The ability of the human malaria parasite Plasmodium falciparum to remodel its host red blood cell (RBC) is central to the parasite’s ability to survive within the circulation of its host and cause disease. Following invasion, the parasite exports proteins into the RBC cytoplasm where they remodel the membrane skeleton and alter the cell’s biophysical properties. Many of the exported proteins are trafficked through parasite derived structures called Maurer’s clefts in the cytoplasm of the RBC. These clefts act as an intermediate compartment for proteins en route to the RBC membrane such as the major virulence protein, Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). From the Maurer’s clefts, PfEMP1 is delivered to the RBC membrane through a poorly understood process, which may involve trafficking via a physical connection and/or vesicle mediated trafficking. Electron-tomography of 3D7 infected RBCs reveals a tube-like structure, called the tether, that connects the Maurer’s clefts to the RBC membrane. To date, the formation and function of these tether structures is unclear, with only one protein, the Membrane Associated Histidine Rich Protein 2 (MAHRP2) known to locate at these structures. In this study, we show that deletion of MAHRP2 leads to a loss of tethers, reduced Maurer’s cleft immobilization at the RBC membrane and an increase in Maurer’s cleft size. Lastly, we show that deletion of MAHRP2 impacts PTP2 related vesicle formation, and a reduced ability to cytoadhere to the endothelial ligands CD36.

Type I interferon (IFN) signalling promotes development of type 1 regulatory (Tr1) CD4+ T cells, which suppress inflammation but may limit protective immunity in malaria by constraining effector Th1 cells and antibody-promoting T follicular helper (Tfh) cells. We tested whether transiently blocking IFN-driven JAK/STAT signalling with ruxolitinib would reduce Tr1 cell development and enhance protective Th1 and Tfh cell responses. Using controlled human malaria infection, we investigated the impact of ruxolitinib on CD4+ T cell activation and antigen-specific immunity. Whole blood stimulation with IFNβ, IL-2, or PMA/Ionomycin revealed that ruxolitinib suppressed pSTAT1, pSTAT3, and pSTAT5 in CD4+ T cells following treatment. However, antigen-specific CD4+ T cell responses were preserved. Further, ruxolitinib enhanced recall CD4+ T cell responses during a second infection, with increased frequencies of antigen-specific Th1, Tfh, and Tr1 cell subsets. Transcriptional analysis revealed overlapping gene signatures and clonal sharing between Th1, Tfh and Tr1 cells. These findings show transient JAK inhibition can modulate development of Th1, Tfh and Tr1 cell subsets in malaria, suppressing inhibitory signalling while enhancing the magnitude and durability of the CD4+ T cell recall response following a second infection. Targeting IFN-driven Tr1 cell activity therefore represents a promising host-directed strategy to improve malaria immunity.

Hookworms infect nearly half a billion people, causing approximately 2.1 million disability-adjusted life-years lost. While anthelmintic drugs are available, they do not prevent reinfection. A vaccine could provide long-term immunity, but none currently exists. We recently demonstrated that human subjects could be safely vaccinated with irradiated hookworm larvae, eliciting humoral and cellular immune responses that induce partial protection against challenge infection with non-irradiated parasites. Using immunomic approaches, we identified IgG targets in vaccinated individuals by screening a proteome microarray containing recombinant proteins sourced from the secretome of Necator americanus. We characterized the most immunoreactive N. americanus proteins and identified their orthologs in the rodent hookworm Nippostrongylus brasiliensis. These N. brasiliensis proteins were expressed recombinantly and tested for efficacy in a mouse challenge model. Our lead vaccine candidate NBR17057 - a homolog of Na_11335 provided robust protection against infection, evidenced by >90% reductions in intestinal worm burden and fecal egg counts, and elevated antigen-specific IgG levels. Vaccination also stimulated IgA+ B-cells, IgG1+/IgA+ plasma cells, and IgG1+/IgA+ germinal centre B-cells in inguinal lymph nodes, indicating gut immunity. Additionally, high levels of IgG1+/IgA+ B-cells, germinal centre B-cells, and plasma cells in the spleen suggest protection against systemic larval migration. The presence of IgG1+/IgA+ memory B-cells in both the lymph nodes and spleen indicates potential long-term immunity against reinfection. Our findings suggest that a N. brasiliensis L3 larval-stage protein NBR17057 and its homolog in N. americanus Na_11335 could serve as a promising subunit vaccine candidate targeting the infective larval stage of human hookworms.

