Biological and genetic determinants of glycolysis: Phosphofructokinase isoforms boost energy status of stored red blood cells and transfusion outcomes.

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes.

Ferroptosis regulates hemolysis in stored murine and human red blood cells.

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. Here, we leveraged a diversity outbred mouse population to map the genetic drivers of fresh/stored RBC metabolism and extravascular hemolysis upon storage and transfusion in 350 mice. We identify the ferrireductase Steap3 as a critical regulator of a ferroptosis-like process of lipid peroxidation. Steap3 polymorphisms were associated with RBC iron content, in vitro hemolysis, and in vivo extravascular hemolysis both in mice and 13,091 blood donors from the Recipient Epidemiology and Donor evaluation Study. Using metabolite Quantitative Trait Loci analyses, we identified a network of gene products (FADS1/2, EPHX2 and LPCAT3) - enriched in donors of African descent - associated with oxylipin metabolism in stored human RBCs and related to Steap3 or its transcriptional regulator, the tumor protein TP53. Genetic variants were associated with lower in vivo hemolysis in thousands of single-unit transfusion recipients. Highlights: Steap3 regulates lipid peroxidation and extravascular hemolysis in 350 diversity outbred miceSteap3 SNPs are linked to RBC iron, hemolysis, vesiculation in 13,091 blood donorsmQTL analyses of oxylipins identified ferroptosis-related gene products FADS1/2, EPHX2, LPCAT3Ferroptosis markers are linked to hemoglobin increments in transfusion recipients.

Performance of a rapid recency assay for detection of early HIV infection.

The Asanté HIV-1 Rapid Recency assay's 'verification' line detected HIV infection a median of 18 days later than a nucleic acid detection assay and performed similarly to 19 other existing rapid HIV antibody tests. Pending regulatory approval, the assay could be an option with other rapid tests in national HIV-1 testing algorithms, which would allow collection of HIV recency data as part of a national screening program without requiring additional testing.

Circulating plasma-derived extracellular vesicles expressing bone and kidney markers are associated with neurocognitive impairment in people living with HIV.

Background: Although effective antiretroviral therapy (ART) has improved the life expectancy of people with HIV (PWH), the prevalence of milder forms of HIV-associated neurocognitive disorders (HAND) persist, and it is associated with systemic and neuro-inflammatory processes that could impact other organ systems. However, the complex signaling mechanisms between the bone-kidney systems and the brain in HAND remain unknown. Extracellular vesicles (EVs) play a potential role in inter-organ communication and are involved in regulating cell activity in distant tissues. In this study, we examined whether levels of EVs from bone-and kidney-related cells associate with cognitive dysfunction and explored the relationship between kidney-bone EV axis in PWH experiencing cognitive deficits. Methods: EV subtypes were characterized in plasma from 61 PWH with either cognitive impairment (CI, n = 53) or normal cognition (NC, n = 8) based on the American Academy of Neurology criteria for HIV-associated dementia (HAD, n = 11), minor cognitive motor disorder (MCMD, n = 25) or asymptomatic neurocognitive impairment (ANI, n = 17) by spectral flow cytometry. EVs were profiled with markers reflecting bone and kidney cell origin. A support vector machine learning-based model was employed for analyses of EV phenotypes to predict the cognitive dysfunction. Results: Plasma-EVs expressing osteocalcin, sclerostin, and nephrin were significantly higher in the cognitive impairment group compared to the normal cognition group. EVs bearing kidney cell markers correlated significantly with bone-derived EVs. A machine learning-based model, comprised of osteocalcin+, nephrin+, and CD24+ EVs predicted cognitive impairment in PWH on ART. Conclusion: Our study reveals that neurocognitive impairment in PWH is associated with increased levels of plasma EVs enriched with the bone markers osteocalcin and sclerostin and the kidney marker nephrin, suggesting that these EV subtypes may be novel candidate biomarkers for disease-spanning neurocognitive dysfunction. Moreover, the relationship between bone-derived EVs with kidney-derived EVs may suggest their role in mediating inter-organ crosstalk in the pathogenesis of HIV-associated cognitive impairment.

