An effective malaria vaccine will save many lives and lift the global burden of this disease. Among the next-generation approaches that build on the recent success of vaccines for COVID-19 prevention are mRNA vaccines delivered in nanoparticles 36,37. Our prime-and-trap approach combines two strategies that exhibit modest efficacy individually, but confer promising levels of protection when combined. This approach also has key logistical advantages over other current candidates. We summarize all the mouse immunization and challenge studies performed in this manuscript with Py wild-type parasites in Supplemental Table 1. We have shown that a homologous LION/repRNA-PyCS vaccine (prime-boost) is highly immunogenic at all doses evaluated, eliciting strong antibody responses when given in a 2-dose immunization regimen 2 weeks apart. Antibody levels are modest after the priming dose but increase subsequent to the booster dose. We have also demonstrated that homologous LION/repRNA-CS vaccination alone was insufficient to prevent blood-stage infection and did not provide protection in mice. To overcome these issues and enhance the practicability of vaccinating people in low-resource environments, our efforts have focused on minimizing the interval between injections, lowering dosages, and simplifying storage and preparation of the agents. We believe an effective vaccine based on a strategy such as that described herein, administered in a single clinic visit, will help to 1) enhance the logistics of the vaccination schedule, and 2) reduce the cost of goods by combining a repRNA priming dose and a single dose of attenuated spz.
Our results suggest that the priming dose of LION/repRNA-PyCS is highly immunogenic. We observed induction of a strong humoral response in BALB/cJ and C57Bl/6 mice when they were immunized on the 14-day, 5-day, or same-day regimens. We also observed a strain-specific CD8 + T-cell response in BALB/cJ mice. There was a 2-3-day delay in the onset of blood-stage parasitemia compared to the control groups. Compared with RAS-only immunization strategies, our prime-and-trap approach has the advantage of inducing both humoral and cellular immunities without compromising the generation of protective liver-resident CD8+ T cells.
Previous studies have shown that RAS vaccines can induce liver CD8+ Trm cells in animal models, but they can only achieve high levels of sterile protection in humans with three or more IV doses 22,51,52. The requirement for several IV doses compounds the time, cost, and logistical problems inherent to WO vaccines29,30. Others studies employing later-arresting GAP vaccines 50,51,53 have shown that presenting more antigens leads to improve protection over early-arresting GAP or RAS18,54. Likewise, immunization with wild-type (WT) WO spz administered under blood-stage drug chemoprophylaxis (known as CPS, (PfSPZ-CVac)24,55) allows full liver-stage development and can induce high levels of protection at reduced doses, although CPS involves co-administration of drugs with the vaccine, presenting practical and regulatory challenges24. These well-established immunizations with WO SPZ RAS 56,57, GAP 18,50,53,58, or PfSPZ-CVac24,55 are at least in part reliant on the generation of liver-resident memory CD8+ T cell (Trm) responses 21 33,59–61.
In the present study, we show the contribution of CS-specific liver-resident memory CD8+ T cells induced by our prime-and-trap regime with LION/repRNA-PyCSP. Given that the initial dose of RAS administered to naïve individuals appears to be the most immunogenic and effective in inducing liver Trm cells, a vaccination strategy employing a single dose of RAS34 may be the most efficient and cost-effective means of using this valuable resource.
Production of Pf SPZ (RAS or WT) in mosquitoes is an expensive and tedious process. However notable progress in in vitro culture of Pf SPZ under current Good Manufacturing Practices has been made by Sanaria®. It is hoped that their cGMP mosquito-dissected spz, known as Pf SPZ vaccine (irradiated PfSPZ) or PfSPZ-CVac (PfSPZ used with chemoprophylaxis vaccination), will soon be available for human clinical trials 3,22,62,63. Current clinical trials involving injection of several doses of cryopreserved Sanaria PfSPZ have proven that large-scale implementation in endemic areas using liquid-nitrogen storage conditions is achievable 29,30,64,65. The combination of our repRNA/LION-based priming dose with a single dose of in vitro-produced spz may yield a highly effective, convenient, and cost-effective malaria vaccine.