BACKGROUND:Understanding the influence of gametocyte dynamics on transmission of Plasmodium falciparum from humans to mosquitoes may support malaria elimination initiatives. METHODS:Longitudinal parasitemia, gametocytemia and mosquito transmission data from 91 participants in 9 controlled human malaria infection studies conducted in Brisbane, Australia (n=8) and Nijmegen, Netherlands (n=1) were analysed to explore the relationship of parasite and participant characteristics on gametocytes and transmission. Transmission potential estimated using Poisson probabilities was compared to transmission to Anopheles stephensi mosquitoes assessed using direct and membrane feeding assays across 5 clinical trials and 30 participants. RESULTS:Maximum gametocyte density overtime per participant was positively correlated with asexual parasitemia at time of first treatment (ρ minimum=0.44, maximum=0.89). Median maximum gametocyte densities occurring 19-21 days post inoculation were 1087 gametocytes/mL for female and 219 gametocytes/mL for male gametocytes. Both female and male gametocyte densities were higher among individuals who infected mosquitoes, as were pre-treatment parasite growth rates and parasite densities. Infectivity rates varied between 53-54% for direct skin, 29-33% for direct membrane, 54-100% for membrane after serum replacement and 89-100% for enriched membrane feeding assays. Potential for transmission was correctly predicted for 71-74% of direct and 50-78% of membrane after serum replacement feeding assays. CONCLUSIONS:This study confirms that transmissible gametocyte densities can be consistently achieved in controlled infections. Higher asexual densities and growth rates were associated with higher gametocyte densities and higher transmission. Detailed understanding of how low level gametocytemia contributes to transmission will guide both experimental infection studies and interventions to interrupt transmission.

JAK/STAT signalling is a key regulator of CD4+ T cell activation and function, influencing immune responses during infection. Ruxolitinib, a JAK1/2 inhibitor, transiently suppresses JAK/STAT signalling, but its impact on antigen-specific CD4+ T cell responses in malaria is unknown. In this study, we assessed the effects of ruxolitinib on CD4+ T cell cytokine signalling and immune responses using a controlled human malaria infection model. Whole blood was stimulated with IFNβ, IL-2, or PMA/Ionomycin, and phosphorylated STAT (pSTAT) expression was analysed. AIM assays and CyTOF were used to examine antigen-specific and memory CD4+ T cell responses during primary and secondary infections. Ruxolitinib significantly reduced pSTAT1, pSTAT3, and pSTAT5 expression in CD4+ T cells during treatment but did not impair antigen-specific responses. Instead, it enhanced recall immunity during secondary infection, particularly in AIM+ T follicular helper (Tfh), T helper (Th)1, and type 1 regulatory (Tr1) cell subsets. Furthermore, increased frequencies of circulating Th1 and Tfh cells, but not Tr1 cells correlated with improved parasite control. These findings suggest that transient JAK inhibition modulates immune responses in a manner that could be leveraged as a host-directed therapy for malaria. Further research is needed to explore its potential applications in immune modulation for infectious diseases. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial ACTRN12621000866808 ### Funding Statement This study was funded by the Australian National Health and Medical Research Council, with support from Medicines for Malaria and the Bill and Melinda Gates Foundation. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The work described in this manuscript was approved by the QIMR Berghofer Clinical Trial Protocol Committee (CTPC), and ratified by the QIMR Berghofer Human Research Ethics Committee (HREC). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

Introduction Gonorrhoea is a sexually transmissable infection caused by Neisseria gonorrhoeae that causes a significant global burden of disease. Urogenital infection can result in long-term impacts on reproductive, perinatal, and neonatal health. Little progress has been made in the public health control of gonorrhoea, and novel preventative strategies are urgently needed. Furthermore, future gonorrhoea management is threatened by increasing antimicrobial resistance (AMR). Oropharyngeal gonorrhoea is usually asymptomatic; likely plays an important role in development of AMR; and is a high-risk site for treatment failure. Here, we describe a protocol for an oropharyngeal gonorrhoea controlled human infection model (CHIM) that has been designed to maximize participant safety, with the aim of developing this as a platform to accelerate prevention and treatment strategies. Methods and analysis This dose-escalation CHIM study will enrol 20-35 healthy adult volunteers aged 18 to 50 years who were assigned male at birth and only have sex with people assigned male at birth. The primary objectives are to determine i) the safety and tolerability of an oropharyngeal gonorrhoea CHIM; and, ii) the minimum infectious dose of isolate AUSMDU00053933 required for 60–80% of participants to develop oropharyngeal N. gonorrhoeae infection. Secondary and exploratory endpoints include description of clinical, immunological, microbiological and pharmacometric responses. Participants will be monitored daily as outpatients during the five-day experimental infection phase. All participants will be treated with antibiotics, and followed up for three months. Statistical analysis and dose escalation/de-escalation decisions will follow a model-based continual reassessment method in a Bayesian statistical framework. Ethics and dissemination After scientific peer review of this provisional protocol, a detailed protocol will be submitted for human research ethics committee assessment. Protocol development was informed by feedback from community engagement. Study findings will be disseminated in peer-reviewed journals and at scientific meetings, with summaries provided to relevant stakeholders.

BACKGROUND:The in-vivo plasma concentration of penicillin needed to prevent Streptococcus pyogenes pharyngitis, recurrent acute rheumatic fever, and progressive rheumatic heart disease is not known. We used a human challenge model to assess the minimum penicillin concentration required to prevent streptococcal pharyngitis. METHODS:In CHIPS, a randomised, double-blind, placebo-controlled, human challenge trial, healthy adult volunteers were randomly assigned by a computer-generated random sequence to target steady-state penicillin plasma concentrations (placebo, 3, 6, 9, 12, or 20 ng/mL). The study was a single-centre trial held in Perth, WA, Australia. Participants had to be healthy adults, aged 18-40 years, at low risk of complicated S pyogenes disease, and without high type-specific IgG antibodies against the emm75 S pyogenes challenge strain. Participants and staff involved in clinical care remained masked to treatment allocation for the duration of the study. Individualised 5-day continuous intravenous infusions of penicillin were commenced 12 h before direct pharyngeal application of the emm75 challenge strain. The primary endpoint was clinical pharyngitis. This trial is registered on the Australian New Zealand Clinical Trials Registry, ACTRN12621000751875, and is completed. FINDINGS:Between Aug 23, 2022, and July 31, 2023, 60 participants were randomly assigned (35 [58%] were female and 25 [42%] were male), with 57 included in the analysis. The clinical pharyngitis endpoint was met in eight (57%) of 14 in the placebo group, four (44%) of nine in the 3 ng/mL target steady-state penicillin plasma concentration group, four (44%) of nine in the 6 ng/mL group, none of eight in the 9 ng/mL group, none of eight in the 12 ng/mL group, and none of nine in the 20 ng/mL group. No severe or serious adverse events occurred. Using Bayesian concentration-response modelling, the minimum steady-state plasma concentration of penicillin for which 90% of participants would avoid clinical pharyngitis was 8·1 ng/mL (95% credible interval 6·1-10·9). INTERPRETATION:When steady-state penicillin concentrations are greater than 9 ng/mL, few people will develop experimental emm75 S pyogenes pharyngitis. These data will inform efforts to improve long-acting penicillin preparations and dosage regimens to prevent recurrent rheumatic fever and rheumatic heart disease. FUNDING:The National Health and Medical Research Council of Australia.

Background Neisseria gonorrhoeae is a human pathogen of major public health importance due to its increasing global prevalence and antimicrobial resistance (AMR). Evidence suggests that oropharyngeal infection plays a key role in N gonorrhoeae transmission and AMR; however, our understanding of oropharyngeal gonorrhoea pathogenesis is poor. A controlled human infection model (CHIM) for oropharyngeal gonorrhoea will improve understanding of infection and accelerate urgently needed novel gonorrhoea prevention and therapeutic strategies. As the first step in the development of this CHIM, we describe a systematic approach to CHIM strain selection that leverages genomics and clinical data. Methods In this genomics-based analysis, we applied a systematic N gonorrhoeae challenge strain selection strategy incorporating genomic and clinical data to a primary dataset of clinical isolates of N gonorrhoeae collected from adult patients in Victoria, Australia, between Jan 1 and Dec 31, 2017, and July 1, 2019, and June 30, 2021. This selection strategy used clinical, phenotypic, and genomic characteristics to define a set of eight criteria that aimed to ensure the contemporary global clinical relevance of the candidate strains; select strains that would be applicable for the assessment of current and future gonorrhoea vaccines; and maximise participant safety by reducing the risk of disseminated gonococcal infection and clinically significant AMR. We applied these criteria to our primary dataset to generate a panel of potential challenge strains. From this final dataset of potential challenge strains, we predetermined that we would select up to ten isolates to proceed to the next stage of detailed phenotypic characterisation for final N gonorrhoeae CHIM strain selection. Findings 5881 isolates comprised the primary dataset. After application of the selection criteria, most of the isolates (5795 [98·6%] of 5881) were excluded, mostly due to having clinically significant AMR and poor contemporary global clinical relevance. The remaining 86 N gonorrhoeae challenge strain candidates comprised five multilocus sequence types and six N gonorrhoeae multiantigen sequence types, many of which were represented by a single isolate. Of these 86 strains, five isolates were selected to maximise coverage of the phylogenetically distinct groups within the 86 candidate challenge strains and ensure representation of strains collected from various anatomical sites. Interpretation We transparently describe a novel, systematic, and rational genomics-based strategy for oropharyngeal gonorrhoea CHIM strain selection that improves the efficiency and transparency of CHIM strain selection and enables identification of contemporary and clinically relevant potential challenge strains. A final N gonorrhoeae challenge strain will be selected from the subset of five shortlisted candidates after detailed phenotypic assessment. Funding Medical Research Future Fund, Australian National Health and Medical Research Council and Australian Government Research Training Program.

Infection with Neisseria gonorrhoeae, the causative agent of gonorrhea, causes significant morbidity worldwide and can have long-term impacts on reproductive health. The greatest global burden of gonorrhea occurs in low- and middle-income settings. Global public health significance is increasing due to rising antimicrobial resistance, which threatens future gonorrhea management. The oropharynx is an important asymptomatic reservoir for gonorrhea transmission and a high-risk site for development of antimicrobial resistance and treatment failure. Controlled human infection model (CHIM) studies using N gonorrhoeae may provide a means to accelerate the development of urgently needed therapeutics, vaccines, and other biomedical prevention strategies. A gonorrhea urethritis CHIM has been used since the 1980s with no reported serious adverse events. Here, we describe the rationale for an oropharyngeal gonorrhea CHIM, including analysis of potential ethical issues that should inform the development of this novel study design.

Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of NF-ƙB ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL), is increasingly recognized as a marker of poor prognosis in cardiovascular disease. Here, we demonstrate that in adults with falciparum and vivax malaria, OPG is increased, and its ligands TRAIL and RANKL decreased, in association with validated markers of disease severity. Using longitudinal samples from malaria volunteer infection studies, we demonstrate that TRAIL is unexpectedly increased in early infection, suggesting binding of OPG to RANKL prior to TRAIL. Finally, in addition to its known vascular origin, we show that P. falciparum stimulates B cells to produce OPG in vitro, and that B cell OPG production is increased ex vivo in patients with falciparum, vivax, and knowlesi malaria. Our findings provide evidence of the importance of the OPG/RANKL/TRAIL pathway in pathogenesis of acute systemic inflammatory and microvascular diseases, with implications for adjunctive therapies.

Although benzylpenicillin (penicillin G) is listed by the World Health Organization as an Essential Medicine, dose optimization is a persistent challenge, especially for long-acting intramuscular formulations. Maintaining sustained antibiotic exposure at target concentrations is crucial for secondary chemoprophylaxis of rheumatic heart disease and treatment of syphilis. This study compared the pharmacokinetic profile of continuous low-dose benzylpenicillin infusions with a standard-dose bolus and evaluated which renal function marker (serum creatinine, cystatin C, or combined e-glomerular filtration rate [eGFR]) best predicted clearance. Healthy adult volunteers received a single 600 mg IV benzylpenicillin bolus followed by randomization to continuous infusions targeting steady-state concentrations of 3, 6, 9, 12, or 20 ng/mL. Plasma benzylpenicillin concentrations were measured by liquid chromatography-mass spectrometry. Population pharmacokinetic analysis was performed using NONMEM by incorporating both bolus and infusion data, and various GFR estimations were evaluated as covariates for clearance. Data from 72 participants were analyzed, including 504 bolus and 389 continuous infusion samples. A two-compartment model improved fit when the ratio of central volume of distribution between bolus and low-dose infusion was incorporated, and clearance differences at steady state plasma concentration of 3 ng/mL were accounted for. Of the GFR estimations, cystatin C-based eGFR significantly enhanced model fit compared with creatinine-based equations. Benzylpenicillin pharmacokinetics at very low concentrations demonstrated both a higher volume of distribution and increased clearance. Cystatin C-based eGFR may more accurately predict benzylpenicillin clearance, enabling precision dosing for long-acting preparations used for treatment of syphilis and prevention of rheumatic fever.

BackgroundInfection with Mycobacterium ulcerans causes slowly progressive skin lesions known as Buruli ulcer (BU). An M. ulcerans controlled human infection model (MuCHIM) is likely to accelerate our understanding of this otherwise neglected disease, and may be an efficient platform for testing vaccines and other interventions. The aim of this study was to understand perceptions of this model across a range of key stakeholders in an endemic Australian community setting.MethodsWe recruited young adults who live near an Australian BU endemic area but without a personal history of BU, clinicians involved in the management of BU, young adults with a personal history of a small, treated BU, and participants of any age with a demonstrated interest in public advocacy related to their personal BU lived experience. Participants reviewed an abridged version of the provisional protocol. A series of three focus groups were then conducted by video, and the transcribed text was analysed using reflexive thematic analysis to generate themes for exploration.ResultsParticipants universally valued the outcomes that MuCHIM might deliver. The predominant theme was that informed consent required fully transparent communication with potential participants regarding what their participation would involve, how it would impact their lives, and both the expected outcome and 'worst-case scenario'. They also offered actionable recommendations on how best to communicate the tension between the expected outcome and the 'worst-case scenario' of disease associated with delayed diagnosis and comorbidity, as typically portrayed by the media. Participants recommended including images and testimonials from people who have had BU to support the conditions for informed consent. Focus groups also gave a clear sense of who they believed would volunteer for this type of research.ConclusionsThis study offers valuable guidance regarding the content and presentation of information to inform potential participants, with focus group participants suggesting a multimodal approach of communication, including lived experience testimonials and clinical images of the expected outcome. This information will inform development of materials for enrolment to adequately communicate risks and expectations to potential study participants.

Antibodies induced by infection or vaccination are essential mediators of protection. Induction of these protective responses is mediated by T-follicular CD4 T (Tfh) cells, and targeting these cells may be a strategy to boost antibody mediate protection. In humans, Tfh cells are analysed based on expression of CXCR3 and CCR6, with different subsets of Tfh (Tfh1, Tfh2, Tfh17) associated with antibody induction in a context-dependent manner. Here we dissected Tfh cells heterogeneity in healthy donors and individuals during controlled human malaria infection using scRNAseq. We identified two distinct Tfh1-like subsets with functional relevance, defined based on CCR7 expression. CCR7 neg Tfh1 cells express markers of cytotoxicity, while CCR7 pos Tfh1 cells produce reduced inflammatory cytokines and similar IL-21 resulting in unique cytokine milieu. In controlled human malaria infection, both CCR7 pos and CCR7 neg Tfh1, along with Tfh2 cells, clonally expanded and were transcriptionally and phenotypically activated. However, only CCR7 pos Tfh1 and Tfh2 cells associated with antibody development, suggesting a role for both these Tfh subsets in promoting humoral immunity to malaria. Data identify specific protective Tfh subsets that can be targeted to improve antibody mediated protection to malaria induced, and provide a frame work to dissect the role of Tfh subsets in other disease contexts.