Genetic regulation of carnitine metabolism controls lipid damage repair and aging RBC hemolysis in vivo and in vitro.

ABSTRACT: Recent large-scale multiomics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on 2 separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured l-carnitine and acyl-carnitines in 13 091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation study. Genome-wide association studies against 879 000 polymorphisms identified critical genetic factors contributing to interdonor heterogeneity in end-of-storage carnitine levels, including common nonsynonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, and SLC16A9); carnitine synthesis (FLVCR1 and MTDH) and metabolism (CPT1A, CPT2, CRAT, and ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying 2 alleles of the rs12210538 SLC22A16 single-nucleotide polymorphism exhibited the lowest l-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice, and Percoll density gradients of human RBCs, showed age-dependent depletions of l-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process after chemically induced membrane lipid damage. Supplementation of stored murine RBCs with l-carnitine boosted posttransfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.

Patient and immunological factors associated with delayed clearance of mucosal SARS-CoV-2 RNA and symptom persistence.

Serial blood and mucosal samples were characterized for 102 participants enrolled a median of 7.0 days post-COVID-19 diagnosis. Mucosal RNA was detectable a median 31.5 (95% CI 20.5 - 63.5) days, with persistence ≥1 month associated with obesity (BMI ≥30, OR 3.9, 95% CI 1.2 - 13.8) but not age, sex, or chronic conditions. Fifteen participants had likely reinfection; lower serum anti-S IgG levels were associated with reinfection risk. Nearly half of participants (47%) reported symptoms lasting ≥2-3 months; persistence ≥3 months was associated with BMI ≥30 (OR = 4.2 95% CI 1.1 - 12.8) and peak anti-S and anti-NC antibody levels.

Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes.

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in the blood bank. Here we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify an association between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs), hexokinase 1, ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice, and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine levels - and the genetic traits linked to them - were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes. Highlights: Blood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers;Ancestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis;Modeled PFKP effects relate to preventing loss of the total AXP pool in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

Clinical outcomes in hospitalized plasma and platelet transfusion recipients prior to and following widespread blood donor SARS-CoV-2 infection and vaccination.

BACKGROUND: The safety of transfusion of SARS-CoV-2 antibodies in high plasma volume blood components to recipients without COVID-19 is not established. We assessed whether transfusion of plasma or platelet products during periods of increasing prevalence of blood donor SARS-CoV-2 infection and vaccination was associated with changes in outcomes in hospitalized patients without COVID-19. METHODS: We conducted a retrospective cohort study of hospitalized adults who received plasma or platelet transfusions at 21 hospitals during pre-COVID-19 (3/1/2018-2/29/2020), COVID-19 pre-vaccine (3/1/2020-2/28/2021), and COVID-19 post-vaccine (3/1/2021-8/31/2022) study periods. We used multivariable logistic regression with generalized estimating equations to adjust for demographics and comorbidities to calculate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: Among 21,750 hospitalizations of 18,584 transfusion recipients without COVID-19, there were 697 post-transfusion thrombotic events, and oxygen requirements were increased in 1751 hospitalizations. Intensive care unit length of stay (n = 11,683) was 3 days (interquartile range 1-5), hospital mortality occurred in 3223 (14.8%), and 30-day rehospitalization in 4144 (23.7%). Comparing the pre-COVID, pre-vaccine and post-vaccine study periods, there were no trends in thromboses (OR 0.9 [95% CI 0.8, 1.1]; p = .22) or oxygen requirements (OR 1.0 [95% CI 0.9, 1.1]; p = .41). In parallel, there were no trends across study periods for ICU length of stay (p = .83), adjusted hospital mortality (OR 1.0 [95% CI 0.9-1.0]; p = .36), or 30-day rehospitalization (p = .29). DISCUSSION: Transfusion of plasma and platelet blood components collected during the pre-vaccine and post-vaccine periods of the COVID-19 pandemic was not associated with increased adverse outcomes in transfusion recipients without COVID-19.

Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

Genetic polymorphisms and expression of Rhesus blood group RHCE are associated with 2,3-bisphosphoglycerate in humans at high altitude.

