Leigh M. Howard

A Cell-Based Systems Biology Assessment of Human Blood to Monitor Immune Responses after Influenza Vaccination

Systems biology is an approach to comprehensively study complex interactions within a biological system. Most published systems vaccinology studies have utilized whole blood or peripheral blood mononuclear cells (PBMC) to monitor the immune response after vaccination. Because human blood is comprised of multiple hematopoietic cell types, the potential for masking responses of under-represented cell populations is increased when analyzing whole blood or PBMC. To investigate the contribution of individual cell types to the immune response after vaccination, we established a rapid and efficient method to purify human T and B cells, natural killer (NK) cells, myeloid dendritic cells (mDC), monocytes, and neutrophils from fresh venous blood. Purified cells were fractionated and processed in a single day. RNA-Seq and quantitative shotgun proteomics were performed to determine expression profiles for each cell type prior to and after inactivated seasonal influenza vaccination. Our results show that transcriptomic and proteomic profiles generated from purified immune cells differ significantly from PBMC. Differential expression analysis for each immune cell type also shows unique transcriptomic and proteomic expression profiles as well as changing biological networks at early time points after vaccination. This cell type-specific information provides a more comprehensive approach to monitor vaccine responses.

Cell-Based Systems Biology Analysis of Human AS03-Adjuvanted H5N1 Avian Influenza Vaccine Responses: A Phase I Randomized Controlled Trial

Background Vaccine development for influenza A/H5N1 is an important public health priority, but H5N1 vaccines are less immunogenic than seasonal influenza vaccines. Adjuvant System 03 (AS03) markedly enhances immune responses to H5N1 vaccine antigens, but the underlying molecular mechanisms are incompletely understood. Objective and Methods We compared the safety (primary endpoint), immunogenicity (secondary), gene expression (tertiary) and cytokine responses (exploratory) between AS03-adjuvanted and unadjuvanted inactivated split-virus H5N1 influenza vaccines. In a double-blinded clinical trial, we randomized twenty adults aged 18–49 to receive two doses of either AS03-adjuvanted (n = 10) or unadjuvanted (n = 10) H5N1 vaccine 28 days apart. We used a systems biology approach to characterize and correlate changes in serum cytokines, antibody titers, and gene expression levels in six immune cell types at 1, 3, 7, and 28 days after the first vaccination. Results Both vaccines were well-tolerated. Nine of 10 subjects in the adjuvanted group and 0/10 in the unadjuvanted group exhibited seroprotection (hemagglutination inhibition antibody titer > 1:40) at day 56. Within 24 hours of AS03-adjuvanted vaccination, increased serum levels of IL-6 and IP-10 were noted. Interferon signaling and antigen processing and presentation-related gene responses were induced in dendritic cells, monocytes, and neutrophils. Upregulation of MHC class II antigen presentation-related genes was seen in neutrophils. Three days after AS03-adjuvanted vaccine, upregulation of genes involved in cell cycle and division was detected in NK cells and correlated with serum levels of IP-10. Early upregulation of interferon signaling-related genes was also found to predict seroprotection 56 days after first vaccination. Conclusions Using this cell-based systems approach, novel mechanisms of action for AS03-adjuvanted pandemic influenza vaccination were observed. Trial Registration ClinicalTrials.gov NCT01573312

Health literacy predicts pediatric dosing accuracy for liquid zidovudine.

Objectives:Little is known about adult caregivers’ ability to accurately dose pediatric antiretroviral medications. We aimed to characterize the frequency of dosing errors for liquid zidovudine using two dosing devices and to evaluate the association between HIV literacy and dosing errors in adults living with HIV infection. Design:Cross-sectional study enrolling 316 adults receiving combination antiretroviral therapy (cART) for HIV infection in Maputo Province, Mozambique. Methods:Participants were administered the HIV Literacy Test (HIV-LT) and asked to measure 2.5 ml of liquid zidovudine using both a cup and syringe. Dosing measurement errors for liquid zidovudine were defined as ‘any error’ (≥20% deviation from reference dose) and ‘major error’ (≥40% deviation from reference dose). Results:Dosing errors were common using the cup (any error: 50%, major error: 28%) and syringe (any error: 48% of participants, major error: 28%). There were no significant differences in proportions of any dosing error (P = 0.61) or major dosing errors (P = 0.82) between dosing instruments. In multivariable models, associations (P ⩽0.03) were found between higher HIV-LT score and dosing errors for both the cup [any error adjusted odds ratio, AOR: 0.91 (0.84–0.99), major error AOR: 0.84 (0.75–0.92)] and syringe [any error AOR: 0.82 (0.75–0.90), major error AOR: 0.88 (0.80–0.97)]. Conclusion:Liquid antiretroviral medications are critical for prevention and treatment of pediatric HIV infections, yet dosing errors were exceedingly common in this population and were significantly associated with lower HIV literacy levels. Targeted interventions are needed to improve HIV knowledge and skills for pediatric medication dosing, particularly for caregivers with limited literacy.

