Baculovirus-Induced Fast-Acting Innate Immunity Kills Liver-Stage Plasmodium.

Baculovirus (BV), an enveloped insect virus with a circular dsDNA genome, possesses unique characteristics that induce strong innate immune responses in mammalian cells. In this study, we show that BV administration in BALB/c mice not only provides complete protection against a subsequent Plasmodium berghei sporozoite infection for up to 7 d after the injection but also eliminates existing liver-stage parasites completely. The elimination of sporozoites by BV was superior to that by primaquine, and this effect occurred in a TLR9-independent manner. At 6 h after BV administration, IFN-α and IFN-γ were robustly produced in the serum, and RNA transcripts of IFN-stimulated genes were markedly upregulated in the liver compared with control mice. The in vivo passive transfer of serum after BV administration effectively eliminated liver-stage parasites, and IFN-α neutralization abolished this effect, indicating that the BV liver-stage parasite-killing mechanism is downstream of the type I IFN signaling pathway. These findings provide evidence that BV-induced, fast-acting innate immunity completely kills liver-stage parasites and, thus, may lead to new malaria drug and vaccine strategies.

Down-selecting circumsporozoite protein-based malaria vaccine: A comparison of malaria sporozoite challenge model.

Plasmodium falciparum circumsporozoite protein (PfCSP) is the main target antigen in development of pre-erythrocytic malaria vaccines. To evaluate PfCSP vaccines in animal models, challenge by intravenous sporozoite injection is preferentially used. However, in clinical trials, vaccinated human volunteers are exposed to the bites of malaria-infected mosquitoes. In this study, we down-selected Escherichia coli-produced full-length PfCSP (PfCSP-F) and its three truncated PfCSPs based on their abilities to elicit immune response and protection in mice against two challenge models. We showed that immunization with three doses of PfCSP-F elicited high anti-PfCSP antibody titres and 100% protection against the bites of infected mosquitoes. Meanwhile, three-dose truncated PfCSP induced 60%-70% protection after immunization with each truncated PfCSP. Heterologous prime-boost immunization regimen with adenovirus-PfCSP-F and R32LR greatly induced complete protection against intravenous sporozoite injection. Our results suggest that Abs to both anti-repeat and anti-nonrepeat regions induced by PfCSP-F are required to confer complete protection against challenge by the bites of infected mosquitoes, whereas anti-repeat Abs play an important role in protection against intravenous sporozoite injection. Our findings provide a potential clinical application that PfCSP-F vaccine induces potent Abs capable of neutralizing sporozoites in the dermis inoculated by infected mosquitoes and subsequently sporozoites in the blood circulation.

The potential for zoonotic malaria transmission in five areas of Indonesia inhabited by non-human primates.

BACKGROUND: Indonesia is home to many species of non-human primates (NHPs). Deforestation, which is still ongoing in Indonesia, has substantially reduced the habitat of NHPs in the republic. This has led to an intensification of interactions between NHPs and humans, which opens up the possibility of pathogen spillover. The aim of the present study was to determine the prevalence of malarial parasite infections in NHPs in five provinces of Indonesia in 2022. Species of the genus Anopheles that can potentially transmit malarial pathogens to humans were also investigated. METHODS: An epidemiological survey was conducted by capturing NHPs in traps installed in several localities in the five provinces, including in the surroundings of a wildlife sanctuary. Blood samples were drawn aseptically after the NHPs had been anesthetized; the animals were released after examination. Blood smears were prepared on glass slides, and dried blood spot tests on filter paper. Infections with Plasmodium spp. were determined morphologically from the blood smears, which were stained with Giemsa solution, and molecularly through polymerase chain reaction and DNA sequencing using rplU oligonucleotides. The NHPs were identified to species level by using the mitochondrial cytochrome c oxidase subunit I gene and the internal transcribed spacer 2 gene as barcoding DNA markers. Mosquito surveillance included the collection of larvae from breeding sites and that of adults through the human landing catch (HLC) method together with light traps. RESULTS: Analysis of the DNA extracted from the dried blood spot tests of the 110 captured NHPs revealed that 50% were positive for Plasmodium, namely Plasmodium cynomolgi, Plasmodium coatneyi, Plasmodium inui, Plasmodium knowlesi and Plasmodium sp. Prevalence determined by microscopic examination of the blood smears was 42%. Species of the primate genus Macaca and family Hylobatidae were identified by molecular analysis. The most common mosquito breeding sites were ditches, puddles and natural ponds. Some of the Anopheles letifer captured through HLC carried sporozoites of malaria parasites that can cause the disease in primates. CONCLUSIONS: The prevalence of malaria in the NHPs was high. Anopheles letifer, a potential vector of zoonotic malaria, was identified following its collection in Central Kalimantan by the HLC method. In sum, the potential for the transmission of zoonotic malaria in several regions of Indonesia is immense.

