Double peroxidase and histone acetyltransferase AgTip60 maintain innate immune memory in primed mosquitoes.

Immune priming in Anopheles gambiae is mediated by the systemic release of a hemocyte differentiation factor (HDF), a complex of lipoxin A4 bound to Evokin, a lipid carrier. HDF increases the proportion of circulating granulocytes and enhances mosquito cellular immunity. Here, we show that Evokin is present in hemocytes and fat-body cells, and messenger RNA (mRNA) expression increases significantly after immune priming. The double peroxidase (DBLOX) enzyme, present in insects but not in vertebrates, is essential for HDF synthesis. DBLOX is highly expressed in oenocytes in the fat-body tissue, and these cells increase in number in primed mosquitoes. We provide direct evidence that the histone acetyltransferase AgTip60 (AGAP001539) is also essential for a sustained increase in oenocyte numbers, HDF synthesis, and immune priming. We propose that oenocytes may function as a population of cells that are reprogrammed, and orchestrate and maintain a broad, systemic, and long-lasting state of enhanced immune surveillance in primed mosquitoes.

Plasmodium falciparum evades immunity of anopheline mosquitoes by interacting with a Pfs47 midgut receptor.

The surface protein Pfs47 allows Plasmodium falciparum parasites to survive and be transmitted by making them "undetectable" to the mosquito immune system. P. falciparum parasites express Pfs47 haplotypes compatible with their sympatric vectors, while those with incompatible haplotypes are eliminated by the mosquito. We proposed that Pfs47 serves as a "key" that mediates immune evasion by interacting with a mosquito receptor "the lock," which differs in evolutionarily divergent anopheline mosquitoes. Recombinant Pfs47 (rPfs47) was used to identify the mosquito Pfs47 receptor protein (P47Rec) using far-Western analysis. rPfs47 bound to a single 31-kDa band and the identity of this protein was determined by mass spectrometry. The mosquito P47Rec has two natterin-like domains and binds to Pfs47 with high affinity (17 to 32 nM). P47Rec is a highly conserved protein with submicrovillar localization in midgut cells. It has structural homology to a cytoskeleton-interacting protein and accumulates at the site of ookinete invasion. Silencing P47Rec expression reduced P. falciparum infection, indicating that the interaction of Pfs47 with the receptor is critical for parasite survival. The binding specificity of P47Rec from distant anophelines (Anopheles gambiae, Anopheles dirus, and Anopheles albimanus) with Pfs47-Africa (GB4) and Pfs47-South America (7G8) haplotypes was evaluated, and it is in agreement with the previously documented compatibility between P. falciparum parasites expressing different Pfs47 haplotypes and these three anopheline species. Our findings give further support to the role of Pfs47 in the adaptation of P. falciparum to different vectors.

Design of Dissolvable Microneedles for Delivery of a Pfs47-Based Malaria Transmission-Blocking Vaccine.

The development of effective malaria vaccines remains a global health priority. In addition to an effective vaccine, there is urgent demand for effective delivery technologies that can be easily deployed. The need for effective vaccine delivery tools is particularly pertinent in resource-poor settings where access to healthcare is limited. Microneedles are micron-scale structures that offer distinct advantages for vaccine delivery by efficiently targeting skin-resident immune cells, eliminating injection-associated pain, and improving patient compliance. Here, we developed and characterized a candidate malaria vaccine loaded and deployed using dissolvable microneedle arrays. Of note, a newly indicated human-relevant antigen was employed, Plasmodium falciparum surface protein P47. P47 and a potent toll-like receptor (TLR9) agonist vaccine adjuvant, CpG, were fabricated into microneedles using a gelatin polymer. Protein binding, ELISA, and fluorescence analysis confirmed the molecular structure, and the function of the P47 antigen and CpG was maintained after fabrication, storage, and release from microneedles. In cell culture, the cargo released from the microneedle arrays triggered TLR9 signaling and activated primary dendritic cells at levels similar to native, unincorporated vaccine components. Together, these studies demonstrate the potential of microneedles as an easily deployable strategy for a P47-based malaria vaccine.

Integrating Biomaterials and Immunology to Improve Vaccines Against Infectious Diseases.

Despite the success of vaccines in preventing many infectious diseases, effective vaccines against pathogens with ongoing challenges - such as HIV, malaria, and tuberculosis - remain unavailable. The emergence of new pathogen variants, the continued prevalence of existing pathogens, and the resurgence of yet other infectious agents motivate the need for new, interdisciplinary approaches to direct immune responses. Many current and candidate vaccines, for example, are poorly immunogenic, provide only transient protection, or create risks of regaining pathogenicity in certain immune-compromised conditions. Recent advances in biomaterials research are creating new potential to overcome these challenges through improved formulation, delivery, and control of immune signaling. At the same time, many of these materials systems - such as polymers, lipids, and self-assembly technologies - may achieve this goal while maintaining favorable safety profiles. This review highlights ways in which biomaterials can advance existing vaccines to safer, more efficacious technologies, and support new vaccines for pathogens that do not yet have vaccines. Biomaterials that have not yet been applied to vaccines for infectious disease are also discussed, and their potential in this area is highlighted.

