Biology subjects

Minns, A. M.

Publications and source records attributed to Minns, A. M..

6 recordsLinked to original sources

bioRxiv2023

Exogenous 4-1BB co-stimulation enhances memory B cell response during malaria.

T-dependent germinal center (GC) output, comprising plasma cells (PC) and memory B cells (MBC), is crucial for the clearance of Plasmodium infection and protection against reinfection. In this study, we examined the effect of an agonistic antibody targeting 4-1BB (CD137), a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), during experimental malaria. Here we show that exogenous 4-1BB stimulation, despite delaying the effector GC response, surprisingly enhanced humoral memory recall and protection from reinfection. Single cell RNA and ATAC sequencing of MBCs from mice that received 4-1BB stimulation revealed distinct populations with transcriptional and epigenetic signatures indicative of superior recall and proliferative potential. Importantly, our results indicate that the effects of 4-1BB stimulation are dependent on IL-9R signaling in B cells but independent of parasite load during primary infection. Our study proposes an immunomodulatory approach to enhance the quality of the MBC pool, providing superior protection during infection and vaccination, particularly in the context of malaria.

immunology↗

bioRxiv2023

Comparative proteomics of vesicles essential for the egress of Plasmodium falciparum gametocytes from red blood cells

Transmission of malaria parasites to the mosquito is mediated by sexual precursor cells, the gametocytes. Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside-out mode during which the membrane of the parasitophorous vacuole ruptures prior to the erythrocyte membrane. Membrane rupture requires the exocytosis of specialized secretory vesicles of the parasites; i.e. the osmiophilic bodies (OBs) involved in rupturing the parasitophorous vacuole membrane, and vesicles (here termed g-exonemes) that harbour the perforin-like protein PPLP2 required for erythrocyte lysis. While several OB proteins are known, like G377 and MDV1/Peg3, the protein composition of the g-exonemes remains unclear. Here, we used high- resolution imaging and BioID methods to study the two types of egress vesicles in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the g-exonemes and that exocytosis of the g-exonemes, but not of the OBs, is calcium-sensitive. Further, the two types of vesicles exhibit distinct proteomes. In addition to known egress-related proteins, our analyses revealed novel components of OBs and g-exonemes, including proteins involved in vesicle trafficking. Our data provide insight into the immense molecular machinery required for the inside-out egress of P. falciparum gametocytes.

molecular biology↗

bioRxiv2022

Intranasal virus-particle mimicking vaccine enhances SARS-CoV-2 clearance in the Syrian hamster model

Multiple vaccines have been developed and licensed for SARS-CoV-2. While these vaccines reduce disease severity, they do not prevent infection, and SARS-CoV-2 continues to spread and evolve. To prevent infection and limit transmission, vaccines must be developed that induce immunity in the respiratory tract. Therefore, we performed proof-of-principle vaccination studies with an intranasal nanoparticle vaccine against SARS-CoV-2. The vaccine candidate consisted of the self-assembling 60-subunit I3-01 protein scaffold covalently decorated with the SARS-CoV-2 receptor binding domain (RBD) using the SpyCatcher-SpyTag system. We verified the intended antigen display features by reconstructing the I3-01 scaffold to 3.4A using cryo-EM, and then demonstrated that the scaffold was highly saturated when grafted with RBD. Using this RBD-grafted SpyCage scaffold (RBD+SpyCage), we performed two unadjuvanted intranasal vaccination studies in the "gold-standard" preclinical Syrian hamster model. Hamsters received two vaccinations 28 days apart, and were then challenged 28 days post-boost with SARS-CoV-2. The initial study focused on assessing the immunogenicity of RBD+SpyCage, which indicated that vaccination of hamsters induced a non-neutralizing antibody response that enhanced viral clearance but did not prevent infection. In an expanded study, we demonstrated that covalent bonding of RBD to the scaffold was required to induce an antibody response. Consistent with the initial study, animals vaccinated with RBD+SpyCage more rapidly cleared SARS-CoV-2 from both the upper and lower respiratory tract. These findings demonstrate the intranasal SpyCage vaccine platform can induce protection against SARS-CoV-2 and, with additional modifications to improve immunogenicity, is a versatile platform for the development of intranasal vaccines targeting respiratory pathogens. IMPORTANCEDespite the availability of efficacious COVID vaccines that reduce disease severity, SARS-CoV-2 continues to spread. To limit SARS-CoV-2 transmission, the next generation of vaccines must induce immunity in the mucosa of the upper respiratory tract. Therefore, we performed proof-of-principle, unadjuvanted intranasal vaccination studies with a recombinant protein nanoparticle scaffold, SpyCage, decorated with the receptor-binding domain (RBD) of the S protein (SpyCage+RBD). We show that SpyCage+RBD was immunogenic and enhanced SARS-CoV-2 clearance from the nose and lungs of Syrian hamsters. Moreover, covalent grafting of the RBD to the scaffold was required to induce an immune response when given via the intranasal route. These proof-of-concept findings indicate that with further enhancements to immunogenicity (e.g., adjuvant incorporation, antigen optimization), the SpyCage scaffold has potential as a versatile, intranasal vaccine platform for respiratory pathogens.

immunology↗

bioRxiv2020

Limited window for donation of convalescent plasma with high live-virus neutralizing antibodies for COVID-19 immunotherapy