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.

Comparison between Neutralization Capacity of Antibodies Elicited by COVID-19 Natural Infection and Vaccination in Indonesia: A Prospective Cohort.

BACKGROUND: To fight the COVID-19 pandemic, immunity against SARS-CoV-2 should be achieved not only through natural infection but also by vaccination. The effect of COVID-19 vaccination on previously infected persons is debatable. METHODS: A prospective cohort was undergone to collect sera from unvaccinated survivors and vaccinated persons-with and without COVID-19 pre-infection. The sera were analyzed for the anti-receptor binding domain (RBD) titers by ELISA and for the capacity to neutralize the pseudovirus of the Wuhan-Hu-1 strain by luciferase assays. RESULTS: Neither the antibody titers nor the neutralization capacity was significantly different between the three groups. However, the correlation between the antibody titers and the percentage of viral neutralization derived from sera of unvaccinated survivors was higher than that from vaccinated persons with pre-infection and vaccinated naïve individuals (Spearman correlation coefficient (r) = -0.8558; 95% CI, -0.9259 to -0.7288), p < 0.0001 vs. -0.7855; 95% CI, -0.8877 to -0.6096, p < 0.0001 and -0.581; 95% CI, -0.7679 to -0.3028, p = 0.0002, respectively), indicating the capacity to neutralize the virus is most superior by infection alone. CONCLUSIONS: Vaccines induce anti-RBD titers as high as the natural infection with lower neutralization capacity, and it does not boost immunity in pre-infected persons.

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.

A Viral-Vectored Multi-Stage Malaria Vaccine Regimen With Protective and Transmission-Blocking Efficacies.

Malaria parasites undergo several stages in their complex lifecycle. To achieve reductions in both the individual disease burden and malaria transmission within communities, a multi-stage malaria vaccine with high effectiveness and durability is a more efficacious strategy compared with a single-stage vaccine. Here, we generated viral-vectored vaccines based on human adenovirus type 5 (AdHu5) and adeno-associated virus serotype 1 (AAV1) expressing a fusion protein of the pre-erythrocytic stage Plasmodium falciparum circumsporozoite protein (PfCSP) and the transmission-blocking sexual stage P25 protein (Pfs25). A two-dose heterologous AdHu5-prime/AAV1-boost immunization regimen proved to be highly effective for both full protection and transmission-blocking activity against transgenic P. berghei parasites expressing the corresponding P. falciparum antigens in mice. Remarkably, the immunization regimen induced antibody responses to both PfCSP and Pfs25 for over 9 months after the boosting and also maintained high levels of transmission-reducing activity (TRA: >99%) during that period, as evaluated by a direct feeding assay. If similar efficacies on P. falciparum can be shown following vaccination of humans, we propose that this multi-stage malaria vaccine regimen will be a powerful tool for malaria control, providing greater overall protection and cost-effectiveness than single-stage vaccines.

Anopheline antiplatelet protein from mosquito saliva regulates blood feeding behavior.