Inhibiting the host inflammatory response to malaria represents a potential strategy to improve clinical outcomes and inhibit immunoregulatory pathways that underlie suboptimal development of antiparasitic immunity. Ruxolitinib is a JAK 1/2 inhibitor that reduces inflammatory biomarkers when used in myeloproliferative disorders, and inhibits type-1 interferons and enhances CD4+ T cell immunity when combined with anti-parasitic drugs in animal models. Here we report the results of a double-blind randomised placebo-controlled trial evaluating the ability of ruxolitinib to reduce inflammatory responses and boost anti-parasitic immunity in malaria-naive volunteers inoculated with blood-stage Plasmodium falciparum. Twenty participants were inoculated, and randomized on day 8 to receive artemether-lumefantrine with either ruxolitinib or placebo. Ninety days later, participants who remained eligible were re-inoculated with a second infection. Ruxolitinib was safe and well-tolerated, and attenuated the host inflammatory response to the initial infection, with reduced post-treatment increases in the inflammatory biomarker CRP, as well as markers of disease severity including angiopoietin-2 and ICAM-1. Further, ruxolitinib enhanced the immune memory response following a second inoculation, with increased plasma levels of HLA-DR and CXCL13, indicating enhanced immune activation and germinal centre responses, respectively. These data support the further evaluation of ruxolitinib as an adjunctive treatment to improve clinical outcomes and boost anti-parasitic immunity in clinical malaria. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial Australian New Zealand Clinical Trials Registry (ACTRN12621000866808). ### Funding Statement This study was funded by the Australian National Health and Medical Research Council (NHMRC); Ideas Grant to BEB and MJB (GNT2002957). BEB and JCM are supported by NHMRC Investigator Grants (2016792 and 2016396 respectively); MJB is supported by a Snow Medical Fellowship (2022/SF167) and CSL Centenary Fellowship. The QIMR Berghofer Clinical Malaria group receives core funding from Medicines for Malaria Venture (MMV) to support the conduct of malaria volunteer infection studies. Work in the Stanford Human Immune Monitoring Center was supported by the Gates Foundation [INV-008378]. The conclusions and opinions expressed in this work are those of the author(s) alone and shall not be attributed to the Foundation. Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. Please note works submitted as a preprint have not undergone a peer review process. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the QIMR Berghofer Medical Research Institute Human Research Ethics Committee (P3696) and ethically reviewed in minimising duplication of ethics by the Australian Departments of Defence and Veterans Affairs Human Research Ethics Committee. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. NUlisa and phospho CyTOF whole blood data is publicly available: DOI 10.5281/zenodo.15080321 and DOI 10.5281/zenodo.14872946

Latent cytomegalovirus (CMV) infection has widely reported immunomodulatory effects on the immune system, leading to altered responses to vaccination or infection with other pathogens. However, the impact of CMV on the immune response to the malaria parasite Plasmodium falciparum is unknown. Here we assessed antibody and cellular responses in malaria-naive volunteers during Controlled Human Malaria Infection (CHMI) with blood stage P. falciparum parasites. Latent CMV infection was associated with reduced induction of parasite specific IgG, IgG1, and functional antibodies following infection. Within the T-follicular helper (Tfh) cell compartment, CMV was associated with a skew towards Tfh1 cell subsets before and after malaria, and reduced activation of Tfh2 cells which have been previously associated with antibody induction during malaria. Taken together, CMV latent infection impacts the phenotype of Tfh cells required for robust antibody induction to malaria and may have particular importance in malaria endemic countries where CMV infection is almost universal and acquired early in life. ### Competing Interest Statement The authors have declared no competing interest.

Hookworm, a parasitic infection, retains a considerable burden of disease, affecting the most underprivileged segments of the general population in endemic countries and remains one of the leading causes of mild to severe anemia in Low and Middle Income Countries (LMICs), particularly in pregnancy and children under 5. Despite repeated large scale Preventive Chemotherapy (PC) interventions since more than 3 decades, there is broad consensus among scholars that elimination targets set in the newly launched NTD roadmap will require additional tools and interventions. Development of a vaccine could constitute a promising expansion of the existing arsenal against hookworm. Therefore, we have evaluated the biological and implementation feasibility of the vaccine development as well as the added value of such a novel tool. Based on pipeline landscaping and the current knowledge on key biological aspects of the pathogen and its interactions with the host, we found biological feasibility of development of a hookworm vaccine to be moderate. Also, our analysis on manufacturing and regulatory issues as well as potential uptake yielded moderate implementation feasibility. Modelling studies suggest a that introduction of a vaccine in parallel with ongoing integrated interventions (PC, WASH, shoe campaigns), could substantially reduce burden of disease in a cost – saving mode. Finally a set of actions are recommended that might impact positively the likelihood of timely development and introduction of a hookworm vaccine.

Maintaining high-affinity antibodies after vaccination may be important for long-lasting immunity to malaria, but data on induction and kinetics of affinity is lacking. In a phase 1 malaria vaccine trial, antibody affinity increased following a second vaccination but declined substantially over 12 months, suggesting poor maintenance of high-affinity antibodies.Clinical Trials Registration Australian New Zealand Clinical Trials Registry ACTRN12607000552482. Maintaining high-affinity antibodies after vaccination may be important for long-lasting immunity to malaria, but knowledge is lacking. In a phase 1 malaria vaccine trial, affinity of vaccine antibodies declined substantially over 12 months, suggesting poor maintenance of high-affinity antibodies.