Red blood cell (RBC) metabolic reprogramming upon exposure to high altitude contributes to physiological human adaptations to hypoxia, a multifaceted process critical to health and disease. To delve into the molecular underpinnings of this phenomenon, first, we performed a multi-omics analysis of RBCs from six lowlanders after exposure to high-altitude hypoxia, with longitudinal sampling at baseline, upon ascent to 5,100 m and descent to sea level. Results highlighted an association between erythrocyte levels of 2,3-bisphosphoglycerate (BPG), an allosteric regulator of hemoglobin that favors oxygen off-loading in the face of hypoxia, and expression levels of the Rhesus blood group RHCE protein. We then expanded on these findings by measuring BPG in RBCs from 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. These data informed a genome-wide association study using BPG levels as a quantitative trait, which identified genetic polymorphisms in the region coding for the Rhesus blood group RHCE as critical determinants of BPG levels in erythrocytes from healthy human volunteers. Mechanistically, we suggest that the Rh group complex, which participates in the exchange of ammonium with the extracellular compartment, may contribute to intracellular alkalinization, thus favoring BPG mutase activity.

Local manufacturing processes contribute to variability in human mesenchymal stromal cell expansion while growth media supplements contribute to variability in gene expression and cell function: a Biomedical Excellence for Safer Transfusion (BEST) collaborative study.

BACKGROUND AIMS: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods, source material and culture media. The purpose of this multicenter study was to assess the impact on MSC expansion, gene expression and other characteristics when different laboratories expanded MSCs from cultures initiated with bone marrow-MSC aliquots derived from the same donor source material yet with different growth media. METHODS: Eight centers expanded MSCs using four human platelet lysate (HPL) and one fetal bovine serum (FBS) products as media supplements. The expanded cells were taken through two passages then assessed for cell count, viability, doubling time, immunophenotype, cell function, immunosuppression and gene expression. Results were analyzed by growth media and by center. RESULTS: Center methodologies varied by their local seeding density, feeding regimen, inoculation density, base media and other growth media features (antibiotics, glutamine, serum). Doubling times were more dependent on center than on media supplements. Two centers had appropriate immunophenotyping showing all MSC cultures were positive for CD105, CD73, CD90 and negative for CD34, CD45, CD14, HLA-DR. MSCs cultured in media supplemented with FBS compared with HPL featured greater T-cell inhibition potential. Gene expression analysis showed greater impact of the type of media supplement (HPL versus FBS) than the manufacturing center. Specifically, nine genes were decreased in expression and six increased when combining the four HPL-grown MSCs versus FBS (false discovery rate [FDR] <0.01), however, without significant difference between different sources of HPL (FDR <0.01). CONCLUSIONS: Local manufacturing process plays a critical role in MSC expansion while growth media may influence function and gene expression. All HPL and FBS products supported cell growth.

Prevalence of SARS-CoV-2 Viremia in Presymptomatic Blood Donors in the Delta and Omicron Variant Eras.

Presymptomatic plasma samples from 1596 donors reporting coronavirus disease 2019 infection or symptoms after blood donation were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and anti-S and anti-N antibodies. Prior infection and vaccination both protected from developing SARS-CoV-2 RNAemia and from symptomatic infection. RNAemia rates did not differ in the Delta and Omicron variant eras.

COVID-19 convalescent plasma boosts early antibody titer and does not influence the adaptive immune response.

Multiple randomized, controlled clinical trials have yielded discordant results regarding the efficacy of convalescent plasma in outpatients, with some showing an approximately 2-fold reduction in risk and others showing no effect. We quantified binding and neutralizing antibody levels in 492 of the 511 participants from the Clinical Trial of COVID-19 Convalescent Plasma in Outpatients (C3PO) of a single unit of COVID-19 convalescent plasma (CCP) versus saline infusion. In a subset of 70 participants, peripheral blood mononuclear cells were obtained to define the evolution of B and T cell responses through day 30. Binding and neutralizing antibody responses were approximately 2-fold higher 1 hour after infusion in recipients of CCP compared with saline plus multivitamin, but levels achieved by the native immune system by day 15 were almost 10-fold higher than those seen immediately after CCP administration. Infusion of CCP did not block generation of the host antibody response or skew B or T cell phenotype or maturation. Activated CD4+ and CD8+ T cells were associated with more severe disease outcome. These data show that CCP leads to a measurable boost in anti-SARS-CoV-2 antibodies but that the boost is modest and may not be sufficient to alter disease course.