The optimal timeframe for donating convalescent plasma to be used for COVID-19 immunotherapy is unknown. To address this important knowledge deficit, we determined in vitro live-virus neutralizing capacity and persistence of IgM and IgG antibody responses against the receptor-binding domain and S1 ectodomain of the SARS-CoV-2 spike glycoprotein in 540 convalescent plasma samples obtained from 175 COVID-19 plasma donors for up to 142 days post-symptom onset. Robust IgM, IgG, and viral neutralization responses to SARS-CoV-2 persist, in the aggregate, for at least 100 days post-symptom onset. However, a notable acceleration in decline in virus neutralization titers [≥]160, a value suitable for convalescent plasma therapy, was observed starting 60 days after first symptom onset. Together, these findings better define the optimal window for donating convalescent plasma useful for immunotherapy of COVID-19 patients and reveal important predictors of an ideal plasma donor, including age and COVID-19 disease severity score. One Sentence SummaryEvaluation of SARS-CoV-2 anti-spike protein IgM, IgG, and live-virus neutralizing titer profiles reveals that the optimal window for donating convalescent plasma for use in immunotherapy is within the first 60 days of symptom onset.

immunology↗

bioRxiv2019

Extensive Transcriptional and Translational Regulation Occur During the Maturation of Malaria Parasite Sporozoites

Plasmodium sporozoites are transmitted from an infected mosquito to mammals in which they infect the liver. The infectivity profile of sporozoites changes as they egress from oocysts on the mosquito midgut into the hemocoel, and then invade the salivary glands, where they maintain a poised and infectious state until transmission occurs. Upon transmission, the sporozoite must then navigate the host skin, vasculature, and liver. All of these feats require distinct repertoires of proteins and capabilities that are coordinated in an appropriate temporal manner. Here, we report the comprehensive and dynamic transcriptomes and proteomes of both oocyst sporozoite and salivary gland sporozoite stages in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. These data robustly define mRNAs and proteins that are Upregulated in Oocyst Sporozoites (UOS) or Upregulated in Infectious Sporozoites (UIS), which include critical gene products for sporozoite functions, as well as many of unknown importance that are similarly regulated. Moreover, we found that Plasmodium uses two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate specific genes necessary to successfully navigate the mosquito vector and mammalian host environments. Finally, gene-specific validation experiments of selected, translationally repressed transcripts in P. yoelii confirmed the interpretations of the global transcriptomic and proteomic datasets. Together, these data indicate that two waves of translational repression are implemented and relieved at different times in sporozoite maturation to promote its successful life cycle progression.

microbiology↗

bioRxiv2018

Plasmodium male gametocyte development and transmission are critically regulated by general and transmission-specific members of the CAF1/CCR4/NOT complex

With relatively few known specific transcription factors to control the abundance of specific mRNAs, Plasmodium parasites also regulate the stability and turnover of transcripts to provide more comprehensive gene regulation. Plasmodium transmission stages impose translational repression on specific transcripts in part to accomplish this. However, few proteins are known to participate in this process, and those that are characterized primarily affect female gametocytes. We have identified and characterized PyCCR4-1, a putative deadenylase, which plays a role in the development and activation of male gametocytes, regulates the abundance of specific mRNAs in gametocytes, and ultimately increases the efficiency of host-to-vector transmission. We find that when pyccr4-1 is deleted or its protein made catalytically inactive, there is a loss in the initial coordination of male gametocyte maturation and a reduction of parasite infectivity of the mosquito. Expression of only the N-terminal CAF1 domain of the essential CAF1 deadenylase, which prevents PyCCR4-1 association with the complex, leads to a similar phenotype. Comparative RNA-seq revealed that PyCCR4-1 affects transcripts important for transmission-related functions that are associated with male or female gametocytes, some of which directly associate with the immunoprecipitated complex. Finally, circular RT-PCR of one of the bound, dysregulated transcripts showed that PyCCR4-1 does not have gross changes in UTR or poly(A) tail length. We conclude that general and transmission-specialized members of the CAF1/CCR4/NOT complex play critical and intertwined roles in gametocyte maturation and transmission.\n\nAUTHOR SUMMARYMalaria is a disease caused by Plasmodium parasites, which are transmitted during an infectious blood meal by anopheline mosquitoes. Transmission of the sexual stages of the parasite to mosquitoes requires the proper regulation of specific mRNAs. While much work has been done to characterize regulation of mRNAs in female gametocytes, little has been done to assess this regulation in male gametocytes. Here, we demonstrate that PyCCR4-1, a member of the CAF1/CCR4/NOT RNA metabolic complex, acts upon transcripts both directly and indirectly in both male and female parasites, and results in a reduction of male gametocytemia. In gametocytes lacking PyCCR4-1, as well as those expressing a catalytically dead variant, the initial coordinated wave of male gametocyte activation is lost, and these parasites are less able to productively infect mosquitoes. We find that PyCCR4-1 requires its association with PyCAF1 and by proxy, the rest of the complex, in order to perform its functions based upon experiments in both Plasmodium yoelii and Plasmodium falciparum. We also find that the CAF1/CCR4/NOT complex is directly binding some of these transcripts and is likely acting both directly and indirectly to modulate transcript abundance. These findings demonstrate that the combined effects of the CAF1/CCR4/NOT complex upon specific mRNAs are important for both male and female gametocytes, and that this regulation is required for efficient transmission to the mosquito vector.

microbiology↗