Respiratory Viral Detections During Symptomatic and Asymptomatic Periods in Young Andean Children.

Background: Viruses are commonly detected in children with acute respiratory illnesses (ARIs) and in asymptomatic children. Longitudinal studies of viral detections during asymptomatic periods surrounding ARI could facilitate interpretation of viral detections but are currently scant. Methods: We used reverse transcription polymerase chain reaction to analyze respiratory samples from young Andean children for viruses during asymptomatic periods within 8–120 days of index ARI (cough or fever). We compared viral detections over time within children and explored reverse transcription polymerase chain reaction cycle thresholds (CTs) as surrogates for viral loads. Results: At least 1 respiratory virus was detected in 367 (43%) of 859 samples collected during asymptomatic periods, with more frequent detections in periods with rhinorrhea (49%) than those without (34%, P < 0.001). Relative to index ARI with human rhinovirus (HRV), adenovirus (AdV), respiratory syncytial virus (RSV) and parainfluenza virus detected, the same viruses were also detected during 32, 22, 10 and 3% of asymptomatic periods, respectively. RSV was only detected 8–30 days after index RSV ARI, whereas HRV and AdV were detected throughout asymptomatic periods. Human metapneumovirus and influenza were rarely detected during asymptomatic periods (<3%). No significant differences were observed in the CT for HRV or AdV during asymptomatic periods relative to ARI. For RSV, CTs were significantly lower during ARI relative to the asymptomatic period (P = 0.03). Conclusions: These findings indicate that influenza, human metapneumovirus, parainfluenza virus and RSV detections in children with an ARI usually indicate a causal relationship. When HRV or AdV is detected during ARI, the causal relationship is less certain.

Molecular Epidemiology of Rhinovirus Detections in Young Children

Background. Human rhinoviruses (HRVs) are frequently detected in children with acute respiratory illnesses (ARIs) but also in asymptomatic children. We compared features of ARI with HRV species (A, B, C) and determined genotypes associated with repeated HRV detections within individuals. Methods. We used clinical data and respiratory samples obtained from children <3 years old during weekly active household-based surveillance. A random subset of samples in which HRV was detected from individuals during both ARI and an asymptomatic period within 120 days of the ARI were genotyped. Features of ARI were compared among HRV species. Concordance of genotype among repeated HRV detections within individuals was assessed. Results. Among 207 ARI samples sequenced, HRV-A, HRV-B, and HRV-C were detected in 104 (50%), 20 (10%), and 83 (40%), respectively. Presence of fever, decreased appetite, and malaise were significantly higher in children with HRV-B. When codetections with other viruses were excluded (n = 155), these trends persisted, but some did not reach statistical significance. When 58 paired sequential HRV detections during asymptomatic and ARI episodes were sequenced, only 9 (16%) were identical genotypes of HRV. Conclusions. Clinical features may differ among HRV species. Repeated HRV detections in young children frequently represented acquisition of new HRV strains.

Nasopharyngeal Pneumococcal Density and Evolution of Acute Respiratory Illnesses in Young Children, Peru, 2009-2011

We examined nasopharyngeal pneumococcal colonization density patterns surrounding acute respiratory illnesses (ARI) in young children in Peru. Pneumococcal densities were dynamic, gradually increasing leading up to an ARI, peaking during the ARI, and decreasing after the ARI. Rhinovirus co-infection was associated with higher pneumococcal densities.

