Faculty Bibliography

Liquid-chromatography mass spectrometry is commonly used to identify and quantify metabolites from biological samples to gain insight into human physiology and pathology. Metabolites and their abundance in biological samples are labile and sensitive to variations in collection conditions, handling and processing. Variations in sample handling could influence metabolite levels in ways not related to biology, ultimately leading to the misinterpretation of results. For example, anticoagulants and preservatives modulate enzyme activity and metabolite oxidization. Temperature may alter both enzymatic and non-enzymatic chemistry. The potential for variation induced by collection conditions is particularly important when samples are collected in remote locations without immediate access to specimen processing. Data are needed regarding the variation introduced by clinical sample collection processes to avoid introducing artifact biases. In this study, we used metabolomics and lipidomics approaches paired with univariate and multivariate statistical analyses to assess the effects of anticoagulant, temperature, and time on healthy human plasma samples collected to provide guidelines on sample collection, handling, and processing for vaccinology. Principal component analyses demonstrated clustering by sample collection procedure and that anticoagulant type had the greatest effect on sample metabolite variation. Lipids such as glycerophospholipids, acylcarnitines, sphingolipids, diacylglycerols, triacylglycerols, and cholesteryl esters are significantly affected by anticoagulant type as are amino acids such as aspartate, histidine, and glutamine. Most plasma metabolites and lipids were unaffected by storage time and temperature. Based on this study, we recommend samples be collected using a single anticoagulant (preferably EDTA) with sample processing at <24 h at 4 °C.

Background/UNASSIGNED:Clinical, virologic, and immunologic characteristics of Zika virus (ZIKV) infections in US patients are poorly defined. Methods/UNASSIGNED:US subjects with suspected ZIKV infection were enrolled. Clinical data and specimens were prospectively collected for ZIKV RNA detection and serologic and cellular assays. Confirmed ZIKV infection (cases) and ZIKV-negative (controls) subjects were compared. Dengue-experienced and dengue-naïve cases were also compared. Results/UNASSIGNED:= .046). In intracellular cytokine staining assays, the ZIKV proteins targeted most often by peripheral blood mononuclear cells from cases were structural proteins C and E for CD4+ T cells and nonstructural proteins NS3, NS5, and NS4B for CD8+ T cells. Conclusions/UNASSIGNED:ZIKV RNA detection was more frequent and prolonged in whole-blood specimens. Immunoglobulin G (IgG) and neutralizing antibodies, but not IgM, were influenced by prior dengue infection. Robust cellular responses to E and nonstructural proteins have potential vaccine development implications.

The re-emergence of Zika virus (ZIKV) in the western hemisphere has most significantly affected dengue virus (DENV) endemic regions. Due to the geographical overlap between these two closely related flaviviruses, numerous individuals who suffered ZIKV infection during recent outbreaks may have also previously been exposed to DENV. As such, the impact of pre-existing dengue immunity on immune responses to ZIKV has been an area of focused research and interest. To understand how B cell responses to a ZIKV infection may be modulated by prior dengue exposures, we compared and contrasted plasmablast repertoire and specificity between two ZIKV-infected individuals, one dengue-naïve (ZK018) and the other dengue-experienced (ZK016). In addition to examining serological responses, we generated 59 patient plasmablast-derived monoclonal antibodies (mAbs) to define the heterogeneity of the early B cell response to ZIKV. Both donors experienced robust ZIKV-induced plasmablast expansions early after infection, with comparable mutational frequencies in their antibody variable genes. However, notable differences were observed in plasmablast clonality and functional reactivity. Plasmablasts from the dengue-experienced donor ZK016 included cells with shared clonal origin, while ZK018 mAbs were entirely clonally unrelated. Both at the mAb and plasma level, ZK016 antibodies displayed extensive cross-reactivity to DENV1-4, and preferentially neutralized DENV compared to ZIKV. In contrast, the neutralization activity of ZK018 mAbs was primarily directed towards ZIKV, and fewer mAbs from this donor were cross-reactive, with the cross-reactive phenotype largely limited to fusion loop-specific mAbs. ZK016 antibodies caused greater enhancement of DENV2 infection of FcRγ-expressing cells overall compared to ZK018, with a striking difference at the plasma level. Taken together, these data strongly suggest that the breadth and protective capacity of the initial antibody responses after ZIKV infection may depend on the dengue immune status of the individual. These findings have implications for vaccine design, given the likelihood that future epidemics will involve both dengue-experienced and naïve populations.

