Adeno-associated virus-based malaria booster vaccine following attenuated replication-competent vaccinia virus LC16m8Δ priming.

We previously demonstrated that boosting with adeno-associated virus (AAV) type 1 (AAV1) can induce highly effective and long-lasting protective immune responses against malaria parasites when combined with replication-deficient adenovirus priming in a rodent model. In the present study, we compared the efficacy of two different AAV serotypes, AAV1 and AAV5, as malaria booster vaccines following priming with the attenuated replication-competent vaccinia virus strain LC16m8Δ (m8Δ), which harbors the fusion gene encoding both the pre-erythrocytic stage protein, Plasmodium falciparum circumsporozoite (PfCSP) and the sexual stage protein (Pfs25) in a two-dose heterologous prime-boost immunization regimen. Both regimens, m8Δ/AAV1 and m8Δ/AAV5, induced robust anti-PfCSP and anti-Pfs25 antibodies. To evaluate the protective efficacy, the mice were challenged with sporozoites twice after immunization. At the first sporozoite challenge, m8Δ/AAV5 achieved 100% sterile protection whereas m8Δ/AAV1 achieved 70% protection. However, at the second challenge, 100% of the surviving mice from the first challenge were protected in the m8Δ/AAV1 group whereas only 55.6% of those in the m8Δ/AAV5 group were protected. Regarding the transmission-blocking efficacy, we found that both immunization regimens induced high levels of transmission-reducing activity (>99%) and transmission-blocking activity (>95%). Our data indicate that the AAV5-based multistage malaria vaccine is as effective as the AAV1-based vaccine when administered following an m8Δ-based vaccine. These results suggest that AAV5 could be a viable alternate vaccine vector as a malaria booster vaccine.

Sterile protection and transmission blockade by a multistage anti-malarial vaccine in the pre-clinical study.

The Malaria Vaccine Technology Roadmap 2013 (World Health Organization) aims to develop safe and effective vaccines by 2030 that will offer at least 75% protective efficacy against clinical malaria and reduce parasite transmission. Here, we demonstrate a highly effective multistage vaccine against both the pre-erythrocytic and sexual stages of Plasmodium falciparum that protects and reduces transmission in a murine model. The vaccine is based on a viral-vectored vaccine platform, comprising a highly-attenuated vaccinia virus strain, LC16m8Δ (m8Δ), a genetically stable variant of a licensed and highly effective Japanese smallpox vaccine LC16m8, and an adeno-associated virus (AAV), a viral vector for human gene therapy. The genes encoding P. falciparum circumsporozoite protein (PfCSP) and the ookinete protein P25 (Pfs25) are expressed as a Pfs25-PfCSP fusion protein, and the heterologous m8Δ-prime/AAV-boost immunization regimen in mice provided both 100% protection against PfCSP-transgenic P. berghei sporozoites and up to 100% transmission blocking efficacy, as determined by a direct membrane feeding assay using parasites from P. falciparum-positive, naturally-infected donors from endemic settings. Remarkably, the persistence of vaccine-induced immune responses were over 7 months and additionally provided complete protection against repeated parasite challenge in a murine model. We propose that application of the m8Δ/AAV malaria multistage vaccine platform has the potential to contribute to the landmark goals of the malaria vaccine technology roadmap, to achieve life-long sterile protection and high-level transmission blocking efficacy.

Liver-Directed AAV8 Booster Vaccine Expressing Plasmodium falciparum Antigen Following Adenovirus Vaccine Priming Elicits Sterile Protection in a Murine Model.

Hepatocyte infection by malaria sporozoites is a bottleneck in the life-cycle of Plasmodium spp. including P. falciparum, which causes the most lethal form of malaria. Therefore, developing an effective vaccine capable of inducing the strong humoral and cellular immune responses necessary to block the pre-erythrocytic stage has potential to overcome the spatiotemporal hindrances pertaining to parasite biology and hepatic microanatomy. We recently showed that when combined with a human adenovirus type 5 (AdHu5)-priming vaccine, adeno-associated virus serotype 1 (AAV1) is a potent booster malaria vaccine vector capable of inducing strong and long-lasting protective immune responses in a rodent malaria model. Here, we evaluated the protective efficacy of a hepatotropic virus, adeno-associated virus serotype 8 (AAV8), as a booster vector because it can deliver a transgene potently and rapidly to the liver, the organ malaria sporozoites initially infect and multiply in following sporozoite injection by the bite of an infected mosquito. We first generated an AAV8-vectored vaccine expressing P. falciparum circumsporozoite protein (PfCSP). Intravenous (i.v.) administration of AAV8-PfCSP to mice initially primed with AdHu5-PfCSP resulted in a hepatocyte transduction rate ~2.5 times above that seen with intramuscular (i.m.) administration. This immunization regimen provided a better protection rate (100% sterile protection) than that of the i.m. AdHu5-prime/i.m. AAV8-boost regimen (60%, p < 0.05), i.m. AdHu5-prime/i.v. AAV1-boost (78%), or i.m. AdHu5-prime/i.m. AAV1-boost (80%) against challenge with transgenic PfCSP-expressing P. berghei sporozoites. Compared with the i.m. AdHu5-prime/i.v. AAV1-boost regimen, three other regimens induced higher levels of PfCSP-specific humoral immune responses. Importantly, a single i.v. dose of AAV8-PfCSP recruited CD8+ T cells, especially resident memory CD8+ T cells, in the liver. These data suggest that boost with i.v. AAV8-PfCSP can improve humoral and cellular immune responses in BALB/c mice. Therefore, this regimen holds great promise as a next-generation platform for the development of an effective malaria vaccine.