The impact of Plasmodium-driven immunoregulatory networks on immunity to malaria.

Malaria, caused by infection with Plasmodium parasites, drives multiple regulatory responses across the immune landscape. These regulatory responses help to protect against inflammatory disease but may in some situations hamper the acquisition of adaptive immune responses that clear parasites. In addition, the regulatory responses that occur during Plasmodium infection may negatively affect malaria vaccine efficacy in the most at-risk populations. Here, we discuss the specific cellular mechanisms of immunoregulatory networks that develop during malaria, with a focus on knowledge gained from human studies and studies that involve the main malaria parasite to affect humans, Plasmodium falciparum. Leveraging this knowledge may lead to the development of new therapeutic approaches to increase protective immunity to malaria during infection or after vaccination.

The role of CD4+ T cells in visceral leishmaniasis; new and emerging roles for NKG7 and TGFß.

Visceral leishmaniasis is a potentially devastating neglected tropical disease caused by the protozoan parasites Leishmania donovani and L. infantum (chagasi). These parasites reside in tissue macrophages and survive by deploying a number of mechanisms aimed at subverting the host immune response. CD4+ T cells play an important role in controlling Leishmania parasites by providing help in the form of pro-inflammatory cytokines to activate microbiocidal pathways in infected macrophages. However, because these cytokines can also cause tissue damage if over-produced, regulatory immune responses develop, and the balance between pro-inflammatory and regulatory CD4+ T cells responses determines the outcomes of infection. Past studies have identified important roles for pro-inflammatory cytokines such as IFNγ and TNF, as well as regulatory co-inhibitory receptors and the potent anti-inflammatory cytokine IL-10. More recently, other immunoregulatory molecules have been identified that play important roles in CD4+ T cell responses during VL. In this review, we will discuss recent findings about two of these molecules; the NK cell granule protein Nkg7 and the anti-inflammatory cytokine TGFß, and describe how they impact CD4+ T cell functions and immune responses during visceral leishmaniasis.

Apolipoprotein E Is Upregulated in Blood and Circulating Monocytes of Indian Patients With Visceral Leishmaniasis.

Apolipoprotein E (ApoE) has been associated with several diseases including Parkinson's disease, Alzheimer's and multiple sclerosis. ApoE also has documented immunomodulatory functions. We investigated gene expression in circulating monocytes and in bone marrows of patients with visceral leishmaniasis (VL) living in an endemic area in Bihar, India, and contrasted these with control healthy subjects or other diagnostic bone marrows from individuals in the same region. Samples from VL patients were obtained prior to initiating treatment. Our study revealed significant upregulated expression of the apoE transcript in patients with VL. Furthermore, the levels of ApoE protein were elevated in serum samples of subjects with VL compared with healthy endemic controls. These observations may provide clues regarding the complex interactions between lipid metabolism and immunoregulation of infectious and inflammatory diseases.

Phenotypic and functional characteristics of monocyte subsets in the blood and bone marrow of Indian subjects with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a potentially fatal parasitic infection caused by Leishmania donovani in India. L. donovani is an obligate intracellular protozoan residing mostly in macrophages of the reticuloendothelial system throughout chronic infection. Monocytic phagocytes are critical in the pathogenesis of different forms of leishmaniasis. Subsets of monocytes are distinguished by their surface markers into CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes, and CD16++CD14low non-classical monocyte subsets. During cutaneous leishmaniasis (CL), intermediate monocyte are reported to be a source of inflammatory cytokines IL-1ß and TNF, and they express CCR2 attracting them to sites of inflammatory pathology. We examined monocyte subsets in the blood and bone marrow of patients with VL from an endemic site in Bihar, India, and found these contrasted with the roles of monocytes in CL. During VL, intermediate and non-classical CD16+ monocyte subsets expressed instead a non-inflammatory phenotype with low CCR2, high CX3CR1 and low microbicidal oxidant generation, making them more similar to patrolling monocytes than inflammatory cells. Bone marrow CD16+ monocyte subsets expressed a phenotype that might be more similar to the inflammatory subsets of CL, although our inability to obtain bone marrow from healthy donors in the endemic region hampered this interpretation Overall the data suggest that CD16+ intermediate monocyte subsets in VL patients express a phenotypes that contributes to an immunosuppressed pathologic immune state, but in contrast to CL, these do not mediate localized inflammatory responses.

Altered IL-7 signaling in CD4+ T cells from patients with visceral leishmaniasis.

BACKGROUND: CD4+ T cells play a central role in control of L. donovani infection, through IFN-γ production required for activation of macrophages and killing of intracellular parasites. Impaired control of parasites can in part be explained by hampered CD4+ T cells effector functions in visceral leishmaniasis (VL) patients. In a recent studies that defined transcriptional signatures for CD4+ T cells from active VL patients, we found that expression of the IL-7 receptor alpha chain (IL-7Rα; CD127) was downregulated, compared to CD4+ T cells from endemic controls (ECs). Since IL-7 signaling is critical for the survival and homeostatic maintenance of CD4+ T cells, we investigated this signaling pathway in VL patients, relative to ECs. METHODS: CD4+ T cells were enriched from peripheral blood collected from VL patients and EC subjects and expression of IL7 and IL7RA mRNA was measured by real time qPCR. IL-7 signaling potential and surface expression of CD127 and CD132 on CD4+ T cell was analyzed by multicolor flow cytometry. Plasma levels of soluble IL-7 and sIL-7Rα were measured by ELISA. RESULT: Transcriptional profiling data sets generated previously from our group showed lower IL7RA mRNA expression in VL CD4+ T cells as compared to EC. A significant reduction was, however not seen when assessing IL7RA mRNA by RT-qPCR. Yet, the levels of soluble IL-7Rα (sIL-7Rα) were reduced in plasma of VL patients compared to ECs. Furthermore, the levels of soluble IL-7 were higher in plasma from VL patients compared to ECs. Interestingly, expression of the IL-7Rα protein was higher on VL patient CD4+ T cells as compared to EC, with activated CD38+ CD4+ T cells showing higher surface expression of IL-7Rα compared to CD38- CD4+ T cells in VL patients. CD4+ T cells from VL patients had higher signaling potential baseline and after stimulation with recombinant human IL-7 (rhIL-7) compared to EC, as measured by phosphorylation of STAT5 (pSTAT5). Interestingly, it was the CD38 negative cells that had the highest level of pSTAT5 in VL patient CD4+ T cells after IL-7 stimulation. Thus, despite unaltered or potentially lowered IL7RA mRNA expression by CD4+ T cells from VL patients, the surface expression of the IL-7Rα was higher compared to EC and increased pSTAT5 was seen following exposure to rhIL-7. Accordingly, IL-7 signaling appears to be functional and even enhanced in VL CD4+ T cells and cannot explain the impaired effector function of VL CD4+ T cells. The enhanced plasma IL-7 may serve as part of homeostatic feedback mechanism regulating IL7RA expression in CD4+ T cells.

Single cell transcriptomics shows that malaria promotes unique regulatory responses across multiple immune cell subsets.

Plasmodium falciparum malaria drives immunoregulatory responses across multiple cell subsets, which protects from immunopathogenesis, but also hampers the development of effective anti-parasitic immunity. Understanding malaria induced tolerogenic responses in specific cell subsets may inform development of strategies to boost protective immunity during drug treatment and vaccination. Here, we analyse the immune landscape with single cell RNA sequencing during P. falciparum malaria. We identify cell type specific responses in sub-clustered major immune cell types. Malaria is associated with an increase in immunosuppressive monocytes, alongside NK and γδ T cells which up-regulate tolerogenic markers. IL-10-producing Tr1 CD4 T cells and IL-10-producing regulatory B cells are also induced. Type I interferon responses are identified across all cell types, suggesting Type I interferon signalling may be linked to induction of immunoregulatory networks during malaria. These findings provide insights into cell-specific and shared immunoregulatory changes during malaria and provide a data resource for further analysis.

A molecular signature for IL-10-producing Th1 cells in protozoan parasitic diseases.

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.

STING activation promotes autologous type I interferon-dependent development of type 1 regulatory T cells during malaria.

The development of highly effective malaria vaccines and improvement of drug-treatment protocols to boost antiparasitic immunity are critical for malaria elimination. However, the rapid establishment of parasite-specific immune regulatory networks following exposure to malaria parasites hampers these efforts. Here, we identified stimulator of interferon genes (STING) as a critical mediator of type I interferon production by CD4+ T cells during blood-stage Plasmodium falciparum infection. The activation of STING in CD4+ T cells by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) stimulated IFNB gene transcription, which promoted development of IL-10- and IFN-γ-coproducing CD4+ T (type I regulatory [Tr1]) cells. The critical role for type I IFN signaling for Tr1 cell development was confirmed in vivo using a preclinical malaria model. CD4+ T cell sensitivity to STING phosphorylation was increased in healthy volunteers following P. falciparum infection, particularly in Tr1 cells. These findings identified STING expressed by CD4+ T cells as an important mediator of type I IFN production and Tr1 cell development and activation during malaria.

Post kala-azar dermal leishmaniasis in the Indian sub-continent: challenges and strategies for elimination.

Visceral leishmaniasis (VL) is a severe and often fatal form of leishmaniasis caused by Leishmania donovani in the Indian sub-continent. Post Kala-azar Dermal Leishmaniasis (PKDL) is a late cutaneous manifestation of VL, typically occurring after apparent cure of VL, but sometimes even without a prior history of VL in India. PKDL serves as a significant yet neglected reservoir of infection and plays a crucial role in the transmission of the disease, posing a serious threat to the VL elimination program in the Indian sub-continent. Therefore, the eradication of PKDL should be a priority within the current VL elimination program aimed at achieving a goal of less than 1 case per 10,000 in the population at the district or sub-district levels of VL endemic areas. To accomplish this, a comprehensive understanding of the pathogenesis of PKDL is essential, as well as developing strategies for disease management. This review provides an overview of the current status of diagnosis and treatment options for PKDL, highlighting our current knowledge of the immune responses underlying disease development and progression. Additionally, the review discusses the impact of PKDL on elimination programs and propose strategies to overcome this challenge and achieve the goal of elimination. By addressing the diagnostic and therapeutic gaps, optimizing surveillance and control measures, and implementing effective intervention strategies, it is possible to mitigate the burden of PKDL and facilitate the successful elimination of VL in the Indian sub-continent.

IL-10-producing Th1 cells possess a distinct molecular signature in malaria

Control of intracellular parasites responsible for malaria requires host IFN-γ+T-bet+CD4+ T cells (Th1 cells) with IL-10 produced by Th1 cells to mitigate the pathology induced by this inflammatory response. However, these IL-10-producing Th1 (induced type I regulatory [Tr1]) cells can also promote parasite persistence or impair immunity to reinfection or vaccination. Here, we identified molecular and phenotypic signatures that distinguished IL-10-Th1 cells from IL-10+Tr1 cells in Plasmodium falciparum-infected people who participated in controlled human malaria infection studies, as well as C57BL/6 mice with experimental malaria caused by P. berghei ANKA. We also identified a conserved Tr1 cell molecular signature shared between patients with malaria, dengue, and graft-versus-host disease. Genetic manipulation of primary human CD4+ T cells showed that the transcription factor cMAF played an important role in the induction of IL-10, while BLIMP-1 promoted the development of human CD4+ T cells expressing multiple coinhibitory receptors. We also describe heterogeneity of Tr1 cell coinhibitory receptor expression that has implications for targeting these molecules for clinical advantage during infection. Overall, this work provides insights into CD4+ T cell development during malaria that offer opportunities for creation of strategies to modulate CD4+ T cell functions and improve antiparasitic immunity.

Novel antiinflammatory biologics shaped by parasite-host coevolution.

Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host's immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm's excretory-secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen in vivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude in vitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases.

Increased amphiregulin expression by CD4+ T cells from individuals with asymptomatic Leishmania donovani infection.

Objectives: There is an urgent need to be able to identify individuals with asymptomatic Leishmania donovani infection, so their risk of progressing to VL and transmitting parasites can be managed. This study examined transcriptional markers expressed by CD4+ T cells that could distinguish asymptomatic individuals from endemic controls and visceral leishmaniasis (VL) patients. Methods: CD4+ T cells were isolated from individuals with asymptomatic L. donovani infection, endemic controls and VL patients. RNA was extracted and RNAseq employed to identify differentially expressed genes. The expression of one gene and its protein product during asymptomatic infection were evaluated. Results: Amphiregulin (AREG) was identified as a distinguishing gene product in CD4+ T cells from individuals with asymptomatic L. donovani infection, compared to VL patients and healthy endemic control individuals. AREG levels in plasma and antigen-stimulated whole-blood assay cell culture supernatants were significantly elevated in asymptomatic individuals, compared to endemic controls and VL patients. Regulatory T (Treg) cells were identified as an important source of AREG amongst CD4+ T-cell subsets in asymptomatic individuals. Conclusion: Increased Treg cell AREG expression was identified in individuals with asymptomatic L. donovani infection, suggesting the presence of an ongoing inflammatory response in these individuals required for controlling infection and that AREG may play an important role in preventing inflammation-induced tissue damage and subsequent disease in asymptomatic individuals.

NKG7 Is Required for Optimal Antitumor T-cell Immunity.

Tumor antigen-specific CD8+ T cells play a critical role in antitumor immunity. Clinical trials reinvigorating the immune system via immune checkpoint blockade (ICB) have shown remarkable clinical promise. Numerous studies have identified an association between NKG7 expression and patient outcome across different malignancies. However, aside from these correlative observations, very little is known about NKG7 and its role in antitumor immunity. Herein, we utilized single-cell RNA sequencing (scRNA-seq) datasets, NKG7-deficient mice, NKG7-reporter mice, and mouse tumor models to investigate the role of NKG7 in neoantigen-mediated tumor rejection and ICB immunotherapy. scRNA-seq of tumors from patients with metastatic melanoma or head and neck squamous cell carcinoma revealed that NKG7 expression is highly associated with cytotoxicity and specifically expressed by CD8+ T cells and natural killer (NK) cells. Furthermore, we identified a key role for NKG7 in controlling intratumor T-cell accumulation and activation. NKG7 was upregulated on intratumor antigen-specific CD8+ T cells and NK cells and required for the accumulation of T cells in the tumor microenvironment. Accordingly, neoantigen-expressing mouse tumors grew faster in Nkg7-deficient mice. Strikingly, efficacy of single or combination ICB was significantly reduced in Nkg7-deficient mice.See related article by Wen et al., p. 162.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Coadministered Ruxolitinib and Artemether-Lumefantrine in Healthy Adults.

Despite repeated malaria infection, individuals living in areas where malaria is endemic remain vulnerable to reinfection. The Janus kinase (JAK1/2) inhibitor ruxolitinib could potentially disrupt the parasite-induced dysfunctional immune response when administered with antimalarial therapy. This randomized, single-blind, placebo-controlled, single-center phase 1 trial investigated the safety, tolerability, and pharmacokinetic and pharmacodynamic profile of ruxolitinib and the approved antimalarial artemether-lumefantrine in combination. Ruxolitinib pharmacodynamics were assessed by inhibition of phosphorylation of signal transducer and activator of transcription 3 (pSTAT3). Eight healthy male and female participants ages 18 to 55 years were randomized to either ruxolitinib (20 mg) (n = 6) or placebo (n = 2) administered 2 h after artemether-lumefantrine (80/480 mg) twice daily for 3 days. Mild adverse events occurred in six participants (four ruxolitinib; two placebo). The combination of artemether-lumefantrine and ruxolitinib was well tolerated, with adverse events and pharmacokinetics consistent with the known profiles of both drugs. The incidence of adverse events and artemether, dihydroartemisinin (the major active metabolite of artemether), and lumefantrine exposure were not affected by ruxolitinib coadministration. Ruxolitinib coadministration resulted in a 3-fold-greater pSTAT3 inhibition compared to placebo (geometric mean ratio = 3.01 [90% confidence interval = 2.14 to 4.24]), with a direct and predictable relationship between ruxolitinib plasma concentrations and %pSTAT3 inhibition. This study supports the investigation of the combination of artemether-lumefantrine and ruxolitinib in healthy volunteers infected with Plasmodium falciparum malaria. (This study has been registered at ClinicalTrials.gov under registration no. NCT04456634.).

Amphiregulin in cellular physiology, health, and disease: Potential use as a biomarker and therapeutic target.

Amphiregulin (AREG), which acts as one of the ligands for epidermal receptor growth factor receptor (EGFR), plays a crucial role in tissue repair, inflammation, and immunity. AREG is synthesized as membrane-anchored pre-protein, and is excreted after proteolytic cleavage, and serves as an autocrine or paracrine factor. After engagement with the EGFR, AREG triggers a cascade of signaling events required for many cellular physiological processes including metabolism, cell cycle, and proliferation. Under different inflammatory and pathogenic conditions, AREG is expressed by various activated immune cells that orchestrate both tolerance and host resistance mechanisms. Several factors including xenobiotics, cytokines, and inflammatory lipids have been shown to trigger AREG gene expression and release. In this review, we discuss the structure, function, and regulation of AREG, its role in tissue repair, inflammation, and homeostasis as well as the potential of AREG as a biomarker and therapeutic target.

Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine.

BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).

Vaccination of human participants with attenuated Necator americanus hookworm larvae and human challenge in Australia: a dose-finding study and randomised, placebo-controlled, phase 1 trial.

BACKGROUND: Control of human hookworm infection would be greatly aided by the development of an effective vaccine. We aimed to develop a live attenuated human hookworm vaccine. METHODS: This was a two-part clinical trial done at Q-Pharm in Brisbane (QLD, Australia) using a live ultraviolet C (UVC)-attenuated Necator americanus larvae vaccine. Part one was an open-label, dose-finding study using 50 L3 larvae suspended in water to a volume of 200 µL, attenuated with UVC exposure of 700 µJ (L3-700) or 1000 µJ (L3-1000). Part two was a randomised, double-blind, placebo-controlled, challenge study, in which participants were randomly assigned 2:1 to the vaccine group or placebo group. Healthy hookworm-naive adults aged 18-65 years with body-mass index 18-35 kg/m2 received two doses of either placebo (Tabasco sauce) or vaccine (50 L3-700) on day 1 and day 42, followed by challenge with 30 unattenuated L3 larvae to both groups. All participants received a single oral dose of 400 mg albendazole 4 weeks after each inoculation and a 3-day course (400 mg orally daily) initiated on day 161 after the challenge phase, to eliminate any remaining infection. The primary outcome of part 1 was the level of larval attenuation the resulted in a grade 2 or 3 dermal adverse event. The primary outcome of part 2 was safety and tolerability, assessed by frequency and severity of adverse events in all randomly assigned participants. Prespecified exploratory outcomes in the challenge study were faecal N americanus DNA concentration, the number of N americanus larvae recovered per g of faeces cultured, hookworm antigen-specific serum IgG antibody responses, and hookworm antigen-specific peripheral blood cytokine responses. The trial is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12617001007325). FINDINGS: Between Sept 19, 2017, and Oct 24, 2018, seven participants were enrolled into three cohorts in part one (two participants in cohort 1, who received L3-700; two participants in cohort 2, who received L3-700; and three participants in cohort 3, who received L3-1000) and a further 15 were enrolled into part two. There were no serious adverse events in part one or part two. In part one, a greater number of skin penetration sites were observed after administration of L3-700 than L3-1000 (mean 15·75 [95% CI 11·18 to 20·32] with L3-700 vs 4·33 [-1·40 to 10·07] with L3-1000). Similarly, greater erythema (median 225 mm2 [IQR 150 to 325] vs 25 mm2 [12·5 to 80]) and a longer duration of the dermal reaction (median 8·0 days [IQR 3·5 to 11·5] vs 2·0 days [2·0 to 4·5]) were observed after L3-700 than L3-1000. The mean number of adverse events per participant did not differ between the groups (3·25 [95% CI 1·48 to 5·02] vs 3·00 [1·04 to 4·96]). Thus, L3-700 was used for vaccination in part two. In part two, ten participants were randomly assigned to receive L3-700 and five to placebo. Significantly more adverse events occurred after vaccination with attenuated larvae than with placebo (incident rate ratio [IRR] 2·13 [95% CI 2·09 to 5·51]; p=0·0030). There was no difference between groups in the frequency of adverse events after challenge (IRR 1·25 [0·78 to 2·01]; p=0·36). Most adverse events were mild in severity, with only one severe adverse event reported (erythematous and indurated pruritic rash >100 mm in a vaccine group participant after challenge). The eosinophil count increased in all participants after challenge, with a significantly greater increase among vaccinated participants than placebo participants (1·55 × 109 cells per L [IQR 0·92 to 1·81] in the vaccine group vs 0·49 × 109 cells per L [0·43 to 0·63] in the placebo group; p=0·014). Vaccinated participants had an IgG response to larval extract after challenge that was higher than that in placebo participants (increase in IgG titre 0·22 [IQR 0·10 to 0·41] vs 0·03 [-0·40 to 0·06]; p=0·020). Significantly fewer larvae per g of faeces were recovered in the vaccine group than in the placebo group after challenge (median larvae per g 0·8 [IQR 0·00 to 3·91] vs 10·2 [5·1 to 18·1]; p=0·014). The concentration of N americanus DNA in faeces was not significantly different between the vaccinated group and the placebo group (log10 DNA intensity 4·28 [95% CI 3·92 to 4·63] vs 4·88 [4·31 to 5·46]; p=0·14). Peripheral blood mononuclear cells from vaccinated participants exhibited significantly greater cytokine production at day 112 than placebo participants for IFNγ, TNFα, IL-2, IL-4, and IL-5 (p<0·05), but not IL-10. INTERPRETATION: Vaccination with UVC-attenuated N americanus larvae is well tolerated, induces humoral and cellular responses to hookworm antigens, and reduces larval output after challenge with unattenuated larvae. Larger studies are required to confirm protective efficacy. FUNDING: National Health and Medical Research Council of Australia.