Herpes zoster (HZ) (shingles) is the clinical manifestation of varicella-zoster virus (VZV) reactivation. HZ typically develops as people age, due to decreased cell-mediated immunity. However, the importance of antibodies for immunity against HZ prevention remains to be understood. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated HZ vaccine (Zostavax). Direct enumeration of VZV-specific antibody-secreting cells (ASCs) via enzyme-linked immunosorbent spot assay (ELISPOT assay) showed that Zostavax can induce both IgG and IgA ASCs 7 days after vaccination but not IgM ASCs. The VZV-specific ASCs range from 33 to 55% of the total IgG ASCs. Twenty-five human VZV-specific monoclonal antibodies (MAbs) were cloned and characterized from single-cell-sorted ASCs of five subjects (>60 years old) who received Zostavax. These MAbs had an average of ∼20 somatic hypermutations per VH gene, similar to those seen after seasonal influenza vaccination. Fifteen of the 25 MAbs were gE specific, whereas the remaining MAbs were gB, gH, or gI specific. The most potent neutralizing antibodies were gH specific and were also able to inhibit cell-to-cell spread of the virus in vitro Most gE-specific MAbs were able to neutralize VZV, but they required the presence of complement and were unable to block cell-to-cell spread. These data indicate that Zostavax induces a memory B cell recall response characterized by anti-gE > anti-gI > anti-gB > anti-gH antibodies. While antibodies to gH could be involved in limiting the spread of VZV upon reactivation, the contribution of anti-gE antibodies toward protective immunity after Zostavax needs further evaluation.IMPORTANCE Varicella-zoster virus (VZV) is the causative agent of chickenpox and shingles. Following infection with VZV, the virus becomes latent and resides in nerve cells. Age-related declines in immunity/immunosuppression can result in reactivation of this latent virus, causing shingles. It has been shown that waning T cell immunity correlates with an increased incidence of VZV reactivation. Interestingly, serum with high levels of VZV-specific antibodies (VariZIG; IV immunoglobulin) has been administered to high-risk populations, e.g., immunocompromised children, newborns, and pregnant women, after exposure to VZV and has shown some protection against chickenpox. However, the relative contribution of antibodies against individual surface glycoproteins toward protection from shingles in elderly/immunocompromised individuals has not been established. Here, we examined the breadth and functionality of VZV-specific antibodies after vaccination with the live attenuated VZV vaccine Zostavax in humans. This study will add to our understanding of the role of antibodies in protection against shingles.

New tools are needed to understand human immunodeficiency virus central nervous system involvement. Testing 15 cerebrospinal fluid (CSF) samples for p24 antigen, using a high-sensitivity assay, we found a strong correlation trend between CSF p24 concentration and worse neuropsychological performance.

Licensure of an HIV vaccine could reduce or eliminate HIV among vulnerable populations. However, vaccine effectiveness could be undermined by risk compensation (RC), defined by an increase in risky behavior due to a belief that the vaccine will confer protection. Interest in an HIV vaccine for reasons indicative of RC may serve as an indicator of actual RC in a postlicensure era. This study assessed factors associated with interest in an HIV vaccine for reasons indicative of RC among African American women aged 18 to 55 years, recruited from a hospital-based family planning clinic in Atlanta, Georgia ( N = 321). Data were collected using audio-computer-assisted surveys. Survey items were guided by risk homeostasis theory and social cognitive theory. Multivariable logistic regression was used to assess determinants of interest in an HIV vaccine for reasons indicative of RC. Thirty-eight percent of the sample expressed interest in an HIV vaccine for at least one reason indicative of RC. In the final model, interest in an HIV vaccine for reasons indicative of RC was positively associated with higher impulsivity, perceived benefits of sexual risk behaviors, and perceived benefits of HIV vaccination; it was negatively associated with having at least some college education, positive future orientation, and self-efficacy for sex refusal. Results suggest that demographic, personality, and theory-based psychosocial factors are salient to wanting an HIV vaccine for reasons indicative of RC, and underscore the need for risk-reduction counseling alongside vaccination during the eventual rollout of an HIV vaccine.

Antigen-specific CD4 and CD8 T cells are important components of the immune response toMycobacterium tuberculosis, yet little information is currently known regarding how the breadth, specificity, phenotype, and function ofM. tuberculosis-specific T cells correlate withM. tuberculosisinfection outcome in humans. To facilitate evaluation of humanM. tuberculosis-specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay requiring low blood sample volumes. We describe here the optimization and standardization of a microtiter plate-based, diluted whole blood stimulation assay utilizing overlapping peptide pools corresponding to a functionally diverse panel of 60M. tuberculosisAgs. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50 μl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and interassay variability, and implemented testing of the assay in diverse cohorts ofM. tuberculosis-unexposed healthy adults, foreign-born adults with latentM. tuberculosisinfection residing in the United States, and tuberculosis household contacts with latentM. tuberculosisinfection in a tuberculosis-endemic setting in Kenya. TheM. tuberculosis-specific T cell response spectrum assay further enhances the immunological toolkit available for evaluatingM. tuberculosis-specific T cell responses across different states ofM. tuberculosisinfection, and can be readily implemented in resource-limited settings. Moreover, application of the assay to longitudinal cohorts will facilitate evaluation of treatment- or vaccine-induced changes in the breadth and specificity of Ag-specific T cell responses, as well as identification ofM. tuberculosis-specific T cell responses associated withM. tuberculosisinfection outcomes.