Plasma extracellular vesicles and cell-free mitochondrial DNA are associated with cognitive dysfunction in treated older adults with HIV.

Extracellular vesicles (EVs) are nanoparticles with a role in intercellular communication. Cell-free mitochondrial DNA (cf-mtDNA) has been associated with cognitive dysfunction in people with HIV (PWH). We conducted a nested case-control study to test the hypothesis that plasma EVs are associated with cf-mtDNA and cognitive dysfunction in older PWH. A machine learning-based model identified total EVs, including select EV subpopulations, as well as urine cf-mtDNA and 4-meter walk time carry power to predict the neurocognitive impairment. These features resulted in an AUC-ROC of 0.845 + / - 0.109 (0.615, 1.00).

Risk of HLA antibody generation after receipt of Mirasol versus standard platelets in the MIPLATE randomized trial.

BACKGROUND: Human leukocyte antigen (HLA) alloimmunization can occur after platelet transfusion. These antibodies can complicate future platelet transfusions or organ transplantation. Animal data suggest that Mirasol pathogen reduction treatment (PRT) can prevent alloimmunization after transfusion. STUDY DESIGN AND METHODS: The MIPLATE trial enrolled 330 of a planned 660 participants with hematological malignancies at risk for grade 2 or greater bleeding. The study was halted early for futility after a planned interim analysis. Participants were randomized to receive PRT versus standard control platelets. Serum samples were collected from participants at baseline (pretransfusion), weekly for the first 4 weeks, then at days 42 and 56. HLA antibody levels were determined using a commercial multianalyte bead-based assay. HLA antibody levels were analyzed using low, medium, and high cutoffs based on prior studies. RESULTS: The rate of alloimmunization was low in both arms of the study, particularly at the high HLA antibody cutoff (total of 6 of 277 subjects at risk, or 2.2%). The risk of alloimmunization did not differ between study arms, nor did the risk of immune refractoriness to platelet transfusion. CONCLUSIONS: The data do not support the conclusion that Mirasol exerted a protective effect against alloimmunization after platelet transfusion in the MIPLATE trial.

A Stable Dried Tube Specimen for Quality Assurance and Training Programs for HIV Rapid Test for Recent Infection.

The HIV epidemic is still one of the world's most serious public health challenges, affecting about 38 million people worldwide, especially in sub-Saharan African and Southeast Asian countries. In recent years, tests have been developed to discriminate recent from long-term infection in HIV-infected populations, and these tools can help identify new outbreaks and networks of transmission and target prevention and treatment plans. New rapid tests for recent infection are being deployed in point-of-care settings; however, quality assurance programs need to be implemented to ensure consistency and reliability of the results. We have developed a dried tube specimen (DTS) stabilized with disaccharide trehalose as a quality control reagent for rapid recency testing that can be stored unrefrigerated prior to reconstitution at temperatures up to 37°C for up to 12 weeks. Analysis of 10 trehalose-stabilized DTSs showed that they maintained the same recency classification in all of the samples stored at 4°C and 37°C up to 12 weeks and at 56°C for 2 weeks, while the DTSs prepared without trehalose changed their classification from long-term to recent or recent to negative after storage at 37°C for 12 weeks. Development of DTS quality control reagents will facilitate proficiency and training programs, particularly in settings without cold chain capability in field environments. IMPORTANCE Implementation of stabilized dried tube specimens (DTSs) for quality control and training would facilitate HIV recency programs, especially in point-of-care settings without cold chain availability. This study shows that addition of the disaccharide trehalose to DTSs prior to drying the samples increased stability of the samples across a range of temperatures. This finding provides an affordable way to increase the availability of these key reagents for quality control in resource-constrained settings.

