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Summary Background WHO recently approved a partially effective vaccine that reduces clinical malaria in children, but increased vaccine activity is required to pursue malaria elimination. A phase 1 clinical trial was done in Mali, west Africa, to assess the safety, immunogenicity, and protective efficacy of a three-dose regimen of Plasmodium falciparum sporozoite (PfSPZ) Vaccine (a metabolically active, non-replicating, whole malaria sporozoite vaccine) against homologous controlled human malaria infection (CHMI) and natural P falciparum infection. Methods We recruited healthy non-pregnant adults aged 18–50 years in Doneguebougou, Mali, and surrounding villages (Banambani, Toubana, Torodo, Sirababougou, Zorokoro) for an open-label, dose-escalation pilot study and, thereafter, a randomised, double-blind, placebo-controlled main trial. Pilot study participants were enrolled on an as-available basis to one group of CHMI infectivity controls and three staggered vaccine groups receiving: one dose of 4·5 × 105, one dose of 9 × 105, or three doses of 1·8 × 106 PfSPZ via direct venous inoculation at approximately 8 week intervals, followed by homologous CHMI 5 weeks later with infectious PfSPZ by direct venous inoculation (PfSPZ Challenge). Main cohort participants were stratified by village and randomly assigned (1:1) to receive three doses of 1·8 × 106 PfSPZ or normal saline at 1, 13, and 19 week intervals using permuted block design by the study statistician. The primary outcome was safety and tolerability of at least one vaccine dose; the secondary outcome was vaccine efficacy against homologous PfSPZ CHMI (pilot study) or against naturally transmitted P falciparum infection (main study) measured by thick blood smear. Combined artesunate and amodiaquine was administered to eliminate pre-existing parasitaemia. Outcomes were analysed by modified intention to treat (mITT; including all participants who received at least one dose of investigational product; safety and vaccine efficacy) and per protocol (vaccine efficacy). This trial is registered with ClinicalTrials.gov , number NCT02627456 . Findings Between Dec 20, 2015, and April 30, 2016, we enrolled 56 participants into the pilot study (five received the 4·5 × 105 dose, five received 9 × 105, 30 received 1·8 × 106, 15 were CHMI controls, and one withdrew before vaccination) and 120 participants into the main study cohort with 60 participants assigned PfSPZ Vaccine and 60 placebo in the main study. Adverse events and laboratory abnormalities post-vaccination in all dosing groups were few, mainly mild, and did not differ significantly between vaccine groups (all p>0·05). Unexpected severe transaminitis occured in four participants: one participant in pilot phase that received 1·8 × 106 PfSPZ Vaccine, one participant in main phase that received 1·8 × 106 PfSPZ Vaccine, and two participants in the main phase placebo group. During PfSPZ CHMI, approximately 5 weeks after the third dose of 1·8 × 106 PfSPZ, none of 29 vaccinees and one of 15 controls became positive on thick blood smear; subsequent post-hoc PCR analysis for submicroscopic blood stage infections detected P falciparum parasites in none of the 29 vaccine recipients and eight of 15 controls during CHMI. In the main trial, 32 (58%) of 55 vaccine recipients and 42 (78%) of 54 controls became positive on thick blood smear during 24-week surveillance after vaccination. Vaccine efficacy (1–hazard ratio) was 0·51 per protocol (95% CI 0·20–0·70; log-rank p=0·0042) and 0·39 by mITT (0·04–0·62; p=0·033); vaccine efficacy (1–risk ratio) was 0·24 per-protocol (0·02–0·41; p=0·031) and 0·22 mITT (0·01–0·39; p=0·041). Interpretation A three-dose regimen of PfSPZ Vaccine was safe, well tolerated, and conferred 51% vaccine efficacy against intense natural P falciparum transmission, similar to 52% vaccine efficacy reported for a five-dose regimen in a previous trial. Funding US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Sanaria. Translation For the French translation of the abstract see Supplementary Materials section.

Malaria transmission-blocking vaccines candidates based on Pfs25 and Pfs230 have advanced to clinical studies. Exoprotein A (EPA) conjugate of Pfs25 in Alhydrogel® developed functional immunity in humans, with limited durability. Pfs230 conjugated to EPA (Pfs230D1-EPA) with liposomal adjuvant AS01 is currently in clinical trials in Mali. Studies with these conjugates revealed that non-human primates are better than mice to recapitulate the human immunogenicity and functional activity. Here, we evaluated the effect of ALFQ, a liposomal adjuvant consisting of TLR4 agonist and QS21, on the immunogenicity of Pfs25-EPA and Pfs230D1-EPA in Rhesus macaques. Both conjugates generated strong antibody responses and functional activity after two vaccinations though activity declined rapidly. A third vaccination of Pfs230D1-EPA induced functional activity lasting at least 9 months. Antibody avidity increased with each vaccination and correlated strongly with functional activity. IgG subclass analysis showed induction of Th1 and Th2 subclass antibody levels that correlated with activity.