In vivo Characterization of Plasmodium berghei P47 (Pbs47) as a Malaria Transmission-Blocking Vaccine Target.

An effective vaccine to reduce malaria transmission is central to control and ultimately achieve disease eradication. Recently, we demonstrated that antibodies targeting the Plasmodium falciparum surface protein P47 (Pfs47) reduce parasite transmission to Anopheles gambiae mosquitoes. Here, Plasmodium berghei (Pb) was used as a model to assess the in vivo efficacy of a P47-targeted transmission blocking vaccine (Pbs47). Mice were immunized following a prime/boost regimen and infected with P. berghei. The effect of immunization on infectivity to mosquitoes was evaluated by direct feeding on P. berghei-infected mice. The key region in Pbs47 where antibody binding confers protection was mapped, and the immunogenicity of this protective antigen was enhanced by conjugation to a virus-like particle. Passive immunization with 100 and 50 µg/mL of anti-Pbs47 IgG reduced oocyst density by 77 and 67%, respectively. Furthermore, affinity purified Pbs47-specific IgG significantly reduced oocyst density by 88 and 77%, respectively at doses as low as 10 and 1 µg/mL. These studies suggest that P47 is a promising transmission blocking target and show that antibodies to the same specific region in Pfs47 and Pbs47 confer protection.

Engineering a Virus-Like Particle as an Antigenic Platform for a Pfs47-Targeted Malaria Transmission-Blocking Vaccine.

We recently characterized Pfs47, a protein expressed on the surface of sexual stages and ookinetes of Plasmodium falciparum, as a malaria transmission-blocking vaccine (TBV) target. Mice immunization induced antibodies that conferred strong transmission-reducing activity (TRA) at a concentration of 200 µg/mL. Here, we sought to optimize the Pfs47 vaccine to elicit higher titers of high-affinity antibodies, capable of inducing strong TRA at a lower concentration. We report the development and evaluation of a Pfs47-based virus-like particle (VLP) vaccine generated by conjugating our 58 amino acid Pfs47 antigen to Acinetobacter phage AP205-VLP using the SpyCatcher:SpyTag adaptor system. AP205-Pfs47 complexes (VLP-P47) formed particles of ~22 nm diameter that reacted with polyclonal anti-Pfs47 antibodies, indicating that the antigen was accessible on the surface of the particle. Mice immunized with VLP-P47 followed by a boost with Pfs47 monomer induced significantly higher antibody titers, with higher binding affinity to Pfs47, than mice that received two immunizations with either VLP-P47 (VLP-P47/VLP-P47) or the Pfs47 monomer (P47/P47). Purified IgG from VLP-P47/P47 mice had strong TRA (83-98%) at concentrations as low as 5 µg/mL. These results indicate that conjugating the Pfs47 antigen to AP205-VLP significantly enhanced antigenicity and confirm the potential of Pfs47 as a TBV candidate.

Antibody targeting of a specific region of Pfs47 blocks Plasmodium falciparum malaria transmission.

Transmission-blocking vaccines are based on eliciting antibody responses in the vertebrate host that disrupt parasite development in the mosquito vector and prevent malaria transmission. The surface protein Pfs47 is present in Plasmodium falciparum gametocytes and female gametes. The potential of Pfs47 as a vaccine target was evaluated. Soluble full-length recombinant protein, consisting of three domains, was expressed in E. coli as a thioredoxin fusion (T-Pfs47). The protein was immunogenic, and polyclonal and monoclonal antibodies (mAb) were obtained, but they did not confer transmission blocking activity (TBA). All fourteen mAb targeted either domains 1 or 3, but not domain 2 (D2), and immune reactivity to D2 was also very low in polyclonal mouse IgG after T-Pfs47 immunization. Disruption of the predicted disulfide bond in D2, by replacing cysteines for alanines (C230A and C260A), allowed expression of recombinant D2 protein in E. coli. A combination of mAbs targeting D2, and deletion proteins from this domain, allowed us to map a central 52 amino acid (aa) region where antibody binding confers strong TBA (78-99%). This 52 aa antigen is immunogenic and well conserved, with only seven haplotypes world-wide that share 96-98% identity. Neither human complement nor the mosquito complement-like system are required for the observed TBA. A dramatic reduction in ookinete numbers and ookinete-specific transcripts was observed, suggesting that the antibodies are interacting with female gametocytes and preventing fertilization.