The saliva of hematophagous arthropods is enriched with a complex mixture of antihemostatic molecules, the biological functions of which are largely unknown. Anopheline antiplatelet protein (AAPP) from malaria vector mosquito exhibits strong antiplatelet activity when bound directly to host collagen by its C-terminus and through its N-terminus with Ca2+-binding activity. To investigate the biological functions of AAPP in blood feeding behavior and malaria transmission, we generated transgenic Anopheles stephensi mosquito lines expressing anti-AAPP antibody single-chain fragment (scFv) in their salivary glands. The AAPP-specific collagen-binding activity was completely abolished by AAPP-scFv complex formation in the saliva. Probing and prediuresis time, feeding success, blood meal size, and fecundity, which are all fitness characteristics, were significantly reduced in the transgenic mosquitoes. However, oocysts number in these mosquitoes were not significantly reduced following blood meal intake from Plasmodium berghei-infected mice. These results show that although AAPP plays an important role in mosquito blood feeding, its neutralizing activity did not affect sporogonic development in our laboratory model, but its high fitness cost would pose a survival risk for parasite-infected mosquitoes in nature.

Adeno-Associated Virus as an Effective Malaria Booster Vaccine Following Adenovirus Priming.

An ideal malaria vaccine platform should potently induce protective immune responses and block parasite transmission from mosquito to human, and it should maintain these effects for an extended period. Here, we have focused on vaccine development based on adeno-associated virus serotype 1 (AAV1), a viral vector widely studied in the field of clinical gene therapy that is able to induce long-term transgene expression without causing toxicity in vivo. Our results show the potential utility of AAV1 vectors as an extremely potent booster vaccine to induce durable immunity when combined with an adenovirus-priming vaccine in a rodent malaria model. We generated a series of recombinant AAV1s and human adenovirus type 5 (AdHu5) expressing either Plasmodium falciparum circumsporozoite protein (PfCSP) or P25 (Pfs25) protein. Heterologous two-dose immunization with an AdHu5-prime and AAV1-boost (AdHu5-AAV1) elicited robust and durable PfCSP- or Pfs25-specific functional antibodies over 280 days. Regarding protective efficacy, AdHu5-AAV1 PfCSP achieved high sterile protection (up to 80% protection rate) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. When examining transmission-blocking (TB) efficacy, we found that immunization with AdHu5-AAV1 Pfs25 maintained TB activity in vivo against transgenic P. berghei expressing Pfs25 for 287 days (99% reduction in oocyst intensity, 85% reduction in oocyst prevalence). Our data indicate that AAV1-based malaria vaccines can confer potent and durable protection as well as TB efficacy when administered following an AdHu5 priming vaccine, supporting the further evaluation of this regimen in clinical trials as a next-generation malaria vaccine platform.

Adenovirus-prime and baculovirus-boost heterologous immunization achieves sterile protection against malaria sporozoite challenge in a murine model.

With the increasing prevalence of artemisinin-resistant malaria parasites, a highly efficacious and durable vaccine for malaria is urgently required. We have developed an experimental virus-vectored vaccine platform based on an envelope-modified baculovirus dual-expression system (emBDES). Here, we show a conceptually new vaccine platform based on an adenovirus-prime/emBDES-boost heterologous immunization regimen expressing the Plasmodium falciparum circumsporozoite protein (PfCSP). A human adenovirus 5-prime/emBDES-boost heterologous immunization regimen consistently achieved higher sterile protection against transgenic P. berghei sporozoites expressing PfCSP after a mosquito-bite challenge than reverse-ordered or homologous immunization. This high protective efficacy was also achieved with a chimpanzee adenovirus 63-prime/emBDES-boost heterologous immunization regimen against an intravenous sporozoite challenge. Thus, we show that the adenovirus-prime/emBDES-boost heterologous immunization regimen confers sterile protection against sporozoite challenge by two individual routes, providing a promising new malaria vaccine platform for future clinical use.