap is a recently developed two-step heterologous vaccine strategy that combines priming with DNA encoding CSP followed by a single dose of attenuated SPZ. This strategy aims to induce CD8+ T cells that can eliminate parasites in the liver. Prior data has demonstrated that prime-and-trap with P. yoelii CSP and PySPZ was immunogenic and protective in mice. Here we report preliminary data on the immunogenicity of PfCSP prime and PfSPZ trap vaccine in rhesus macaques. This vaccine induced PfCSP-specific antibodies and T cell responses in all animals. However, response magnitude differed between individuals, suggesting further study is required.

More time to kill: A longer liver stage increases T cell-mediated protection against pre-erythrocytic malaria.

Liver stage (LS) Plasmodia mature in 2-2.5 days in rodents compared to 5-6 days in humans. Plasmodium-specific CD8+ T cell expansion differs across these varied timespans. To mimic the kinetics of CD8+ T cells of human Plasmodium infection, a two-dose challenge mouse model that achieved 4-5 days of LS antigen exposure was developed. In this model, mice were inoculated with a non-protective, low dose of late-arresting, genetically attenuated sporozoites to initiate T cell activation and then re-inoculated 2-3 days later with wild-type sporozoites. Vaccines that partially protected against traditional challenge completely protected against two-dose challenge. During the challenge period, CD8+ T cell frequencies increased in the livers of two-dose challenged mice but not in traditionally challenged mice, further suggesting that this model better recapitulates kinetics of CD8+ T cell expansion in humans during the P. falciparum LS. Vaccine development and antigen discovery efforts may be aided by using the two-dose challenge strategy.

Needle-free, spirulina-produced Plasmodium falciparum circumsporozoite vaccination provides sterile protection against pre-erythrocytic malaria in mice.

Antibodies against the Plasmodium falciparum circumsporozoite protein (PfCSP) can block hepatocyte infection by sporozoites and protect against malaria. Needle-free vaccination strategies are desirable, yet most PfCSP-targeted vaccines like RTS,S require needle-based administration. Here, we evaluated the edible algae, Arthrospira platensis (commonly called 'spirulina') as a malaria vaccine platform. Spirulina were genetically engineered to express virus-like particles (VLPs) consisting of the woodchuck hepatitis B core capsid protein (WHcAg) displaying a (NANP)15 PfCSP antigen on its surface. PfCSP-spirulina administered to mice intranasally followed by oral PfCSP-spirulina boosters resulted in a strong, systemic anti-PfCSP immune response that was protective against subcutaneous challenge with PfCSP-expressing P. yoelii. Unlike male mice, female mice did not require Montanide adjuvant to reach high antibody titers or protection. The successful use of spirulina as a vaccine delivery system warrants further development of spirulina-based vaccines as a useful tool in addressing malaria and other diseases of global health importance.

Gene gun DNA immunization of cattle induces humoral and CD4 T-cell-mediated immune responses against the Theileria parva polymorphic immunodominant molecule.

Theileria parva kills over one million cattle annually in sub-Saharan Africa. Parasite genetic complexity, cellular response immunodominance, and bovine MHC diversity have precluded traditional vaccine development. One potential solution is gene gun (GG) immunization, which enables simultaneous administration of one or more DNA-encoded antigens. Although promising in murine, porcine, and human vaccination trials, bovine GG immunization studies are limited. We utilized the model T. parva antigen, polymorphic immunodominant molecule (PIM) to test bovine GG immunization. GG immunization using a mammalian codon optimized PIM sequence elicited significant anti-PIM antibody and cell-mediated responses in 7/8 steers, but there was no difference between immunized and control animals following T. parva challenge. The results suggest immunization with PIM, as delivered here, is insufficient to protect cattle from T. parva. Nonetheless, the robust immune responses elicited against this model antigen suggest GG immunization is a promising vaccine platform for T. parva and other bovine pathogens.

Beyond Blood Smears: Qualification of Plasmodium 18S rRNA as a Biomarker for Controlled Human Malaria Infections.

18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. We reviewed data from CHMI trials at non-endemic sites that used blood smears and Plasmodium 18S rRNA/rDNA biomarker nucleic acid tests (NATs) for time to positivity. We validated a multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. Literature review demonstrated accelerated NAT-based infection detection compared with blood smears (mean acceleration: 3.2-3.6 days). For prospectively tested trials, the validated Plasmodium 18S rRNA qRT-PCR positivity was earlier (7.6 days; 95% CI: 7.1-8.1 days) than blood smears (11.0 days; 95% CI: 10.3-11.8 days) and significantly preceded the onset of grade 2 malaria-related symptoms (12.2 days; 95% CI: 10.6-13.3 days). Discrepant analysis showed that the risk of a blood smear-positive, biomarker-negative result was negligible. Data modeling predicted that treatment triggered by specific biomarker-defined thresholds can differentiate complete, partial, and non-protective outcomes and eliminate many grade 2 and most grade 3 malaria-related symptoms post-CHMI. Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials.

Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens.

Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens.