Recombinant full-length Plasmodium falciparum circumsporozoite protein-based vaccine adjuvanted with GLA-LSQ: Results of Phase 1 testing with malaria challenge.

INTRODUCTION: Malaria is preventable yet causes >600,000 deaths annually. RTS, S, the first marketed malaria vaccine, has modest efficacy, but improvements are needed for eradication. METHODS: We conducted an open-label, dose escalation Phase 1 study of a recombinant, full-length circumsporozoite protein vaccine (rCSP) administered with adjuvant GLA-LSQ on days 1, 29, and 85 or 1 and 490 to healthy, malaria-naïve adults. Primary endpoints were safety and reactogenicity. Secondary endpoints were antibody responses and Plasmodium falciparum parasitemia after homologous controlled human malaria infection (CHMI). RESULTS: Participants were enrolled into four groups receiving rCSP/GLA-LSQ: 10 µg x 3 (n = 20), 30 µg x 3 (n = 10), 60 µg x 3 (n = 10) or 60 µg x 2 (n = 9); ten participants received 30 µg rCSP alone x 3; and six infectivity controls. Participants experienced no serious adverse events. Rates of solicited and unsolicited adverse events were similar among groups. All 26 participants who underwent CHMI 28 days after final vaccinations developed malaria. Increasing vaccine doses induced higher IgG titers, but did not achieve previously established RTS, S benchmarks. CONCLUSIONS: rCSP/GLA-LSQ had favorable safety results. However, tested regimens did not induce protective immunity. Further investigation could assess if adjuvant or schedule adjustments improve efficacy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03589794.

Profiling of serum factors associated with Staphylococcus aureus skin and soft tissue infections as a foundation for biomarker identification.

Background: People living in close quarters, such as military trainees, are at increased risk for skin and soft tissue infections (SSTI), especially those caused by methicillin-resistant Staphylococcus aureus (MRSA). The serum immune factors associated with the onset of SSTI are not well understood. Methods: We conducted a longitudinal study of SSTIs, enrolling US Army trainees before starting military training and following up for 14 weeks. Samples were collected on Day 0, 56, and 90. Serum chemokines and cytokines among 16 SSTI cases and 51 healthy controls were evaluated using an electro-chemiluminescence based multiplex assay platform. Results: Of 54 tested cytokines, 12 were significantly higher among SSTI cases as compared to controls. Among the cases, there were correlations between factors associated with vascular injury (i.e., VCAM-1, ICAM-1, and Flt1), the angiogenetic factor VEGF, and IL-10. Unsupervised machine learning (Principal Component Analysis) revealed that IL10, IL17A, C-reactive protein, ICAM1, VCAM1, SAA, Flt1, and VGEF were indicative of SSTI. Conclusion: The study demonstrates the power of immunoprofiling for identifying factors predictive of pre-illness state of SSTI thereby identifying early stages of an infection and individuals susceptible to SSTI.

Novel antibody competition binding assay identifies distinct serological profiles associated with protection.

Introduction: Pre-erythrocytic malaria vaccines hold the promise of inducing sterile protection thereby preventing the morbidity and mortality associated with Plasmodium infection. The main surface antigen of P. falciparum sporozoites, i.e., the circumsporozoite protein (CSP), has been extensively explored as a target of such vaccines with significant success in recent years. Systematic adjuvant selection, refinements of the immunization regimen, and physical properties of the antigen may all contribute to the potential of increasing the efficacy of CSP-based vaccines. Protection appears to be dependent in large part on CSP antibodies. However due to a knowledge gap related to the exact correlates of immunity, there is a critical need to improve our ability to down select candidates preclinically before entering clinical trials including with controlled human malaria infections (CHMI). Methods: We developed a novel multiplex competition assay based on well-characterized monoclonal antibodies (mAbs) that target crucial epitopes across the CSP molecule. This new tool assesses both, quality and epitope-specific concentrations of vaccine-induced antibodies by measuring their equivalency with a panel of well-characterized, CSP-epitope-specific mAbs. Results: Applying this method to RTS,S-immune sera from a CHMI trial demonstrated a quantitative epitope-specificity profile of antibody responses that can differentiate between protected vs. nonprotected individuals. Aligning vaccine efficacy with quantitation of the epitope fine specificity results of this equivalency assay reveals the importance of epitope specificity. Discussion: The newly developed serological equivalence assay will inform future vaccine design and possibly even adjuvant selection. This methodology can be adapted to other antigens and disease models, when a panel of relevant mAbs exists, and could offer a unique tool for comparing and down-selecting vaccine formulations.

Retroviral b-Zip protein (HBZ) contributes to the release of soluble and exosomal immune checkpoint molecules in the context of neuroinflammation.

HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive, neuroinflammatory demyelinating condition of the spinal cord. We have previously shown that aberrant expression and activity of immune checkpoint (ICP) molecules such as PD-1 and PD-L1/PD-L2, negatively associates with the cytolytic potential of T cells in individuals with HAM/TSP. Interestingly, ICPs can exist in a soluble cell-free form and can be carried on extracellular vesicles (EVs) and exosomes (small EVs, <300nm) while maintaining their immunomodulatory activity. Therefore, we investigated the role of soluble and exosomal ICPs in HTLV-1 associated neuroinflammation. For the very first time, we demonstrate a unique elevated presence of several stimulatory (CD27, CD28, 4-1BB) and inhibitory (BTLA, CTLA-4, LAG-3, PD-1, PD-L2) ICP receptors in HAM/TSP sera, and in purified exosomes from a HAM/TSP-derived HTLV-1-producing (OSP2) cells. These ICPs were found to be co-localized with the endosomal sorting complex required for transport (ESCRT) pathway proteins and exhibited functional binding with their respective ligands. Viral proteins and cytokines (primarily IFNγ) were found to be present in purified exosomes. IFNγ exposure enhanced the release of ICP molecules while antiretroviral drugs (Azidothymidine and Lopinavir) significantly inhibited this process. HTLV-1 b-Zip protein (HBZ) has been linked to factors that enhance EV release and concurrent knockdown here led to the reduced expression of ESCRT associated genes (eg. Hrs, Vsp4, Alix, Tsg101) as well as abrogated the release of ICP molecules, suggesting HBZ involvement in this process. Moreso, exosomes from OSP2 cells adversely affected CD8 T-cell functions by dimishing levels of cytokines and cytotoxic factors. Collectively, these findings highlight exosome-mediated immunmodulation of T-cell functions with HBZ and ESCRT pathways as an underlying mechanism in the context of HTLV-1-induced neuroinflammation.

Differential Homing Receptor Profiles of Lymphocytes Induced by Attenuated versus Live Plasmodium falciparum Sporozoites.

The onset of an adaptive immune response provides the signals required for differentiation of antigen-specific lymphocytes into effector cells and imprinting of these cells for re-circulation to the most appropriate anatomical site (i.e., homing). Lymphocyte homing is governed by the expression of tissue-specific lymphocyte homing receptors that bind to unique tissue-specific ligands on endothelial cells. In this study, a whole-parasite malaria vaccine (radiation-attenuated sporozoites (RAS)) was used as a model system to establish homing receptor signatures induced by the parasite delivered through mosquito bite to provide a benchmark of desirable homing receptors for malaria vaccine developers. This immunization regimen resulted in the priming of antigen-specific B cells and CD8+ T cells for homing primarily to the skin and T/B cell compartments of secondary lymphoid organs. Infection with live sporozoites, however, triggers the upregulation of homing receptor for the liver and the skin, demonstrating that there is a difference in the signal provided by attenuated vs. live sporozoites. This is the first report on imprinting of homing routes by Plasmodium sporozoites and, surprisingly, it also points to additional, yet to be identified, signals provided by live parasites that prime lymphocytes for homing to the liver. The data also demonstrate the utility of this method for assessing the potential of vaccine formulations to direct antigen-specific lymphocytes to the most relevant anatomical site, thus potentially impacting vaccine efficacy.