Background An estimated 241 million malaria cases and 627,000 malaria deaths occurred worldwide in 2020. Efforts to develop a higher efficacy malaria vaccine are a global priority, but parasites pose a complex biological puzzle for vaccine development. Innovative vaccine strategies include transmission blocking vaccines (TBV) that target the mosquito stages of parasite development to interrupt parasite transmission. The current leading TBV called Pfs230D1-EPA targets Pfs230, a Plasmodium falciparum gamete surface protein that mediates binding of microgametes to red blood cells. Antibodies against Pfs230 bind and lyse gametes in a complement-dependent manner to block transmission. Methods Here, we are exploiting a panel of human monoclonal antibodies (hmAb) generated from human volunteers who received Pfs230D1-EPA formulated in Alhydrogel or AS01 adjuvants, to develop a competitive ELISA platform. This assay will quantify epitope-specific serum antibodies along with their features and effector functions, expanding the conventionally used functional assay of standard membrane feeding (SMFA). Results The competitive ELISA is being developed with single-chain variable fragments (ScFv) to define epitope-specific binding of antibodies in sera from vaccinated individuals. Sera of human volunteers from trials in Mali, Africa will be assayed to assess epitope-specific serum antibody binding that is competed off in the presence of saturating levels of ScFv. Preliminary optimization of the competitive assay has shown concentration-dependent epitope-specific competition between ScFv and hmAbs and confirmation of ScFv binding to Pfs230D1 antigen. Conclusion Further optimization of this assay will characterize the complement-binding and isotype/subclass-specific features of epitope-specific antibodies. These efforts will lay the foundations for standardized assays that can assess correlates of vaccine efficacy and durability of this leading TBV. Disclosures All Authors: No reported disclosures.

Background False positivity may hinder the utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests in sub-Saharan Africa. Methods From 312 Malian samples collected before 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and 4 other betacoronaviruses by enzyme-linked immunosorbent assay (ELISA). In a subset of samples, we assessed antibodies to a panel of Plasmodium falciparum antigens by suspension bead array and functional antiviral activity by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA using SARS-CoV-2 spike protein and receptor-binding domain developed in the United States using Malian positive and negative control samples. To optimize test performance, we compared single- and 2-antigen approaches using existing assay cutoffs and population-specific cutoffs. Results Background reactivity to SARS-CoV-2 antigens was common in prepandemic Malian samples. The SARS-CoV-2 reactivity varied between communities, increased with age, and correlated negligibly/weakly with other betacoronavirus and P falciparum antibodies. No prepandemic samples demonstrated functional activity. Regardless of the cutoffs applied, test specificity improved using a 2-antigen approach. Test performance was optimal using a 2-antigen assay with population-specific cutoffs (sensitivity, 73.9% [95% confidence interval {CI}, 51.6–89.8]; specificity, 99.4% [95% CI, 97.7–99.9]). Conclusions We have addressed the problem of SARS-CoV-2 seroassay performance in Africa by using a 2-antigen assay with cutoffs defined by performance in the target population.

The global decline in malaria has stalled1, emphasizing the need for vaccines that induce durable sterilizing immunity. Here we optimized regimens for chemoprophylaxis vaccination (CVac), for which aseptic, purified, cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ) were inoculated under prophylactic cover with pyrimethamine (PYR) (Sanaria PfSPZ-CVac(PYR)) or chloroquine (CQ) (PfSPZ-CVac(CQ))—which kill liver-stage and blood-stage parasites, respectively—and we assessed vaccine efficacy against homologous (that is, the same strain as the vaccine) and heterologous (a different strain) controlled human malaria infection (CHMI) three months after immunization ( https://clinicaltrials.gov/ , NCT02511054 and NCT03083847). We report that a fourfold increase in the dose of PfSPZ-CVac(PYR) from 5.12 × 104 to 2 × 105 PfSPZs transformed a minimal vaccine efficacy (low dose, two out of nine (22.2%) participants protected against homologous CHMI), to a high-level vaccine efficacy with seven out of eight (87.5%) individuals protected against homologous and seven out of nine (77.8%) protected against heterologous CHMI. Increased protection was associated with Vδ2 γδ T cell and antibody responses. At the higher dose, PfSPZ-CVac(CQ) protected six out of six (100%) participants against heterologous CHMI three months after immunization. All homologous (four out of four) and heterologous (eight out of eight) infectivity control participants showed parasitaemia. PfSPZ-CVac(CQ) and PfSPZ-CVac(PYR) induced a durable, sterile vaccine efficacy against a heterologous South American strain of P. falciparum, which has a genome and predicted CD8 T cell immunome that differs more strongly from the African vaccine strain than other analysed African P. falciparum strains. Two malaria vaccines comprising Plasmodium falciparum sporozoites and treatment with either pyrimethamine or chloroquine induced durable protective responses against both the African vaccine strain and a heterologous South American strain of P. falciparum.

In Kenya, the first trial of the attenuated whole organism PfSPZ Vaccine in infants has shown little efficacy against malaria infection, whereas trials in African adults have repeatedly observed protection. Differences in immune responses offer clues to the possible reasons.

Malaria has been hypothesized as a factor that may have reduced the severity of the COVID-19 pandemic in sub-Saharan Africa. To evaluate the effect of recent malaria on COVID-19 we assessed a subgroup of individuals participating in a longitudinal cohort COVID-19 serosurvey that were also undergoing intensive malaria monitoring as part of antimalarial vaccine trials during the 2020 transmission season in Mali. These communities experienced a high incidence of primarily asymptomatic or mild COVID-19 during 2020 and 2021. In 1314 individuals, 711 were parasitemic during the 2020 malaria transmission season; 442 were symptomatic with clinical malaria and 269 had asymptomatic infection. Presence of parasitemia was not associated with new COVID-19 seroconversion (29.7% (211/711) vs. 30.0% (181/603), p=0.9038) or with rates of reported symptomatic seroconversion during the malaria transmission season. In the subsequent dry season, prior parasitemia was not associated with new COVID-19 seroconversion (30.2% (133/441) vs. 31.2% (108/346), p=0.7499), with symptomatic seroconversion, or with reversion from seropositive to seronegative (prior parasitemia: 36.2% (64/177) vs. no parasitemia: 30.1% (37/119), p=0.3842). After excluding participants with asymptomatic infection, clinical malaria was also not associated with COVID-19 serostatus or symptomatic seroconversion when compared to participants with no parasitemia during the monitoring period. In communities with intense seasonal malaria and a high incidence of asymptomatic or mild COVID-19, we did not demonstrate a relationship between recent malaria and subsequent response to COVID-19. Lifetime exposure, rather than recent infection, may be responsible for any effect of malaria on COVID-19 severity.

Malaria may affect the reliability of SARS-CoV-2 seroassay performance and limit understanding of SARS-CoV-2 epidemiology in malaria-endemic regions. We present our experience conducting SARS-CoV-2 serosurveillance in seasonal malaria-affected communities in Mali and discuss relevant literature regarding the effect of malaria on the performance of SARS-CoV-2 serodiagnostics, including approaches to minimize the effect of malaria-associated assay interference.

Greater Mekong inhabitants are exposed to pathogens, zoonotic and otherwise, that may influence SARS-CoV-2 seroreactivity. A pre-pandemic (2005 to 2011) serosurvey of from 528 malaria-experienced Cambodians demonstrated higher-than-expected (up to 13.8 %) positivity of non-neutralizing IgG to SARS-CoV-2 spike and RBD antigens. These findings have implications for interpreting large-scale serosurveys., Article Summary Line: In the pre-COVID19 pandemic years of 2005 to 2011, malaria-experienced Cambodians from rural settings had higher-than-expected seroreactivity to SARS-CoV-2 spike and receptor binding domain proteins.

Background Seasonal malaria chemoprevention (SMC) is a novel strategy to reduce malaria infections in children. Infection with Plasmodium falciparum results in immune dysfunction characterized by elevated expression of markers associated with exhaustion, such as PD1 and LAG3, and regulatory CD4+FOXP3+ T cells. Methods In the current study, the impact of seasonal malaria chemoprevention on malaria-induced immune dysfunction, as measured by markers associated with exhaustion and regulatory T cells, was explored by flow cytometry. Results Children that received seasonal malaria chemoprevention had fewer malaria episodes and showed significantly lower fold changes in CD4+PD1+ and CD4+PD1+LAG3+ compared to those that did not receive SMC. Seasonal malaria chemoprevention had no observable effect on fold changes in CD8 T cells expressing PD1 or CD160. However, children receiving SMC showed greater increases in CD4+FOXP3+ T regulatory cells compared to children not receiving SMC. Conclusions These results provide important insights into the dynamics of malaria-induced changes in the CD4 T-cell compartment of the immune system and suggest that the reduction of infections due to seasonal malaria chemoprevention may also prevent immune dysfunction. Clinical Trials Registration NCT02504918.

Background Immunity that limits malarial disease is acquired over time, but adults living in endemic areas continue to become infected and can require treatment for clinical illness. Gamma delta (γδ) T cells, particularly the Vδ2+ subset, have been associated with development of clinical malaria in children. In this study, the dynamics of total γδ T cells, Vδ2+ and Vδ2− T cells were measured during a malaria transmission season in Malian adults. Methods This study explored γδ T cell dynamics and Plasmodium falciparum infection outcomes over the course of the malaria transmission season in Malian adults enrolled in the placebo arm of a double-blind randomized vaccine trial. All volunteers were treated with anti-malarial drugs prior to the start of the transmission season and blood smears were assessed for P. falciparum infection every 2 weeks from July 2014 to January 2015. The study participants were stratified as either asymptomatic infections or clinical malaria cases. Vδ2+ and Vδ2− γδ T cell frequencies and activation (as measured by CD38 expression) were measured in all study participants at baseline and then every 2 months using a whole blood flow cytometry assay. Results Forty of the forty-three subjects became infected with P. falciparum and, of those, 21 individuals were diagnosed with clinical malaria at least once during the season. The γδ T cell percentage and activation increased over the duration of the transmission season. Both the Vδ2+ and Vδ2− γδ T cells were activated by P. falciparum infection. Conclusion γδ T cells increased during a malaria transmission season and this expansion was noted in both the Vδ2+ and Vδ2− γδ T cells. However, neither expansion or activation of either γδ T cell subsets discriminated study participants that had asymptomatic infections from those that had clinical malaria cases.

BACKGROUND: The extent of SARS-CoV-2 exposure and transmission in Mali and the surrounding region is not well understood, although infection has been confirmed in nearly 14,000 symptomatic individuals and their contacts since the first case in March 2020. We aimed to estimate the cumulative incidence of SARS-CoV-2 in three Malian communities, and understand factors associated with infection. METHODS: Between 27 July 2020 and 29 January 2021, we collected blood samples along with demographic, social, medical and self-reported symptoms information from residents aged 6 months and older in three study communities at two study visits. SARS-CoV-2 antibodies were measured using a highly specific two-antigen ELISA optimized for use in Mali. We calculated cumulative adjusted seroprevalence for each site and evaluated factors associated with serostatus at each visit by univariate and multivariate analysis. FINDINGS: Overall, 94.8% (2533/2672) of participants completed both study visits. A total of 50.3% (1343/2672) of participants were male, and 31.3% (837/2672) were aged

Background The extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure and transmission in Mali and the surrounding region is not well understood. We aimed to estimate the cumulative incidence of SARS-CoV-2 in 3 communities and understand factors associated with infection. Methods Between July 2020 and January 2021, we collected blood samples and demographic, social, medical, and self-reported symptoms information from residents aged 6 months and older over 2 study visits. SARS-CoV-2 antibodies were measured using a highly specific 2-antigen enzyme-linked immunosorbent assay optimized for use in Mali. We calculated cumulative adjusted seroprevalence for each community and evaluated factors associated with serostatus at each visit by univariate and multivariate analysis. Results Overall, 94.8% (2533/2672) of participants completed both study visits. A total of 31.3% (837/2672) were aged, This study demonstrates a large, previously unquantified burden of SARS-COV-2 infection in the community in West Africa. In this young study population, there was limited evidence of severe illness and seropositivity rates that may approach hypothetical “herd immunity.”

Background Vaccines that block human-to-mosquito Plasmodium transmission are needed for malaria eradication and clinical trials have targeted zygote antigen Pfs25 for decades. We reported that a Pfs25 protein-protein conjugate vaccine formulated in alum adjuvant induced significant serum functional activity in both US and Malian adults. However, antibody titers declined rapidly, and transmission-reducing activity required four vaccine doses. Functional immunogenicity and durability must be improved before advancing TBV further in clinical development. We hypothesized that the pre-fertilization protein Pfs230 alone or in combination with Pfs25 would improve functional activity. Methods Transmission-blocking vaccine candidates based on gamete antigen Pfs230 or Pfs25 were conjugated with Exoprotein A, formulated in Alhydrogel, and administered to mice, rhesus macaques, and humans. Antibody titers were measured by ELISA and transmission-reducing activity was assess by the Standard Membrane Feeding Assay. Results Pfs25-EPA/Alhydrogel and Pfs230D1-EPA/Alhydrogel induced similar serum functional activity in mice, but Pfs230D1-EPA induced significantly greater activity in rhesus monkeys that was enhanced by complement. In U.S. adults, two vaccine doses induced complement-dependent activity in 4 of 5 Pfs230D1-EPA/Alhydrogel recipients but no significant activity in five Pfs25-EPA recipients, and combination with Pfs25-EPA did not increase activity over Pfs230D1-EPA alone. Conclusion The complement-dependent functional immunogenicity of Pfs230D1-EPA represents a significant improvement over Pfs25-EPA in this comparative study. The rhesus model is more predictive of the functional human immune response to Pfs230D1 than is the mouse model. Trial registration ClinicalTrials.gov NCT02334462Funding. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Serological tests are an indispensable tool to understand the epidemiology of the SARS-CoV-2 pandemic, particularly in areas where molecular diagnostics are limited. Poor assay performance may hinder the utility of these tests, including high rates of false-positivity previously reported in sub-Saharan Africa. From 312 Malian samples collected prior to 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and four other betacoronaviruses by ELISA, and assessed functional cross-reactivity in a subset by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA developed in the US, using two-antigen SARS-CoV-2 spike protein and receptor-binding domain. To optimize test performance, we compared single and two-antigen approaches using existing assay cutoffs and population-specific cutoffs for Malian control samples (positive and negative). Background reactivity to SARS-CoV-2 antigens was common in pre-pandemic samples compared to US controls (43.4% (135/311) for spike protein, 22.8% (71/312) for RBD, and 33.9% (79/233) for nucleocapsid protein). SARS-CoV-2 reactivity correlated weakly with other betacoronavirus reactivity, varied between Malian communities, and increased with age. No pre-pandemic samples demonstrated functional activity. Regardless of the cutoffs applied, specificity improved using a two-antigen approach. Test performance was optimal using a two-antigen assay with population-specific cutoffs derived from ROC curve analysis [Sensitivity: 73.9% (51.6-89.8), Specificity: 99.4% (97.7-99.9)]. In the setting of high background reactivity, such as sub-Saharan Africa, SARS-CoV-2 serological assays need careful qualification is to characterize the epidemiology of disease, prevent unnecessary harm, and allocate resources for targeted control measures.

Background: Serological tests are an indispensable tool to understand the epidemiology of the SARS-CoV-2 pandemic, particularly in areas where molecular diagnostics are limited. Poor assay performance may hinder the utility of these tests, including high rates of false-positivity previously reported in sub-Saharan Africa. Methods: From 312 Malian samples collected prior to 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and four other betacoronaviruses by ELISA, and assessed functional cross-reactivity in a subset by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of a two-antigen test developed in the US, using SARS-CoV-2 spike protein and receptor-binding domain ELISA measurements. To optimize test performance, we compared single and two-antigen approaches using existing assay cutoffs and population-specific cutoffs for Malian control samples (positive and negative). Findings: Background reactivity to SARS-CoV-2 antigens was common in pre-pandemic samples compared to US controls (43·4% (135/311) for spike protein, 22·8% (71/312) for RBD, and 33·9% (79/233) for nucleocapsid protein). SARS-CoV-2 reactivity correlated weakly with other betacoronavirus reactivity, varied between Malian communities, and increased with age. No pre-pandemic samples demonstrated functional activity. Regardless of the cutoffs applied, specificity improved using a two-antigen approach. Test performance was optimal using a two-antigen assay with population-specific cutoffs derived from ROC curve analysis [Sensitivity: 73·9% (51·6-89·8), Specificity: 99·4% (97·7-99·9)]. Interpretation: In the setting of high background reactivity, such as sub-Saharan Africa, SARS-CoV-2 serological assays need careful qualification is to characterize the epidemiology of disease, prevent unnecessary harm, and allocate resources for targeted control measures. Funding: This project was funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, as well as the National Cancer Institute, National Institutes of Health. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All pre-pandemic samples were collected during NIH-sponsored studies that were approved by the Malian USTTB FMPOS human research ethics committee and the NIAID/NIH institutional review board. Convalescent PCR-confirmed COVID-19 US positive control samples (n=10) were provided by the Adventist Hospital, Maryland. De-identified residual clinical samples for non-human subject research were obtained in accordance with 45 CFR 46. Convalescent samples were collected as part of a Public Health surveillance activity in collaboration with the Malian Ministry of Health COVID-19 Coordination Unit and with the approval of the USTTB FMOS-FAPH ethics committee (No2020/114/CE/FMOS/FAPH).

Whole-sporozoite vaccines confer sterilizing immunity to malaria-naive individuals by unknown mechanisms. In the first PfSPZ Vaccine trial ever in a malaria-endemic population, Vδ2 γδ T cells were significantly elevated and Vγ9/Vδ2 transcripts ranked as the most upregulated in vaccinees who were protected from Plasmodium falciparum infection. In a mouse model, absence of γδ T cells during vaccination impaired protective CD8 T cell responses and ablated sterile protection. γδ T cells were not required for circumsporozoite protein–specific Ab responses, and γδ T cell depletion before infectious challenge did not ablate protection. γδ T cells alone were insufficient to induce protection and required the presence of CD8α+ dendritic cells. In the absence of γδ T cells, CD8α+ dendritic cells did not accumulate in the livers of vaccinated mice. Altogether, our results show that γδ T cells were essential for the induction of sterile immunity during whole-organism vaccination.

The Malaria Vaccine Symposium occurred at Johns Hopkins University in Baltimore, MD, USA on April 25th, 2017, coinciding with World Malaria Day and the WHO announcement that the RTS,S malaria vaccine would begin pilot implementation programs in Ghana, Kenya, and Malawi in 2018. Scientists from several disciplines reported progress on an array of malaria vaccine concepts and product candidates, including pre-erythrocytic vaccines that prevent infection, blood-stage vaccines that limit infection and disease, and transmission-blocking vaccines that interrupt the spread of infection. Other speakers highlighted the immunological and genetic considerations that must be addressed by vaccinologists to yield the most efficacious vaccines. Here, we highlight the advances in malaria vaccinology that were reported at the symposium.

Summary Background Plasmodium falciparum sporozite (PfSPZ) Vaccine is a metabolically active, non-replicating, whole malaria sporozoite vaccine that has been reported to be safe and protective against P falciparum controlled human malaria infection in malaria-naive individuals. We aimed to assess the safety and protective efficacy of PfSPZ Vaccine against naturally acquired P falciparum in malaria-experienced adults in Mali. Methods After an open-label dose-escalation study in a pilot safety cohort, we did a double-blind, randomised, placebo-controlled trial based in Doneguebougou and surrounding villages in Mali. We recruited 18–35-year-old healthy adults who were randomly assigned (1:1) in a double-blind manner, with stratification by village and block randomisation, to receive either five doses of 2·7 × 10 5 PfSPZ or normal saline at days 0, 28, 56, 84, and 140 during the dry season (January to July inclusive). Participants and investigators were masked to group assignments, which were unmasked at the final study visit, 6 months after receipt of the last vaccination. Participants received combined artemether and lumefantrine (four tablets, each containing 20 mg artemether and 120 mg lumefantrine, given twice per day over 3 days for a total of six doses) to eliminate P falciparum before the first and last vaccinations. We collected blood smears every 2 weeks and during any illness for 24 weeks after the fifth vaccination. The primary outcome was the safety and tolerability of the vaccine, assessed as local and systemic reactogenicity and adverse events. The sample size was calculated for the exploratory efficacy endpoint of time to first P falciparum infection beginning 28 days after the fifth vaccination. The safety analysis included all participants who received at least one dose of investigational product, whereas the efficacy analyses included only participants who received all five vaccinations. This trial is registered at ClinicalTrials.gov, number NCT01988636. Findings Between Jan 18 and Feb 24, 2014, we enrolled 93 participants into the main study cohort with 46 participants assigned PfSPZ Vaccine and 47 assigned placebo, all of whom were evaluable for safety. We detected no significant differences in local or systemic adverse events or laboratory abnormalities between the PfSPZ Vaccine and placebo groups, and only grade 1 (mild) local or systemic adverse events occurred in both groups. The most common solicited systemic adverse event in the vaccine and placebo groups was headache (three [7%] people in the vaccine group vs four [9%] in the placebo group) followed by fatigue (one [2%] person in the placebo group), fever (one [2%] person in the placebo group), and myalgia (one [2%] person in each group). The exploratory efficacy analysis included 41 participants from the vaccine group and 40 from the placebo group. Of these participants, 37 (93%) from the placebo group and 27 (66%) from the vaccine group developed P falciparum infection. The hazard ratio for vaccine efficacy was 0·517 (95% CI 0·313–0·856) by time-to-infection analysis (log-rank p=0·01), and 0·712 (0·528–0·918) by proportional analysis (p=0·006). Interpretation PfSPZ Vaccine was well tolerated and safe. PfSPZ Vaccine showed significant protection in African adults against P falciparum infection throughout an entire malaria season. Funding US National Institutes of Health Intramural Research Program, Sanaria.

Malaria transmission blocking vaccines (TBV) target the mosquito stage of parasite development by passive immunization of mosquitoes feeding on a vaccinated human. Through uptake of vaccine-induced antibodies in a blood meal, mosquito infection is halted and hence transmission to another human host is blocked. Pfs230 is a gametocyte and gamete surface antigen currently under clinical evaluation as a TBV candidate. We have previously shown that chemical conjugation of poorly immunogenic TBV antigens to Exoprotein A (EPA) can enhance their immunogenicity. Here, we assessed Outer Membrane Protein Complex (OMPC), a membrane vesicle derived from Neisseria meningitidis, as a carrier for Pfs230. We prepared Pfs230-OMPC conjugates with varying levels of antigen load and examined immunogenicity in mice. Chemical conjugation of Pfs230 to OMPC enhanced immunogenicity and functional activity of the Pfs230 antigen, and OMPC conjugates achieved 2-fold to 20-fold higher antibody titers than Pfs230-EPA/AdjuPhos® at different doses. OMPC conjugates were highly immunogenic even at low doses, indicating a dose-sparing effect. EPA conjugates induced an IgG subclass profile biased towards a Th2 response, whereas OMPC conjugates induced a strong Th1-biased immune response with high levels of IgG2, which can benefit Pfs230 antibody functional activity, which depends on complement activation. OMPC is a promising carrier for Pfs230 vaccines., Malaria transmission blocking vaccine: How bacteria can help Malaria transmission blocking vaccines (TBV) target Plasmodium stages that transmit between human and mosquitos in order to interrupt the parasite’s life cycle and reduce spread. One TBV antigen currently under clinical development is Pf230, which is expressed on sexual Plasmodium stages. In this study, led by Patrick Duffy from the NIAID, researchers improve immunogenicity of Pf230. They chemically conjugate a part of Pf230 to membrane vesicles derived from bacteria, so-called outer membrane protein complexes (OMPC). Immunization of mice with Pf230-OMPC elicits a higher antibody response and a more balanced IgG subclass profile than control immunizations. Serum from Pf230-OMPC-vaccinated mice efficiently blocks infection of mosquitoes. These results with mice encourage further pre-clinical and clinical characterization of OMPC as a carrier for TBV antigens.

Malaria is an infectious disease caused by the protozoan parasite Plasmodium sp, the most lethal being Plasmodium falciparum. Clinical malaria is associated with the asexual replication cycle of Plasmodium parasites inside the red blood cells (RBCs) and a dysregulated immune response. Although the mechanisms of immune responses to blood—or liver-stage parasites have been extensively studied, this has not led to satisfactory leads for vaccine design. Among innate immune cells responding to infection are the non-conventional gamma-delta T-cells. The Vγ9Vδ2 T-cell subset, found only in primates, is activated in response to non-peptidic phosphoantigens produced by stressed mammalian cells or by microorganisms such as Mycobacteria, E.coli, and Plasmodium. The potential protective role of Vγ9Vδ2 T-cells against infections and cancer progression is of current research interest. Vγ9Vδ2 T-cells have been shown to play a role in the early control of P. falciparum parasitemia and to influence malaria adaptive immunity via cytokine release and antigen presentation. They are activated and expanded during a primary P. falciparum infection in response to malaria phosphoantigens and their activity is modulated upon subsequent infections. Here, we review the wide range of functions by which Vγ9Vδ2 T-cells could both contribute to and protect from malaria pathology, with a particular focus on their ability to induce both innate and adaptive responses. We discuss how the multifunctional roles of these T-cells could open new perspectives on gamma-delta T-cell-based interventions to prevent or cure malaria.

We welcome the comments from McKenna and Tsuji on our recent publication demonstrating a requirement for γδ T cells to induce sterile immunity during irradiated sporozoite vaccinations. In our studies, Malian adults who were resistant to Plasmodium falciparum infection after receiving the

While a significant proportion of HIV-2-infected individuals are asymptomatic and maintain undetectable viral loads (controllers), 15% to 20% progress to AIDS and are predicted by detectable viremia. Identifying immune correlates that distinguish these 2 groups should provide insights into how a potentially pathogenic retrovirus can be naturally controlled. We performed a detailed study of HIV-2-specific cellular responses in a unique community cohort in Guinea-Bissau followed for over 2 decades. T-cell responses were compared between controllers (n = 33) and viremic subjects (n = 27) using overlapping peptides, major histocompatibility complex class I tetramers, and multiparameter flow cytometry. HIV-2 viral control was significantly associated with a high-magnitude, polyfunctional Gag-specific CD8(+) T-cell response but not with greater perforin upregulation. This potentially protective HIV-2-specific response is surprisingly narrow. HIV-2 Gag-specific CD8(+) T cells are at an earlier stage of differentiation than cytomegalovirus-specific CD8(+) T-cells, do not contain high levels of cytolytic markers, and exhibit low levels of activation and proliferation, representing distinct properties from CD8(+) T cells associated with HIV-1 control. These data reveal the potential T-cell correlates of HIV-2 control and the detailed phenotype of virus-specific CD8(+) T cells in a naturally contained retroviral infection.

The role of T regulatory cells (Tregs) in human immunodeficiency virus (HIV)‐1 infection, although not entirely clear, has recently been highlighted. Despite their lack of specificity, fluorochrome‐labeled CD4 and CD25 antibodies are common flow cytometric reagents used to identify these cells with immunosuppressive potential. Cryopreservation has previously been shown to alter the proportions of lymphocytes with certain phenotypes expressed in peripheral blood mononuclear cells (PBMCs). The aim of this study was to assess the effect of cryopreservation on CD4+ CD25+ T cells in PBMCs from HIV‐1+ individuals to guide the design of future studies on Tregs. We recruited 30 HIV‐1+ individuals and nine healthy controls. CD25 expression in CD4+ T cells was compared between fresh and frozen/thawed PBMC samples from the same time point. In this study, cryopreservation significantly decreased the proportion of CD4+ CD25+ T cells in PBMC samples from HIV‐1 infected subjects. This finding suggests that studies of CD4+ CD25+ T cells should be carried out on fresh samples to avoid bias introduced by cryopreservation. J. Clin. Lab. Anal. 22:153–158, 2008. © 2008 Wiley‐Liss, Inc.

While well documented in human immunodeficiency virus (HIV)-1, neurologic sequelae have not been systematically evaluated in HIV-2. After excluding for confounding comorbidities, 67 individuals from a rural cohort in Guinea-Bissau (22 HIV-2 participants, 45 seronegative controls) were evaluated. HIV + individuals were divided into CD4

In spite of recent advances in flow cytometry technology, most cytometry data is still analyzed manually which is labor-intensive for large datasets and prone to bias and inconsistency. We designed an automatic processing tool (APT) to rapidly and consistently define and describe cell populations across large datasets. Image processing, smoothing, and clustering algorithms were used to generate an expert system that automatically reproduces the functionality of commercial manual cytometry processing tools. The algorithms were developed using a dataset collected from CMV-infected infants and combined within a graphical user interface, to create the APT. The APT was used to identify regulatory T-cells in HIV-infected adults, based on expression of FOXP3. Results from the APT were compared directly with the manual analyses of five immunologists and showed close agreement, with a concordance correlation coefficient of 0.96 (95% CI 0.91-0.98). The APT was well accepted by users and able to process around 100 data files per hour. By applying consistent criteria to all data generated by a study, the APT can provide a level of objectivity that is difficult to match using conventional manual analysis.

Background: While a significant proportion of HIV-2-infected individuals are asymptomatic and maintain undetectable viral loads (controllers), 15-20% progress to AIDS and are predicted by detectable viraemia. Identifying immune correlates that distinguish these two groups should provide insights into how a potentially pathogenic retrovirus can be naturally controlled. Methods: We performed a detailed study of HIV-2-specific cellular responses in a unique community cohort in Guinea-Bissau followed for over two decades. T-cell responses were compared between controllers (n 1⁄4 33) and viraemic subjects (n 1⁄4 27) using overlapping peptides, MHC class I tetramers and multi-parameter flow cytometry. Results: HIV-2 viral control was significantly associated with a high magnitude, polyfunctional Gag-specific CD8+ T-cell response, but not with greater perforin upregulation. This potentially protective HIV-2-specific response is surprisingly narrow. HIV-2 Gag-specific CD8+ T-cells are at an earlier stage of differentiation than CMV-specific CD8+ Tcells, do not contain high levels of cytolytic markers and exhibit low levels of activation and proliferation, representing distinct properties from CD8+ T-cells associated with HIV-1 control. Furthermore, comparison of viral capsid sequence data from HIV-1 and HIV-2 infected subjects in this cohort reveal limited T-cell driven changes in HIV-2, despite potent T-cell pressure. Conclusions: These data reveal the potential T-cell correlates of HIV-2 control and the detailed phenotype of virus-specific CD8+ T-cells in a naturally contained retroviral infection, which is distinct from descriptions of CD8+ T-cell populations in HIV-1 infected elite controllers. The lack of potential T-cell escape in HIV-2 may be a significant factor in the benign course of infection seen in most HIV-2 infected subjects. http://dx.doi.org/10.1016/j.jinf.2013.07.006 HIV CONTROL IN POST-PARTUM MOTHERS; A