Background/UNASSIGNED:HVTN 505 was an HIV-1 preventive vaccine efficacy trial of a DNA/recombinant adenovirus serotype 5 (rAd5) vaccine regimen. We assessed antibody responses measured one month post-final vaccination (Month 7) as correlates of HIV-1 acquisition risk. Methods/UNASSIGNED:Binding antibody responses were quantified in serum samples from 25 primary endpoint vaccine cases (diagnosed with HIV-1 infection between Month 7 and 24) and 125 randomly sampled frequency-matched vaccine controls (HIV-1 negative at Month 24). We pre-specified for a primary analysis tier, six antibody response biomarkers that measure IgG and IgA binding to Env proteins and two previously assessed T-cell response biomarkers. Results/UNASSIGNED:Envelope-specific IgG responses were significantly correlated with decreased HIV-1 risk. Moreover, the interaction of IgG responses and Env-specific CD8+ T-cell polyfunctionality score had a highly significant association with HIV-1 risk after adjustment for multiple comparisons. Conclusions/UNASSIGNED:Vaccinees with higher levels of Env IgG have significantly decreased HIV-1 risk when CD8+ T-cell responses are low. Moreover, vaccinees with high CD8+ T-cell responses generally have low risk, and those with low CD8+ T-cell and low Env antibody responses have high risk. These findings suggest the critical importance of inducing a robust IgG Env response when the CD8+ T-cell response is low.

We studied the relationship between varicella-zoster virus (VZV) DNAemia and development of VZV-specific immunity after administration of live-attenuated zoster vaccine. VZV-DNAemia, detected by polymerase chain reaction (PCR), and VZV-specific effector (Teff) and memory (Tmem) T cells, was measured in 67 vaccinees. PCR was positive in 56% (9 direct, 28 nested) on day 1 and in 16% (1 direct, 10 nested) on day 14. Teff progressively increased in direct-PCR-positive vaccinees up to day 30, but Tmem did not. Conversely, Tmem, but not Teff, increased in direct-PCR-negative vaccinees on day 7. The kinetics of these immune responses and VZV DNAemia suggested that direct-PCR sample positive represented viremia.

BACKGROUND:Delayed completion of human papillomavirus vaccination (4vHPV) series is common. We sought to identify factors associated with delay. METHODS:This substudy was part of a large prospective, multi-site study recruiting 9-17 year old girls at the time of their third 4vHPV dose to assess immunogenicity associated with prolonged dosing intervals. At participating sites, parents/legal guardians (caregivers) of all enrolled girls (9-17 years old) and enrolled girls aged 14-17 years were approached for participation. Caregivers completed a questionnaire measuring adolescent and caregiver sociodemographic characteristics, caregiver attitudes and beliefs about on-schedule HPV vaccination and HPV vaccine safety, adolescent's health behaviors, barriers to accessing health care, provider office vaccination practices and a Rapid Estimate of Adult Literacy in Medicine (REALM). Participating girls completed a separate questionnaire measuring their attitudes and beliefs about on-schedule HPV vaccination and HPV vaccine safety. Delay was defined as receiving the third 4vHPV dose >12 months after the first. Bivariate, multinomial logistic regression and multivariate logistic regression analyses were used to identify factors predicting delayed completion. RESULTS:Questionnaires were completed by 482 caregivers and 386 adolescents; 422 caregivers completed a REALM. Delayed 4vHPV dosing occurred in most adolescents (67%). In multivariate analyses, predictors of delayed completion included caregiver demographic factors (self-reported black vs. white race and high school or less education vs. college or more) and an interaction between caregiver's inability to get an immunization appointment as soon as needed and adolescent's type of insurance. CONCLUSIONS:Caregiver's race and educational level, accessibility of immunization appointments, and adolescent's insurance type were found to be related to delays in completion of 4vHPV, but caregiver or adolescent attitudes and beliefs about on-schedule HPV vaccination or HPV vaccine safety were not. Therefore, interventions to improve adherence to recommended vaccination schedules could benefit from a focus on improving access to immunizations. ClinicalTrials.gov (NCT01030562).