Sphingosine 1-phosphate has a negative effect on RBC storage quality.

Blood storage promotes the rapid depletion of red blood cell (RBC) high-energy adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG), which are critical regulators of erythrocyte physiology and function, as well as oxygen kinetics and posttransfusion survival. Sphingosine-1-phosphate (S1P) promotes fluxes through glycolysis. We hypothesized that S1P supplementation to stored RBC units would improve energy metabolism and posttransfusion recovery. We quantified S1P in 1929 samples (n = 643, storage days 10, 23, and 42) from the REDS RBC Omics study. We then supplemented human and murine RBCs from good storer (C57BL6/J) and poor storer strains (FVB) with S1P (1, 5, and 10 µM) before measurements of metabolism and posttransfusion recovery. Similar experiments were repeated for mice with genetic ablation of the S1P biosynthetic pathway (sphingosine kinase 1 [Sphk1] knockout [KO]). Sample analyses included metabolomics at steady state, tracing experiments with 1,2,3-13C3-glucose, proteomics, and analysis of end-of-storage posttransfusion recovery, under normoxic and hypoxic storage conditions. Storage promoted decreases in S1P levels, which were the highest in units donated by female or older donors. Supplementation of S1P to human and murine RBCs boosted the steady-state levels of glycolytic metabolites and glycolytic fluxes, ie the generation of ATP and DPG, at the expense of the pentose phosphate pathway. Lower posttransfusion recovery was observed upon S1P supplementation. All these phenomena were reversed in Sphk1 KO mice or with hypoxic storage. S1P is a positive regulator of energy metabolism and a negative regulator of antioxidant metabolism in stored RBCs, resulting in lower posttransfusion recoveries in murine models.

Frequent detection but lack of infectivity of SARS-CoV-2 RNA in presymptomatic, infected blood donor plasma.

Respiratory viruses such as influenza do not typically cause viremia; however, SARS-CoV-2 has been detected in the blood of COVID-19 patients with mild and severe symptoms. Detection of SARS-CoV-2 in blood raises questions about its role in pathogenesis as well as transfusion safety concerns. Blood donor reports of symptoms or a diagnosis of COVID-19 after donation (post-donation information, PDI) preceded or coincided with increased general population COVID-19 mortality. Plasma samples from 2,250 blood donors who reported possible COVID-19-related PDI were tested for the presence of SARS-CoV-2 RNA. Detection of RNAemia peaked at 9%-15% of PDI donors in late 2020 to early 2021 and fell to approximately 4% after implementation of widespread vaccination in the population. RNAemic donors were 1.2- to 1.4-fold more likely to report cough or shortness of breath and 1.8-fold more likely to report change in taste or smell compared with infected donors without detectable RNAemia. No infectious virus was detected in plasma from RNAemic donors; inoculation of permissive cell lines produced less than 0.7-7 plaque-forming units (PFU)/mL and in susceptible mice less than 100 PFU/mL in RNA-positive plasma based on limits of detection in these models. These findings suggest that blood transfusions are highly unlikely to transmit SARS-CoV-2 infection.

A cytotoxic-skewed immune set point predicts low neutralizing antibody levels after Zika virus infection.

Although generating high neutralizing antibody levels is a key component of protective immunity after acute viral infection or vaccination, little is known about why some individuals generate high versus low neutralizing antibody titers. Here, we leverage the high-dimensional single-cell profiling capacity of mass cytometry to characterize the longitudinal cellular immune response to Zika virus (ZIKV) infection in viremic blood donors in Puerto Rico. During acute ZIKV infection, we identify widely coordinated responses across innate and adaptive immune cell lineages. High frequencies of multiple activated cell types during acute infection are associated with high titers of ZIKV neutralizing antibodies 6 months post-infection, while stable immune features suggesting a cytotoxic-skewed immune set point are associated with low titers. Our study offers insight into the coordination of immune responses and identifies candidate cellular biomarkers that may offer predictive value in vaccine efficacy trials aimed at inducing high levels of antiviral neutralizing antibodies.