IgM, Fc mu Rs, and malarial immune evasion

IgM is an ancestral Ab class found in all jawed vertebrates, from sharks to mammals. This ancient ancestry is shared by malaria parasites (genus Plasmodium) that infect all classes of terrestrial vertebrates with whom they coevolved. IgM, the least studied and most enigmatic of the vertebrate Igs, was recently shown to form an intimate relationship with the malaria parasite Plasmodium falciparum. In this article, we discuss how this association might have come about, building on the recently determined structure of the human IgM pentamer, and how this interaction could affect parasite survival, particularly in light of the just-discovered Fc mu R localized to B and T cell surfaces. Because this parasite may exploit an interaction with IgM to limit immune detection, as well as to manipulate the immune response when detected, a better understanding of this association may prove critical for the development of improved vaccines or vaccination strategies

The Importance of Human FcγRI in Mediating Protection to Malaria

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcγRI. This important finding documents the capacity of FcγRI to mediate potent antimalaria immunity and supports the development of FcγRI-directed therapy for human malaria

T cell responses induced by adenoviral vectored vaccines can be adjuvanted by fusion of antigen to the oligomerization domain of C4b-binding protein.

Viral vectored vaccines have been shown to induce both T cell and antibody responses in animals and humans. However, the induction of even higher level T cell responses may be crucial in achieving vaccine efficacy against difficult disease targets, especially in humans. Here we investigate the oligomerization domain of the α-chain of C4b-binding protein (C4 bp) as a candidate T cell "molecular adjuvant" when fused to malaria antigens expressed by human adenovirus serotype 5 (AdHu5) vectored vaccines in BALB/c mice. We demonstrate that i) C-terminal fusion of an oligomerization domain can enhance the quantity of antigen-specific CD4(+) and CD8(+) T cell responses induced in mice after only a single immunization of recombinant AdHu5, and that the T cells maintain similar functional cytokine profiles; ii) an adjuvant effect is observed for AdHu5 vectors expressing either the 42 kDa C-terminal domain of Plasmodium yoelii merozoite surface protein 1 (PyMSP1(42)) or the 83 kDa ectodomain of P. falciparum strain 3D7 apical membrane antigen 1 (PfAMA1), but not a candidate 128kDa P. falciparum MSP1 biallelic fusion antigen; iii) following two homologous immunizations of AdHu5 vaccines, antigen-specific T cell responses are further enhanced, however, in both BALB/c mice and New Zealand White rabbits no enhancement of functional antibody responses is observed; and iv) that the T cell adjuvant activity of C4 bp is not dependent on a functional Fc-receptor γ-chain in the host, but is associated with the oligomerization of small (<80 kDa) antigens expressed by recombinant AdHu5. The oligomerization domain of C4 bp can thus adjuvant T cell responses induced by AdHu5 vectors against selected antigens and its clinical utility as well as mechanism of action warrant further investigation

Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria.

The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field

Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris.

We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P &lt; 0.001) and PARP inhibitor therapy (P &lt; 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P &lt; 0.018) and WEE1 inhibitor (P &lt; 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P &lt; 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Antibody-and Fc-receptor-based therapeutics for malaria A B S T R A C T

Abs (antibodies) are complex glycoproteins that play a crucial role in protective immunity to malaria, but their effectiveness in mediating resistance can be enhanced by genetically engineered modifications that improve on nature. These Abs also aid investigation of immune mechanisms operating to control the disease and are valuable tools in developing neutralization assays for vaccine design. This review explores how this might be achieved