Clinical and genomic characterization of hypervirulent Klebsiella pneumoniae (hvKp) infections via passive surveillance in Southern California, 2020-2022.

Hypervirulent Klebsiella pneumoniae (hvKp) is more invasive and virulent than classical K. pneumoniae, and requires specialized treatment. To raise clinical awareness, this study determined the prevalence, clinical characteristics, and genomic epidemiology of hvKp infections in Southern California (SoCal) by conducting a passive surveillance in a single large academic medical center. We report here that hvKp infections were more common than expected, accounting for 2.6% of invasive K. pneumoniae infections, and presented with a wide disease spectrum, occasionally mimicking tumors, even co-infecting a COVID-19 patient. Most infections were community acquired with no recent international travel, suggesting hvKp strains are circulating in the community. Genomic analysis revealed genetic diversity, with the K1-ST23 lineage predominating but not clonal, and multiple sequence types of K2 including a SoCal unique K2-ST66 sublineage that had been unrecognized. Our findings highlight the urgency of heightened awareness of hvKp infection in the US, the need for rapid diagnosis of hvKp, and the necessity of implementing robust surveillance programs for hvKp at the institutional or local level.

Susceptibility to Human Immunodeficiency Virus Type 1 Acquisition Linked to Malaria Exposure: A Case-Control Study.

Human immunodeficiency virus (HIV) and malaria infection rates overlap across sub-Saharan Africa, but factors influencing their co-occurrence are unclear. In a case-control study, we investigated whether malaria exposure increases risk of type 1 (HIV-1) acquisition. Prior to seroconverting, HIV-positive cases had significantly higher malaria-associated antibodies compared to HIV-negative controls, linking malaria exposure to HIV-1 acquisition.

Blast Waves Cause Immune System Dysfunction and Transient Bone Marrow Failure in a Mouse Model.

Explosive devices, either conventional or improvised, are common sources of injuries during combat, civil unrest, and terror attacks, resulting in trauma from exposure to blast. A blast wave (BW), a near-instantaneous rise in pressure followed by a negative pressure, propagates through the body in milliseconds and can affect physiology for days/months after exposure. Epidemiological data show that blast-related casualties result in significantly higher susceptibility to wound infections, suggesting long-lasting immune modulatory effects from blast exposure. The mechanisms involved in BW-induced immune changes are poorly understood. We evaluated the effects of BW on the immune system using an established murine model. Animals were exposed to BWs (using an Advanced Blast Simulator), followed by longitudinally sampling for 14 days. Blood, bone marrow, and spleen were analyzed for changes in the 1) complete blood count (CBC), and 2) composition of bone marrow cells (BMC) and splenocytes, and 3) concentrations of systemic cytokines/chemokines. Our data demonstrate that BW results in transient bone marrow failure and long-term changes in the frequency and profile of progenitor cell populations. Viability progressively decreased in hematopoietic stem cells and pluripotent progenitor cells. Significant decrease of CD4+ T cells in the spleen indicates reduced functionality of adaptive immune system. Dynamic changes in the concentrations of several cytokines and chemokines such as IL-1α and IL-17 occurred potentially contributing to dysregulation of immune response after trauma. This work lays the foundation for identifying the potential mechanisms behind BW's immunosuppressive effects to inform the recognition of this compromised status is crucial for the development of therapeutic interventions for infections to reduce recovery time of wounded patients injured by explosive devices.

Assessing Prevalence and Transmission Rates of Malaria through Simultaneous Profiling of Antibody Responses against Plasmodium and Anopheles Antigens.

Reliably assessing exposure to mosquitoes carrying malaria parasites continues to be a challenge due to the lack of reliable, highly sensitive diagnostics with high-throughput potential. Here, we describe an approach that meets these requirements by simultaneously measuring immune responses to both disease vector and pathogen, using an electro-chemiluminescence-based multiplex assay platform. While using the same logistical steps as a classic ELISA, this platform allows for the multiplexing of up to ten antigens in a single well. This simple, reproducible, quantitative readout reports the magnitude, incidence, and prevalence of malaria infections in residents of malaria-endemic areas. By reporting exposure to both insect vectors and pathogen, the approach also provides insights into the efficacy of drugs and/or other countermeasures deployed against insect vectors aimed at reducing or eliminating arthropod-borne diseases. The high throughput of the assay enables the quick and efficient screening of sera from individuals for exposure to Plasmodium even if they are taking drug prophylaxis. We applied this assay to samples collected from controlled malaria infection studies, as well as those collected in field studies in malaria-endemic regions in Uganda and Kenya. The assay was sensitive to vector exposure, malaria infection, and endemicity, demonstrating its potential for use in malaria serosurveillance.

Immunoprofiling Identifies Functional B and T Cell Subsets Induced by an Attenuated Whole Parasite Malaria Vaccine as Correlates of Sterile Immunity.

Immune correlates of protection remain elusive for most vaccines. An identified immune correlate would accelerate the down-selection of vaccine formulations by reducing the need for human pathogen challenge studies that are currently required to determine vaccine efficacy. Immunization via mosquito-delivered, radiation-attenuated P. falciparum sporozoites (IMRAS) is a well-established model for efficacious malaria vaccines, inducing greater than 90% sterile immunity. The current immunoprofiling study utilized samples from a clinical trial in which vaccine dosing was adjusted to achieve only 50% protection, thus enabling a comparison between protective and non-protective immune signatures. In-depth immunoprofiling was conducted by assessing a wide range of antigen-specific serological and cellular parameters and applying our newly developed computational tools, including machine learning. The computational component of the study pinpointed previously un-identified cellular T cell subsets (namely, TNFα-secreting CD8+CXCR3-CCR6- T cells, IFNγ-secreting CD8+CCR6+ T cells and TNFα/FNγ-secreting CD4+CXCR3-CCR6- T cells) and B cell subsets (i.e., CD19+CD24hiCD38hiCD69+ transitional B cells) as important factors predictive of protection (92% accuracy). Our study emphasizes the need for in-depth immunoprofiling and subsequent data integration with computational tools to identify immune correlates of protection. The described process of computational data analysis is applicable to other disease and vaccine models.

Comprehensive serologic profile and specificity of maternal and neonatal cord blood SARS-CoV-2 antibodies.

BACKGROUND: Initial studies on COVID-19 in pregnancy have demonstrated a range of neutralizing activity, but little has been published on the full profile of SARS CoV-2 related antibodies in maternal and cordblood. OBJECTIVE: This study aimed to describe the profile and specificity of maternal and neonatal cord blood antibody profiles in response to SARS-CoV-2 virus exposure. STUDY DESIGN: This was a prospective cohort study of delivering patients at Thomas Jefferson University Hospital from April 2020 to February 2021. The primary objective was to describe unique maternal and fetal antibody epitope titers and specificity in patients with COVID-19 history. Serologic profile was assessed with a multiplex platform. Antigens used were hemagglutinin trimer influenza A (Hong Kong H3); spike trimers for SARS-CoV-2, SARS-CoV-1, Middle East respiratory syndrome coronavirus, and betacoronaviruses HKU-1 and OC43; and spike N-terminal domain, spike receptor-binding domain, and nucleocapsid protein (full length) for SARS-CoV-2. RESULTS: Here, 112 maternal samples and 101 maternal and cord blood pairs were analyzed. Of note, 37 patients had a known history of COVID-19 (positive polymerase chain reaction test) during pregnancy. Of 36 patients, 16 (44%) were diagnosed with COVID-19 within 7 days of delivery. Moreover, 15 of the remaining 76 patients (20%) without a known diagnosis had positive maternal serology. For those with a history of COVID-19, we identified robust immunoglobulin G response in maternal blood to CoV-2 nucleocapsid, spike (full length), and spike (receptor-binding domain) antigens with more modest responses to the spike (N-terminal domain) antigen. In contrast, the maternal blood immunoglobulin M response seemed more specific to spike (full length) epitopes than nucleocapsid, spike (receptor-binding domain), or spike (N-terminal domain) epitopes. There were significantly higher maternal and cord blood immunoglobulin G responses not only to CoV-2 spike (127.1-fold; standard deviation, 2.0; P<.00001) but also to CoV-1 spike (21.1-fold higher; standard deviation, 1.8; P<.00001) and Middle East respiratory syndrome spike (6.9-fold higher; standard deviation, 2.5; P<.00001). In contrast, maternal immunoglobulin M responses were more specific to CoV-2 spike (15.8-fold; standard deviation, 2.1; P<.00001) but less specific to CoV-1 (2.5-fold higher; standard deviation, 0.71; P<.00001) and no significant difference for Middle East respiratory syndrome. Maternal and cord blood immunoglobulin G antibodies were highly correlated for both spike and nucleocapsid (R2=0.96 and 0.94, respectively). CONCLUSION: Placental transfer was efficient, with robust nucleocapsid and spike responses. Both nucleocapsid and spike antibody responses should be studied for a better understanding of COVID-19 immunity. Immunoglobulin G antibodies were cross-reactive with related CoV-1 and Middle East respiratory syndrome spike epitopes, whereas immunoglobulin M antibodies, which cannot cross the placenta to provide neonatal passive immunity, were more SARS-CoV-2 specific. Neonatal cord blood may have significantly different fine specificity than maternal blood, despite the high efficiency of immunoglobulin G transfer.

Multiplex serological assay for establishing serological profiles of polymorphic, closely related peptide antigens.

Profiling of serological responses to establish the landscape of antibody specificities in individuals exposed to pathogens or vaccines is crucial for (a) revealing humoral immune correlates of protection; (b) uncovering markers of pathogen exposure; and (c) identifying antigens and epitopes associated with disease vs. protection. Establishing the antigenic profile of serological responses requires either expensive microarrays or labor- and time-intensive ELISA assays. Multiplex assay platforms are increasingly being evaluated for their usefulness for high-throughput testing of sera or plasma. The methodology described here utilizes a plate-based assay that allows the simultaneous detection of up to ten antigens per well in a 96 well format using an electrochemiluminescence immunoassay (ECLIA).•The newly developed protocol outlines high-throughput profiling of serological responses using a multiplex testing platform with subsequent computational analysis.•The protocol is a modification of the basic assay development manual from the manufacturer of the MESO QuickPlex SQ 120 instrument (MSD, Gaithersburg, MD) and can be used for synthetic peptides as well as full length proteins.•The protocol can be applied to map serological responses to pathogens or pathogen-derived antigens to establish serological profiles in search for biomarkers or immune correlates.

Comprehensive Data Integration Approach to Assess Immune Responses and Correlates of RTS,S/AS01-Mediated Protection From Malaria Infection in Controlled Human Malaria Infection Trials.

RTS,S/AS01 (GSK) is the world's first malaria vaccine. However, despite initial efficacy of almost 70% over the first 6 months of follow-up, efficacy waned over time. A deeper understanding of the immune features that contribute to RTS,S/AS01-mediated protection could be beneficial for further vaccine development. In two recent controlled human malaria infection (CHMI) trials of the RTS,S/AS01 vaccine in malaria-naïve adults, MAL068 and MAL071, vaccine efficacy against patent parasitemia ranged from 44% to 87% across studies and arms (each study included a standard RTS,S/AS01 arm with three vaccine doses delivered in four-week-intervals, as well as an alternative arm with a modified version of this regimen). In each trial, RTS,S/AS01 immunogenicity was interrogated using a broad range of immunological assays, assessing cellular and humoral immune parameters as well as gene expression. Here, we used a predictive modeling framework to identify immune biomarkers measured at day-of-challenge that could predict sterile protection against malaria infection. Using cross-validation on MAL068 data (either the standard RTS,S/AS01 arm alone, or across both the standard RTS,S/AS01 arm and the alternative arm), top-performing univariate models identified variables related to Fc effector functions and titer of antibodies that bind to the central repeat region (NANP6) of CSP as the most predictive variables; all NANP6-related variables consistently associated with protection. In cross-study prediction analyses of MAL071 outcomes (the standard RTS,S/AS01 arm), top-performing univariate models again identified variables related to Fc effector functions of NANP6-targeting antibodies as highly predictive. We found little benefit-with this dataset-in terms of improved prediction accuracy in bivariate models vs. univariate models. These findings await validation in children living in malaria-endemic regions, and in vaccinees administered a fourth RTS,S/AS01 dose. Our findings support a "quality as well as quantity" hypothesis for RTS,S/AS01-elicited antibodies against NANP6, implying that malaria vaccine clinical trials should assess both titer and Fc effector functions of anti-NANP6 antibodies.

Serological profiles of pan-coronavirus-specific responses in COVID-19 patients using a multiplexed electro-chemiluminescence-based testing platform.

Serological assessment of SARS-CoV-2 specific responses are an essential tool for determining the prevalence of past SARS-CoV-2 infections in the population especially when testing occurs after symptoms have developed and limited contact tracing is in place. The goal of our study was to test a new 10-plex electro-chemiluminescence-based assay to measure IgM and IgG responses to the spike proteins from multiple human coronaviruses including SARS-CoV-2, assess the epitope specificity of the SARS-CoV-2 antibody response against full-length spike protein, receptor-binding domain and N-terminal domain of the spike protein, and the nucleocapsid protein. We carried out the assay on samples collected from three sample groups: subjects diagnosed with COVID-19 from the U.S. Army hospital at Camp Humphreys in Pyeongtaek, South Korea; healthcare administrators from the same hospital but with no reported diagnosis of COVID-19; and pre-pandemic samples. We found that the new CoV-specific multiplex assay was highly sensitive allowing plasma samples to be diluted 1:30,000 with a robust signal. The reactivity of IgG responses to SARS-CoV-2 nucleocapsid protein and IgM responses to SARS-CoV-2 spike protein could distinguish COVID-19 samples from non-COVID-19 and pre-pandemic samples. The data from the three sample groups also revealed a unique pattern of cross-reactivity between SARS-CoV-2 and SARS-CoV-1, MERS-CoV, and seasonal coronaviruses HKU1 and OC43. Our findings show that the CoV-2 IgM response is highly specific while the CoV-2 IgG response is more cross-reactive across a range of human CoVs and also showed that IgM and IgG responses show distinct patterns of epitope specificity. In summary, this multiplex assay was able to distinguish samples by COVID-19 status and characterize distinct trends in terms of cross-reactivity and fine-specificity in antibody responses, underscoring its potential value in diagnostic or serosurveillance efforts.

Breadth of humoral immune responses to the C-terminus of the circumsporozoite protein is associated with protective efficacy induced by the RTS,S malaria vaccine.

The circumsporozoite protein (CSP) is the main surface antigen of malaria sporozoites, a prime vaccine target, and is known to have polymorphisms in the C-terminal region. Vaccines using a single allele may have lower efficacy against genotypic variants. Recent studies have found evidence suggesting the efficacy of the CSP-based RTS,S malaria vaccine may be limited against P. falciparum CSP alleles that diverge from the 3D7 vaccine allele, particularly in this polymorphic C-terminal region. In order to assess the breadth of the RTS,S-induced antibody responses against CSP C-terminal antigenic variants, we used a novel multiplex assay to measure reactivity of serum samples from a recent RTS,S study against C-terminal peptides from 3D7 and seven additional CSP alleles that broadly represent the genetic diversity found in circulating P. falciparum field isolates. We found that responses to the variants showed, on average, a ~ 30-fold reduction in reactivity relative to the vaccine-matched 3D7 allele. The extent of this reduction, ranging from 21 to 69-fold, correlated with the number of polymorphisms between the variants and 3D7. We calculated antibody breadth of each sample as the median relative reactivity to the seven CSP variants compared to 3D7. Surprisingly, protection from 3D7 challenge in the RTS,S study was associated with higher C-terminal antibody breadth. These findings suggest CSP C-terminal-specific avidity or fine-specificity may play a role in RTS,S-mediated protection and that breadth of C-terminal CSP-specific antibody responses may be a marker of protection.

Multifunctional Antibodies Are Induced by the RTS,S Malaria Vaccine and Associated With Protection in a Phase 1/2a Trial.

BACKGROUND: RTS,S is the leading malaria vaccine candidate but only confers partial efficacy against malaria in children. RTS,S is based on the major Plasmodium falciparum sporozoite surface antigen, circumsporozoite protein (CSP). The induction of anti-CSP antibodies is important for protection; however, it is unclear how these protective antibodies function. METHODS: We quantified the induction of functional anti-CSP antibody responses in healthy malaria-naive adults (N = 45) vaccinated with RTS,S/AS01. This included the ability to mediate effector functions via the fragment crystallizable (Fc) region, such as interacting with human complement proteins and Fcγ-receptors (FcγRs) that are expressed on immune cells, which promote various immunological functions. RESULTS: Our major findings were (1) RTS,S-induced antibodies mediated Fc-dependent effector functions, (2) functional antibodies were generally highest after the second vaccine dose, (3) functional antibodies targeted multiple regions of CSP, (4) participants with higher levels of functional antibodies had a reduced probability of developing parasitemia following homologous challenge (P < .05), and (5) nonprotected subjects had higher levels of anti-CSP IgM. CONCLUSIONS: Our data suggest a role for Fc-dependent antibody effector functions in RTS,S-induced immunity. Enhancing the induction of these functional activities may be a strategy to improve the protective efficacy of RTS,S or other malaria vaccines. CLINICAL TRIALS REGISTRATION: NCT00075049.

Optimized flow cytometric protocol for the detection of functional subsets of low frequency antigen-specific CD4+ and CD8+ T cells.

Detection of low-frequency cells using flow cytometry is challenging, as the sensitivity of the analysis is dependent on the signal-to-noise ratio, and a cell frequency of 1 in 10,000 cells is accepted as the lower limit of detection for standard flow cytometry. A solution to this problem is to pre-enrich rare cell populations using magnetic-bead conjugated antibodies targeting lineage or activation markers. For measuring vaccine or pathogen induced immune responses, this method drastically increases the signal-to-noise ratio by enriching only activated (i.e., antigen-specific) cells and excluding all other peripheral blood leukocytes from the subsequent analysis. To date, magnetic enrichment of antigen-specific cells has only been described for CD4+ T cells processed for surface staining. The current study significantly expands the methodology to allow detection of antigen-specific CD8+ T cells and analysis of cells that had been processed for intracellular staining.•The protocol described here allows magnetic enrichment of PBMCs after fixation and intracellular staining steps without increasing the non-specific background.•The protocol is adapted to automated enrichment-mode on flow cytometers.•The procedure boosts the sensitivity of the flow cytometry analysis by significantly increasing the sample size of functional antigen-specific cells without skewing the composition of the functional cells pool.

Role of Opsonophagocytosis in Immune Protection against Malaria.

The quest for immune correlates of protection continues to slow vaccine development. To date, only vaccine-induced antibodies have been confirmed as direct immune correlates of protection against a plethora of pathogens. Vaccine immunologists, however, have learned through extensive characterizations of humoral responses that the quantitative assessment of antibody responses alone often fails to correlate with protective immunity or vaccine efficacy. Despite these limitations, the simple measurement of post-vaccination antibody titers remains the most widely used approaches for vaccine evaluation. Developing and performing functional assays to assess the biological activity of pathogen-specific responses continues to gain momentum; integrating serological assessments with functional data will ultimately result in the identification of mechanisms that contribute to protective immunity and will guide vaccine development. One of these functional readouts is phagocytosis of antigenic material tagged by immune molecules such as antibodies and/or complement components. This review summarizes our current understanding of how phagocytosis contributes to immune defense against pathogens, the pathways involved, and defense mechanisms that pathogens have evolved to deal with the threat of phagocytic removal and destruction of pathogens.

Comparison of ELISA with electro-chemiluminescence technology for the qualitative and quantitative assessment of serological responses to vaccination.

BACKGROUND: Profiling immune responses induced by either infection or vaccination can provide insight into identification of correlates of protection. Furthermore, profiling of serological responses can be used to identify biomarkers indicative of exposure to pathogens. Conducting such immune surveillance requires readout methods that are high-throughput, robust, and require small sample volumes. While the enzyme-linked immunosorbent assay (ELISA) is the classical readout method for assessing serological responses, the advent of multiplex assays has significantly increased the throughput and capacity for immunoprofiling. This report describes the development and assay performance (sensitivity, linearity of detection, requirement for multiple dilutions for each sample, intra- and inter-assay variability) of an electro-chemiluminescence (ECLIA)-based multiplex assay. METHODS: The current study describes the development of a multiplex ECLIA-based assay and characterizes the sensitivity, linear range, and inter- and intra-assay variability of the ECLIA platform and its agreement with the traditional ELISA. Special emphasis was placed on potential antigenic competition when testing closely related antigens in the multiplex format. RESULTS: Multiplexing of antigens in ECLIA provides significant practical benefits in terms of reducing sample volume requirements and experimental time. Beyond the practical advantages of multiplexing, the ECLIA provides superior assay performance when compared to the ELISA. Not only does ECLIA show good agreement with the ELISA assay, but the linear range of ECLIA is also sufficiently wide to permit single-dilution measurements of concentration without the need to do serial dilutions. The lack of antigenic competition allows the simultaneous testing of closely related antigens, such as plate antigens representing different alleles of the same protein, which can inform about cross-reactivities-or lack thereof-of serological responses. CONCLUSION: The advantages of the newly developed tool for assessing the antigen profiles of serological responses may ultimately lead to the identification of biomarkers associated with various disease stages and or protection against disease.

Combining immunoprofiling with machine learning to assess the effects of adjuvant formulation on human vaccine-induced immunity.

Adjuvants produce complex, but often subtle, effects on vaccine-induced immune responses that, nonetheless, play a critical role in vaccine efficacy. In-depth profiling of vaccine-induced cytokine, cellular, and antibody responses ("immunoprofiling") combined with machine-learning holds the promise of identifying adjuvant-specific immune response characteristics that can guide rational adjuvant selection. Here, we profiled human immune responses induced by vaccines adjuvanted with two similar, clinically relevant adjuvants, AS01B and AS02A, and identified key distinguishing characteristics, or immune signatures, they imprint on vaccine-induced immunity. Samples for this side-by-side comparison were from malaria-naïve individuals who had received a recombinant malaria subunit vaccine (AMA-1) that targets the pre-erythrocytic stage of the parasite. Both adjuvant formulations contain the same immunostimulatory components, QS21 and MPL, thus this study reveals the subtle impact that adjuvant formulation has on immunogenicity. Adjuvant-mediated immune signatures were established through a two-step approach: First, we generated a broad immunoprofile (serological, functional and cellular characterization of vaccine-induced responses). Second, we integrated the immunoprofiling data and identify what combination of immune features was most clearly able to distinguish vaccine-induced responses by adjuvant using machine learning. The computational analysis revealed statistically significant differences in cellular and antibody responses between cohorts and identified a combination of immune features that was able to distinguish subjects by adjuvant with 71% accuracy. Moreover, the in-depth characterization demonstrated an unexpected induction of CD8+ T cells by the recombinant subunit vaccine, which is rare and highly relevant for future vaccine design.