Antibody-Mediated Natural Killer Cell Function against <i>Plasmodium falciparum</i>

Background: The protective effect of polyclonal immunoglobulins following passive transfer from malaria-immune adults to patients hospitalised with malaria provides strong motivation for successful vaccination. Antibodies against the merozoite stage of the parasite have been associated with clinical protection in immuno-epidemiological studies and controlled human malaria infection (CHMI) trials. Unfortunately, this knowledge has yet to be translated into highly effective malaria vaccines. This failure is partly attributed to the lack of robust functional correlates of vaccine-induced or naturally acquired immunity. This thesis contributes to this knowledge gap by proposing an antibody-mediated natural killer cell (ab-NK) assay as a novel Fc-mediated functional correlate of clinical protection against Plasmodium falciparum (P. falciparum) merozoites. Methods: I used multiparameter flow cytometry to quantify Fc receptor-mediated natural killer (ab-NK) cell degranulation (CD107a) and IFNγ production following co-incubation with anti-merozoite antibodies. I evaluated the utility of this assay as a functional correlate of protection from clinical malaria using samples from two independent prospective studies. The first involved 142 semi-immune Kenyan adults from a controlled human malaria infection study (CHMI-SIKA). In the second, 293 children from Junju village in Kilifi, Kenya, were monitored weekly for clinical malaria episodes over six months. I developed an antigen- specific ab-NK assay to identify potential merozoite targets. Results: The ab-NK cell assay was specific, showed minimal day-to-day variation, was effective against multiple P. falciparum strains and enhanced antibody-mediated invasion inhibition activity in vitro. In CHMI-SIKA, high ab-NK degranulation and IFNγ secretion were strongly correlated with reduced in vivo parasite growth (Hazard Ratio 0.25 and 0.22, respectively, P<0.000). In Junju, ab-NK activity increased with age and was boosted by a recent infection. High ab-NK degranulation and IFNγ secretion were associated with clinical protection (Hazard Ratio 0.27, P<0.000 and 0.54, P<0.040, respectively). Using the antigen- specific approach, I identified eight of the fourteen merozoite antigens tested as potential targets. These included merozoite surface antigens, MSP11, MSP2, and well-characterized malaria vaccine candidates like AMA-1 and MSP3, supporting their interest as potential vaccine candidates. Interestingly, poorly studied antigens like P41 and Pf113 also induced comparable ab-NK responses. Conclusions: These findings identify ab-NK cell responses targeting merozoites as a strong predictor of naturally acquired immunity against P. falciparum malaria. Our antigen-specific ab-NK assay facilitated faster evaluation of ab-NK responses against multiple antigens, enabling the functional analysis of potential vaccine candidates that may be considered in future sub-unit vaccine design

Cord blood IgG and the risk of severe Plasmodium falciparum malaria in the first year of life

Young infants are less susceptible to severe episodes of malaria but the targets and mechanisms of protection are not clear. Cord blood antibodies may play an important role in mediating protection but many studies have examined their association with the outcome of infection or non-severe malaria. Here, we investigated whether cord blood IgG to Plasmodium falciparum merozoite antigens and antibody-mediated effector functions were associated with reduced odds of developing severe malaria at different time points during the first year of life. We conducted a case-control study of well-defined severe falciparum malaria nested within a longitudinal birth cohort of Kenyan children. We measured cord blood total IgG levels against five recombinant merozoite antigens and antibody function in the growth inhibition activity and neutrophil antibody-dependent respiratory burst assays. We also assessed the decay of maternal antibodies during the first 6months of life. The mean antibody half-life range was 2.51months (95% confidence interval (CI): 2.19-2.92) to 4.91months (95% CI: 4.47-6.07). The rate of decline of maternal antibodies was inversely proportional to the starting concentration. The functional assay of antibody-dependent respiratory burst activity predicted significantly reduced odds of developing severe malaria during the first 6months of life (Odds ratio (OR) 0.07, 95% CI: 0.007-0.74, P=0.007). Identification of the targets of antibodies mediating antibody-dependent respiratory burst activity could contribute to the development of malaria vaccines that protect against severe episodes of malaria in early infancy

Mesalazine in the initial management of severely acutely malnourished children with environmental enteric dysfunction : a pilot randomized controlled trial

Background: Environmental enteric dysfunction (EED) is an acquired syndrome of impaired gastrointestinal mucosal barrier function that is thought to play a key role in the pathogenesis of stunting in early life. It has been conceptualized as an adaptive response to excess environmental pathogen exposure. However, it is clinically similar to other inflammatory enteropathies, which result from both host and environmental triggers, and for which immunomodulation is a cornerstone of therapy. Methods: In this pilot double-blind randomized placebo-controlled trial, 44 children with severe acute malnutrition and evidence of EED were assigned to treatment with mesalazine or placebo for 28 days during nutritional rehabilitation. Primary outcomes were safety and acceptability of the intervention. Results: Treatment with mesalazine was safe: there was no excess of adverse events, evidence of deterioration in intestinal barrier integrity or impact on nutritional recovery. There were modest reductions in several inflammatory markers with mesalazine compared to placebo. Depression of the growth hormone – insulin-like growth factor-1 axis was evident at enrollment and associated with inflammatory activation. Increases in the former and decreases in the latter correlated with linear growth. Conclusions Intestinal inflammation in EED is non-essential for mucosal homeostasis and is at least partly maladaptive. Further trials of gut-specific immunomodulatory therapies targeting host inflammatory activation in order to optimize the growth benefits of nutritional rehabilitation and to address stunting are warranted. Funded by The Wellcome Trust

Body composition and maximum alactic anaerobic performance during a one month stay at high altitude

Prolonged altitude exposure usually leads to considerable weight loss of which a large part is from muscle tissue. This loss reduces maximum alactic anaerobic muscle power. It was hypothesized that most of the weight loss may simply be the result of malnutrition due to lack of palatable food in an uncomfortable environment. To test this hypothesis eight healthy male subjects (age 33.7 \ub1 4.6 S.C. yr), well acclimatized to prevent symptoms of acute mountain sickness, were exposed for 4 weeks to an altitude of 5050 m with access to a large choice of palatable food in comfortable conditions. Body weight (with a scale), body composition (from skinfolds), arm muscle plus bone cross-sectional area (Am + b) and muscle plus bone leg volume (Vm + b) (from skinfolds and circumferences), maximum voluntary contraction force of the elbow flexors (MVC, with a load cell) and maximum jumping height (Hmax, on a platform) were measured before departure (SL) and in the first (ALT1), second (ALT2) and fourth week (ALT4) of their altitude sojourn. Three-day dietary records were obtained at SL and at ALT4. Body mass had decreased significantly at ALT2 (-3.8%) and at ALT4 (-4.6%) likely reflecting changes in body water homeostasis. No changes were found in \ufat, Am + b, Vm + b, MVC or Hmax. Average dietary intake at SL was 8.96 \ub1 1.45 MJ and had increased to 13.59 \ub1 3.07 MJ at ALT4. In conclusion, up to an altitude of 5050 m loss of body mass from fat and muscle tissue, and hence impairment of maximum anaerobic muscle power (alactic) appears to be avoidable by food intake matched to energy expenditure. The latter may be achieved simply by proper acclimatization, sufficient comfort and availability of palatable food

IL-15 Overcomes Hepatocellular Carcinoma-Induced NK Cell Dysfunction

NK cells have potent antitumor capacity. They are enriched in the human liver, with a large subset specialized for tissue-residence. The potential for liver-resident versus liver-infiltrating NK cells to populate, and exert antitumor functions in, human liver tumors has not been studied. We examined liver-resident and liver-infiltrating NK cells directly ex vivo from human hepatocellular carcinomas (HCCs) and liver colorectal (CRC) metastases, compared with matched uninvolved liver tissue. We found that NK cells were highly prevalent in both HCC and liver CRC metastases, although at lower frequencies than unaffected liver. Up to 79% of intratumoral NK cells had the CXCR6+CD69+ liver-resident phenotype. Direct ex vivo staining showed that liver-resident NK cells had increased NKG2D expression compared to their non-resident counterparts, but both subsets had NKG2D downregulation within liver tumors compared to uninvolved liver. Proliferation of intratumoral NK cells (identified by Ki67) was selectively impaired in those with the most marked NKG2D downregulation. Human liver tumor NK cells were functionally impaired, with reduced capacity for cytotoxicity and production of cytokines, even when compared to the hypo-functional tissue-resident NK cells in unaffected liver. Coculture of human liver NK cells with the human hepatoma cell line PLC/PRF/5, or with autologous HCC, recapitulated the defects observed in NK cells extracted from tumors, with downmodulation of NKG2D, cytokine production, and target cell cytotoxicity. Transwells and conditioned media confirmed a requirement for cell contact with PLC/PRF/5 to impose NK cell inhibition. IL-15 was able to recover antitumor functionality in NK cells inhibited by in vitro exposure to HCC cell lines or extracted directly from HCC. In summary, our data suggest that the impaired antitumor function of local NK cells reflects a combination of the tolerogenic features inherent to liver-resident NK cells together with additional contact-dependent inhibition imposed by HCC itself. The demonstration that IL-15 can recover hepatic NK cell function following tumor exposure supports its inclusion in immunotherapy strategies

Characterization of a novel Plasmodium falciparum merozoite surface antigen and potential vaccine target.

Peer reviewed: TrueINTRODUCTION: Detailed analyses of genetic diversity, antigenic variability, protein localization and immunological responses are vital for the prioritization of novel malaria vaccine candidates. Comprehensive approaches to determine the most appropriate antigen variants needed to provide broad protection are challenging and consequently rarely undertaken. METHODS: Here, we characterized PF3D7_1136200, which we named Asparagine-Rich Merozoite Antigen (ARMA) based on the analysis of its sequence, localization and immunogenicity. We analyzed IgG and IgM responses against the common variants of ARMA in independent prospective cohort studies in Burkina Faso (N = 228), Kenya (N = 252) and Mali (N = 195) using a custom microarray, Div-KILCHIP. RESULTS: We found a marked population structure between parasites from Africa and Asia. African isolates shared 34 common haplotypes, including a dominant pair although the overall selection pressure was directional (Tajima's D = -2.57; Fu and Li's F = -9.69; P < 0.02). ARMA was localized to the merozoite surface, IgG antibodies induced Fc-mediated degranulation of natural killer cells and strongly inhibited parasite growth in vitro. We found profound serological diversity, but IgG and IgM responses were highly correlated and a hierarchical clustering analysis identified only three major serogroups. Protective IgG and IgM antibodies appeared to target both cross-reactive and distinct epitopes across variants. However, combinations of IgG and IgM antibodies against selected variants were associated with complete protection against clinical episodes of malaria. DISCUSSION: Our systematic strategy exploits genomic data to deduce the handful of antigen variants with the strongest potential to induce broad protection and may be broadly applicable to other complex pathogens for which effective vaccines remain elusive

Phagocytosis of Plasmodium falciparum ring-stage parasites predicts protection against malaria

Ring-infected erythrocytes are the predominant asexual stage in the peripheral circulation but are rarely investigated in the context of acquired immunity against Plasmodium falciparum malaria. Here we compare antibody-dependent phagocytosis of ring-infected parasite cultures in samples from a controlled human malaria infection (CHMI) study (NCT02739763). Protected volunteers did not develop clinical symptoms, maintained parasitaemia below a predefined threshold of 500 parasites/μl and were not treated until the end of the study. Antibody-dependent phagocytosis of both ring-infected and uninfected erythrocytes from parasite cultures was strongly correlated with protection. A surface proteomic analysis revealed the presence of merozoite proteins including erythrocyte binding antigen-175 and −140 on ring-infected and uninfected erythrocytes, providing an additional antibody-mediated protective mechanism for their activity beyond invasion-inhibition. Competition phagocytosis assays support the hypothesis that merozoite antigens are the key mediators of this functional activity. Targeting ring-stage parasites may contribute to the control of parasitaemia and prevention of clinical malaria

Image_3_IL-15 Overcomes Hepatocellular Carcinoma-Induced NK Cell Dysfunction.jpeg

<p>NK cells have potent antitumor capacity. They are enriched in the human liver, with a large subset specialized for tissue-residence. The potential for liver-resident versus liver-infiltrating NK cells to populate, and exert antitumor functions in, human liver tumors has not been studied. We examined liver-resident and liver-infiltrating NK cells directly ex vivo from human hepatocellular carcinomas (HCCs) and liver colorectal (CRC) metastases, compared with matched uninvolved liver tissue. We found that NK cells were highly prevalent in both HCC and liver CRC metastases, although at lower frequencies than unaffected liver. Up to 79% of intratumoral NK cells had the CXCR6⁺CD69⁺ liver-resident phenotype. Direct ex vivo staining showed that liver-resident NK cells had increased NKG2D expression compared to their non-resident counterparts, but both subsets had NKG2D downregulation within liver tumors compared to uninvolved liver. Proliferation of intratumoral NK cells (identified by Ki67) was selectively impaired in those with the most marked NKG2D downregulation. Human liver tumor NK cells were functionally impaired, with reduced capacity for cytotoxicity and production of cytokines, even when compared to the hypo-functional tissue-resident NK cells in unaffected liver. Coculture of human liver NK cells with the human hepatoma cell line PLC/PRF/5, or with autologous HCC, recapitulated the defects observed in NK cells extracted from tumors, with downmodulation of NKG2D, cytokine production, and target cell cytotoxicity. Transwells and conditioned media confirmed a requirement for cell contact with PLC/PRF/5 to impose NK cell inhibition. IL-15 was able to recover antitumor functionality in NK cells inhibited by in vitro exposure to HCC cell lines or extracted directly from HCC. In summary, our data suggest that the impaired antitumor function of local NK cells reflects a combination of the tolerogenic features inherent to liver-resident NK cells together with additional contact-dependent inhibition imposed by HCC itself. The demonstration that IL-15 can recover hepatic NK cell function following tumor exposure supports its inclusion in immunotherapy strategies.</p