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Human Immunodeficiency Virus (HIV) remains a global health challenge, and novel approaches to improve HIV control are significantly important. The cell and gene therapy product AGT103-T was previously evaluated (NCT04561258) for safety, immunogenicity, and persistence in seven patients for up to 180 days post infusion. In this study, we sought to investigate the impact of AGT103-T treatment upon analytical treatment interruptions (ATIs). Six patients previously infused with AGT103-T were enrolled into an ATI study (NCT05540964), wherein they suspended their antiretroviral therapy (ART) until their viral load reached 100,000 copies/mL in two successive visits, or their CD4 count was reduced to below 300 cells/μL. During the ATI, all patients experienced viral rebound followed by a notable expansion in HIV specific immune responses. The participants demonstrated up to a five-fold increase in total CD8 counts over baseline approximately 1–2 weeks followed by the peak viremia. This coincided with a rise in HIV-specific CD8 T cells, which was attributed to the increase in antigen availability and memory recall. Thus, the protocol was amended to include a second ATI with the first ATI serving as an “auto-vaccination.” Four patients participated in a second ATI. During the second ATI, the Gag-specific CD8 T cells were either maintained or rose in response to viral rebound and the peak viremia was substantially decreased. The patients reached a viral set point ranging from 7,000 copies/mL to 25,000 copies/mL. Upon resuming ART, all participants achieved viral control more rapidly than during the first ATI, with CD4 counts remaining within 10% of baseline measurements and without any serious adverse events or evidence of drug resistance. In summary, the rise in CD8 counts and the viral suppression observed in 100% of the study participants are novel observations demonstrating that AGT103-T gene therapy when combined with multiple ATIs, is a safe and effective approach for achieving viral control, with viral setpoints consistently below 25,000 copies/mL and relatively stable CD4 T cell counts. We conclude that HIV cure-oriented cell and gene therapy trials should include ATI and may benefit from designs that include multiple ATIs when induction of CD8 T cells is required to establish viral control.

ABSTRACT The mosquito immune system limits Plasmodium infection and malaria transmission. Plasmodium falciparum evades the mosquito defense response by expressing the surface protein P. falciparum P47 (Pfs47) that inhibits midgut epithelial nitration by interacting with the mosquito midgut P47 receptor (P47Rec). However, the mechanism by which P47Rec suppresses caspase-mediated nitration is unknown. Here, we show that epithelial invasion by Pfs47 knockout parasites is followed by an extrusion of cells undergoing caspase-mediated apoptosis, which triggers the release of hemocyte-derived microvesicles (HdMv’s), known to promote complement activation and ookinete lysis. In contrast, invasion by P. falciparum parasites that express Pfs47 accelerates the extrusion of damaged cells and disrupts caspase activation and HdMv release. The Anopheles gambiae heat shock protein 70 cognate 3 (Hsc70-3), the ortholog of Drosophila binding immunoglobulin protein, was identified as a molecular partner of P47Rec. Silencing of Hsc70-3 promotes lysis of Pfs47 wild-type parasites through a caspase S2-dependent mechanism. The interaction of P47Rec activates a Hsc70-3-mediated anti-apoptotic response that prevents caspase activation. The interaction of P47Rec with Hsc70-3 is necessary for P. falciparum to evade the mosquito early immune responses that target the ookinete stage. IMPORTANCE Malaria transmission by Anopheles gambiae mosquitoes is very effective, in part because the parasite expresses a surface protein called Pfs47 that allows it to evade the mosquito immune system. Here we investigate how this protein changes the response of mosquito midgut epithelial cells to invasion by the parasite. Pfs47 is known to interact with P47Rec, a mosquito midgut receptor. We found that Pf47Rec inhibits caspase-mediated apoptosis by interacting with the Hsc70-3. This disrupts nitration of midgut epithelial cells invaded by the parasite and the release of hemocyte-derived microvesicles, which are critical for effective activation of the mosquito complement system that eliminates the parasite.

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.

Malaria is a life-threatening disease caused by Plasmodium falciparum parasites that is transmitted through the bites of infected anopheline mosquitoes. P. falciparum dispersal from Africa, as a result of human migration, required adaptation of the parasite to several different indigenous anopheline species. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses that is mediated by the Pfs47 gene. Pfs47 is a polymorphic gene with signatures of diversifying selection and a strong geographic genetic structure at a continental level. Here, we investigated the role of single four amino acid differences between the Pfs47 gene from African (GB4 and NF54) and a New World (7G8) strains that differ drastically in their ability to evade the immune system of A. gambiae L35 refractory mosquitoes. Wild type NF54 and GB4 parasites can survive in this mosquito strain, while 7G8 parasites are eliminated. Our studies indicate that replacement in any of these four single amino acids in Pfs47 from the NF54 strain by those present in 7G8, completely disrupts the ability of NF54 parasites to hide from the mosquito immune system. One of these amino acid replacements had the opposite effect on A. albimanus mosquitoes, and enhanced infection. We conclude that malaria transmission involves a complex interplay between the genetic background of the parasite and the mosquito and that Pfs47 can be critical in this interaction as it mediates Plasmodium immune evasion through molecular interactions that need to be precise in some parasite/vector combinations.

Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3' UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes.

The post genomic era revealed the need for developing better performing, easier to use and more sophisticated genetic manipulation tools for the study of Trypanosoma cruzi, the etiological agent of Chagas disease. In this work a series of plasmids that allow genetic manipulation of this protozoan parasite were developed. First of all we focused on useful tools to establish selection strategies for different strains and which can be employed as expression vectors. On the other hand molecular building blocks in the form of diverse selectable markers, modifiable fluorescent protein and epitope-tag coding sequences were produced. Both types of modules were harboured in backbone molecules conceived to offer multiple construction and sub-cloning strategies. These can be used to confer new properties to already available genetic manipulation tools or as starting points for whole novel designs. The performance of each plasmid and building block was determined independently. For illustration purposes, some simple direct practical applications were conducted.