Nasopharyngeal Pneumococcal Density Is Associated With Viral Activity but Not With Use of Improved Stoves Among Young Andean Children

Abstract Background Indoor smoke exposure is common in developing countries and may influence nasopharyngeal (NP) pneumococcal colonization density and risk of acute respiratory illness. We compared colonization density among Andean children living in households previously enrolled in a randomized controlled trial of a home intervention package including improved stoves to reduce smoke, kitchen sinks, and water disinfection. Methods We enrolled 260 children aged <3 years and made weekly household visits to assess for acute respiratory illness (ARI) and collect nasal swabs for respiratory virus polymerase chain reaction (PCR) testing during ARI. At monthly intervals, NP swabs were collected to determine pneumococcal colonization density through quantitative lytA PCR. We used linear quantile mixed-effects models to compare median log-transformed colonization densities among children in households randomized to the control (n = 129) versus intervention (n = 131) in sequential time points, accounting for random effects of multiple samples from individual children. Other covariates included age, sex, month, antibiotic exposure, and timing of sample collection relative to ARI with and without viral detection. Results Age and sociodemographic characteristics were similar between groups. Although no differences were observed in densities between groups, colonization density varied significantly over time in both groups, with highest densities coinciding with spring months. Time during and after virus-associated ARI was also associated with higher pneumococcal colonization density than time remote from ARIs. Conclusions A home intervention package, including improved stoves, was not associated with changes in pneumococcal densities in young Andean children. However, increasing pneumococcal density was observed with spring season and viral-associated ARIs.

Clinical Features of Human Metapneumovirus Infection in Ambulatory Children Aged 5–13 Years

Abstract We detected human metapneumovirus (HMPV) in 54 (5%) of 1055 children aged 5 to 13 years with acute respiratory illness (ARI) identified by outpatient and emergency department surveillance between November and May 2003–2009. Its clinical features were similar to those of HMPV-negative ARI, except a diagnosis of pneumonia was more likely (13% vs 4%, respectively; P = .005) and a diagnosis of pharyngitis (7% vs 24%, respectively; P = .005) was less likely in patients with HMPV- positive ARI than those with HMPV-negative ARI.

Proteomics show antigen presentation processes in human immune cells after AS03-H5N1 vaccination

Adjuvants enhance immunity elicited by vaccines through mechanisms that are poorly understood. Using a systems biology approach, we investigated temporal protein expression changes in five primary human immune cell populations: neutrophils, monocytes, natural killer cells, T cells, and B cells after administration of either an Adjuvant System 03 adjuvanted or unadjuvanted split‐virus H5N1 influenza vaccine. Monocytes demonstrated the strongest differential signal between vaccine groups. On day 3 post‐vaccination, several antigen presentation‐related pathways, including MHC class I‐mediated antigen processing and presentation, were enriched in monocytes and neutrophils and expression of HLA class I proteins was increased in the Adjuvant System 03 group. We identified several protein families whose proteomic responses predicted seroprotective antibody responses (>1:40 hemagglutination inhibition titer), including inflammation and oxidative stress proteins at day 1 as well as immunoproteasome subunit (PSME1 and PSME2) and HLA class I proteins at day 3 in monocytes. While comparison between temporal proteomic and transcriptomic results showed little overlap overall, enrichment of the MHC class I antigen processing and presentation pathway in monocytes and neutrophils was confirmed by both approaches.

A novel real-time RT-PCR assay for influenza C tested in Peruvian children

Abstract Background Influenza C virus (ICV) is associated with acute respiratory illness. Yet ICV remains under recognized, with most previous studies using only culture to identify cases. Objectives To develop a sensitive and specific real-time RT-PCR assay for ICV that allows for rapid and accurate detection in a clinical or research setting. Study design Multiple ICV sequences obtained from GenBank were analyzed, including 141 hemagglutinin-esterase (HE), 106 matrix (M), and 97 nucleoprotein (NP) sequences. Primers and probes were designed based on conserved regions. Multiple primer-probe sets were tested against multiple ICV strains. Results The ICV M and NP genes offered the most conserved sequence regions. Primers and probes based on newer sequence data offered enhanced detection of ICV, especially for low titer specimens. An NP-targeted assay yielded the best performance and was capable of detecting 10–100 RNA copies per reaction. The NP assay detected multiple clinical isolates of ICV collected in a field epidemiology study conducted in Peru. Conclusions We report a new real-time RT-PCR assay for ICV with high sensitivity and specificity.