Our findings reinforce the concept that CS remains one of the most immunodominant and protective antigens expressed by spz and suggest that its CD8+ T-cell epitope is involved in the protective effect against parasites in the liver of the BALB/cJ mice following RAS immunization 66. However, it is not clear that any single antigen will confer the robust and durable protection needed to eradicate malaria. Given the genetic diversity among parasite strains and the many variables in physiology, environment, and logistical capabilities involved in a broad vaccination campaign, it may be necessary to target multiple antigens from various stages of the parasite life cycle to eliminate malaria. A multi-stage malaria vaccine with high efficacy and durability is more likely to achieve eradication or major reductions in disease burden and transmission than a single-stage vaccine. With this in mind, we are currently evaluating the immunogenicity of repRNA presenting multiples antigens from various stages of the parasite life-cycle.
As seen during the ongoing SARS-CoV-2/COVID-19 pandemic, the use of RNA vaccines has numerous advantages over conventional vaccine approaches. These include the flexibility and the rapidity of the manufacturing processes, which proved important during the SARS-CoV-2/COVID-19 pandemic 55. Another advantage is the ease of production of the antigen-encoding RNA, which, when translated in host cells, yields the antigen that engages the host immune system through MHC class I and II pathways, resulting in T-cell responses and antibody production 49. Despite the advantages of RNA vaccines, however, the method of intracellular delivery of the RNA is also critical. The Pfizer/BioNTech and Moderna mRNA vaccines both employ LNPs to protect the mRNA from extracellular ribonucleases and facilitate cellular uptake via endocytosis 39,40,67. The mRNA-PfCSP LNP vaccine can provide from 40–80% protection in a lethal rodent infection model, depending on the dosage, schedule, and nucleoside-modified sequences of the mRNA, but the protection is short-lived36. Induction of antigen expression mediated by these mRNA-LNP vaccines is rapid but transient, yielding an immune response that is limited in magnitude and duration. While the mRNA-LNP vaccine approach is versatile and its efficacy is promising 36 more robust and durable antigen expression will be needed to reach efficacy objectives.
Our approach of combining repRNA and LION has several advantages over mRNA-LNP vaccines, including a reduced number of doses (2), the use of a single dose of the WO irradiated spz, a shorter (5-day or same-day) administration schedule, and simpler logistics 36. A further advantage of nanoparticle formulations as carriers for nucleic acid delivery is the possibility of optimization for specific target cells and tissue types. The balance of tissue targeting can be shifted by changing the nanoparticle composition and route of delivery (oral, subcutaneous, or IV) 68. One focus of our future work is direct targeting of the liver, in the hope of replacing spz administration with a more practical and cost-effective nanoparticle formulation. Studies to assess the efficacy and durability of our prime-and-trap approach over an interval of two months, to be extended in future work, are underway. In the current study, we used an intramuscular LION formulation that has been distributed in India under the product designation GEMCOVAC-19. This LION is composed of a stable cationic (DOTAP) squalene emulsion embedded in a hydrophobic oil phase, proven to be stable for weeks and highly immunogenic when injected intramuscularly 44,45,69. Of critical importance to any vaccine designed for use in a low-resource environment, GEMCOVAC-19 is manufactured as a lyophilized product and reconstituted prior to use, allowing long-term storage without the freezers and power requirements of other SARS-CoV-2 vaccines. Similarly, we expect to lyophilize our repRNA malaria vaccines to eliminate cold-chain requirements prior to reconstitution.
In summary, we have demonstrated a multi-component vaccination approach that concurrently induces humoral and T-cell immunities, using repRNA-CS formulated with LION nanoparticles and PyRAS targeting the liver. This prime-and-trap approach is currently being broadened in mouse studies to include antigens from other stages of malaria. We are also extending our protection studies to longer intervals to assess the durability of the observed efficacy. Our prime-and-trap approach is also being tested in non-human primate models in the US, potentially leading to immunogenicity and efficacy studies in a CHMI trial in the near future.