Background Tuberculosis causes 3 million deaths annually. The most common site of tuberculosis is pulmonary however; extra-pulmonary forms of the disease also remain prevalent. Restriction of Mycobacterium tuberculosis depends on effective recruitment and subsequent activation of T lymphocytes, mononuclear and polymorphonuclear cells to the site of infection. Tumor necrosis factor (TNF)-α is essential for granuloma formation and is a potent activator of monocyte chemotactic protein (MCP-1, CCL2). CCL2 is essential for recruitment of monocytes and T cells and has been shown to play a role in protection against tuberculosis. Interleukin -8 (CXCL8) is a potent activator of neutrophils. Increased levels of CCL2, CXCL8 and TNFα are reported in tuberculosis but their significance in different forms of tuberculosis is as yet unclear. We have used an ex vivo assay to investigate differences in immune parameters in patients with either pulmonary or extra-pulmonary tuberculosis. Methods Serum levels of CCL2, CXCL8 and TNFα were measured in patients with pulmonary tuberculosis (N = 12), extra-pulmonary tuberculosis (N = 8) and BCG-vaccinated healthy volunteers (N = 12). Whole blood cells were stimulated with non-pathogenic Mycobacterium bovis bacille-Calmette Guerin (BCG) vaccine strain or bacterial lipopolysaccharide (LPS) and cyto/chemokines were monitored in supernatants. Results Circulating serum levels of CXCL8 and TNFα were raised in all tuberculosis patients, while CCL2 levels were not. There was no difference in spontaneous cytokine secretion from whole blood cells between patients and controls. M. bovis BCG-induced ex vivo CCL2 secretion was significantly greater in pulmonary as compared with both extra-pulmonary tuberculosis patients and endemic controls. In response to LPS stimulation, patients with pulmonary tuberculosis showed increased CCL2 and TNFα responses as compared with the extra-pulmonary group. BCG-, and LPS-induced CXCL8 secretion was comparable between patients and controls. Conclusion CCL2 is activated by TNFα and is essential for recruitment of monocytes and T cells to the site of mycobacterial infection. Increased CCL2 activation in pulmonary tuberculosis may result in a stronger cellular response as compared with extra-pulmonary tuberculosis patients, and this may contribute to the localization of infection to the pulmonary site.

Introduction Despite the marked reduction in reported cases of P. falciparum malaria, this disease remains a major global health threat claiming the lives of 655,000 people and putting an estimated 3.3 billion of the world’s population at risk of illness. Although people living in malaria-endemic areas mount a broad range of immune responses to a great variety of parasite antigens, protective immunity to clinical malaria develops only slowly, and some argue the duration of such responses is comparatively short-lived. The concept of original antigenic sin, whereby an initial infection imprints a certain response pattern that prevents the immune system from mounting more efficient responses upon subsequent infections, may explain these observations. Methods In a longitudinal cohort study of Malian children aged 0.5 to 10 years, we investigate whether a child experiencing repeated malaria episodes mounts similar immunological responses each time. We also investigated whether these responses can be grouped into distinct patterns, and whether any distinct patterns associate with disease outcome. Results We are currently characterizing the quality and magnitude of parasite antigen-specific T cell responses after in vitro stimulation of peripheral blood mononuclear cells collected during an acute malaria episode using multi-parameter flow cytometry. Based on cell surface phenotype we classify memory CD4 + and CD8 + T cells into effector memory (CD45RO + CCR7 − ) and central memory (CD45RO + CCR7 + ) T cells and characterize their function by measuring the production of IFN ϒ , TNF α , IL-2 and IL-10. We also quantify cytokine levels in the corresponding supernatants and plasma samples using multi-parameter Bioplex assays. Conclusion The results of this study will enable us to determine whether original antigenic sin influences malaria incidence, and may reveal an association between a specific immunological response pattern and enhanced protection from a subsequent malaria episode, which can be further explored in identifying candidate vaccine antigens.

Objective\ud The factors associated with the development of immune reconstitution inflammatory syndrome in HIV patients commencing antiretroviral therapy have not been fully elucidated. Using a longitudinal study design, this study addressed whether alteration in the levels of T regulatory cells contributed to the development of IRIS in a West African cohort of HIV-1 and HIV-2 patients. Seventy-one HIV infected patients were prospectively recruited to the study and followed up for six months. The patients were categorized as IRIS or non-IRIS cases following published clinical guidelines. The levels of T regulatory cells were measured using flow cytometry at baseline and all follow-up visits. Baseline cytokine levels of IL-2, IL-6, IFN-γ, TNF-α, MIP-1β, IL-1, IL-12, IL-13, and IL-10 were measured in all patients.\ud \ud Results\ud Twenty eight percent of patients (20/71) developed IRIS and were predominantly infected with HIV-1. Patients developing IRIS had lower nadir CD4 T cells at baseline (p = 0.03) and greater CD4 T cell reconstitution (p = 0.01) at six months post-ART. However, the development of IRIS was not influenced by the levels of T regulatory cells. Similarly, baseline cytokine levels did not predict the onset of IRIS.\ud \ud Conclusion\ud The development of IRIS was not associated with differences in levels of T regulatory cells or baseline pro-inflammatory cytokines.