Sutterella and its metabolic pathways positively correlate with vaccine-elicited antibody responses in infant rhesus macaques

IntroductionIt is becoming clearer that the microbiota helps drive responses to vaccines; however, little is known about the underlying mechanism. In this study, we aimed to identify microbial features that are associated with vaccine immunogenicity in infant rhesus macaques.MethodsWe analyzed 16S rRNA gene sequencing data of 215 fecal samples collected at multiple timepoints from 64 nursery-reared infant macaques that received various HIV vaccine regimens. PERMANOVA tests were performed to determine factors affecting composition of the gut microbiota throughout the first eight months of life in these monkeys. We used DESeq2 to identify differentially abundant bacterial taxa, PICRUSt2 to impute metagenomic information, and mass spectrophotometry to determine levels of fecal short-chain fatty acids and bile acids.ResultsComposition of the early-life gut microbial communities in nursery-reared rhesus macaques from the same animal care facility was driven by age, birth year, and vaccination status. We identified a Sutterella and a Rodentibacter species that positively correlated with vaccine-elicited antibody responses, with the Sutterella species exhibiting more robust findings. Analysis of Sutterella-related metagenomic data revealed five metabolic pathways that significantly correlated with improved antibody responses following HIV vaccination. Given these pathways have been associated with short-chain fatty acids and bile acids, we quantified the fecal concentration of these metabolites and found several that correlated with higher levels of HIV immunogen-elicited plasma IgG.DiscussionOur findings highlight an intricate bidirectional relationship between the microbiota and vaccines, where multiple aspects of the vaccination regimen modulate the microbiota and specific microbial features facilitate vaccine responses. An improved understanding of this microbiota–vaccine interplay will help develop more effective vaccines, particularly those that are tailored for early life

CHARACTERIZATION OF HUMORAL RESPONSE TO EPSTEIN-BARR VIRUS (EBV) INFECTION AND PROTEIN KINASES INVOLVED IN VIRAL REPLICATION

Epstein-Barr virus (EBV) is a human herpesvirus that infects more than 95% of the world’s population by adulthood. Like other herpesviruses, EBV has a biphasic infection cycle which includes latent and lytic programs. Upon primary infection, the virus establishes a lifelong latency in memory B lymphocytes characterized by a highly restricted gene expression and lack of virion production. Signals responsible for differentiation of mature B lymphocytes into plasma cells may activate viral lytic program in latently infected cells leading to a coordinated expression of majority of viral genes and production of progeny virions. Although most infections are asymptomatic, the primary clinical manifestation of EBV infection is infectious mononucleosis (IM), which accounts for 125,000 cases each year in the US. In addition, EBV is associated with several malignancies including Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin’s lymphoma and gastric carcinoma, which account for approximately 200,000 new cases and 140,000 deaths each year worldwide. Cellular protein kinases have been reported to play a role in the viral life cycle, but their influence on different stages of EBV life cycle has not been studied extensively. Therefore, in the first part of my dissertation, I investigated the involvement of cellular protein kinases in the EBV lytic program. To this end, signaling pathways mediating Epstein-Barr virus (EBV) reactivation by antigen-bound B-cell receptor (BCR) were analyzed using a panel of 80 protein kinase inhibitors. Broad range protein kinase inhibitors Staurosporin, K252A, and PKC-412 significantly reduced the EBV genome copy numbers measured 48 hours after reactivation, perhaps due to their higher toxicity. In addition, selected inhibitors of the phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and nuclear factor B (NF-B) pathways, glycogen synthase kinase 3 (GSK-3), platelet-derived growth factor receptor-associated tyrosine kinase (PDGFRK), and epidermal growth factor receptor-associated tyrosine kinase (EGFRK) significantly reduced the EBV genome copy numbers as well. Of those, only U0126 and Erbstatin analog, which inhibit MAPK pathway and EGFRK respectively, did not inhibit viral reactivation assessed by expression of the EBV early protein, EA-D. None of the tested compounds, except for K252A, affected the activity of the EBV-encoded protein kinase in vitro. These results show that EBV reactivation induced by BCR signaling is mainly mediated through PI3K and PKC, whereas MAPK might be involved in later stages of viral replication. The goal of the second part of my dissertation was to comprehensively characterize IgG response to EBV infection in humans. Currently, EBV serology is used to confirm the EBV infection status of a patient, but there are no clinical markers available to date to diagnose EBV-associated cancers. Out of the 80 proteins that the virus expresses, EBV serology relies on detection of antibodies against only three antigens: BFRF3 (viral capsid antigen or VCA), BMRF1 (early antigen or EA), and BKRF1 (nuclear antigen or NA). Antibodies against two of these antigens, VCA and NA, are produced by almost all EBV carriers for their entire lifetime, and therefore, detection of these antibodies makes it impossible to differentiate between EBV carriers and EBV-associated cancer patients. To address this deficiency, the current project sought to determine if the human antibody response to EBV significantly differed between asymptomatic EBV carriers versus EBV-associated cancer patients. To this end, FLAG-tagged genes representing 90% of the EBV proteome were cloned into a mammalian expression vector, individually expressed in HEK293 cells, and immunoblotted with a monoclonal FLAG antibody (FLAG mAb), and each of the 55 human serum samples in the panel under study. My data confirmed the efficacy of the currently used markers (BFRF3, BKRF1), and several other previously studied potential markers (BNRF1, BdRF1, BLRF2, EBNA-3A, EBNA-3B, BHRF1, and BZLF1). I have also identified 5 novel immunodominant viral antigens (BPLF1, BKRF4, BRRF2, BLLF3, and BSLF2/BMLF1) that have never been described as major antigens of EBV. In addition, I have confirmed previous observations that the antibody response in sera from EBV-associated NPC patients differs significantly from that in sera from EBV seropositive individuals who did not develop an EBV-associated malignancy. I have identified multiple EBV antigens (BXLF1, BPLF1, BMRF1, BHRF1, BKRF4, BLLF3, BZLF1 and BSLF2/BMLF1) that were detected at higher frequency by sera from NPC patients. One of these antigens, BXLF1, was detected by 90% of sera from NPC patients, but only by 10% of the matched control sera, thus suggesting that this antigen may serve as a biomarker of NPC. These data advocate the feasibility of developing a serological assay that may be able to diagnose EBV-associated malignancies at early stages of the disease. Moreover, further studies are warranted to explore the prognostic value of such biomarkers, which could identify individuals prone to developing EBV-associated malignancies, allow their close monitoring, and early intervention

Human Maternal-Fetal Interface Cellular Models to Assess Antiviral Drug Toxicity during Pregnancy

Pregnancy is a period of elevated risk for viral disease severity, resulting in serious health consequences for both the mother and the fetus; yet antiviral drugs lack comprehensive safety and efficacy data for use among pregnant women. In fact, pregnant women are systematically excluded from therapeutic clinical trials to prevent potential fetal harm. Current FDA-recommended reproductive toxicity assessments are studied using small animals which often do not accurately predict the human toxicological profiles of drug candidates. Here, we review the potential of human maternal-fetal interface cellular models in reproductive toxicity assessment of antiviral drugs. We specifically focus on the 2- and 3-dimensional maternal placental models of different gestational stages and those of fetal embryogenesis and organ development. Screening of drug candidates in physiologically relevant human maternal-fetal cellular models will be beneficial to prioritize selection of safe antiviral therapeutics for clinical trials in pregnant women

Non-invasive estimation of hemoglobin, bilirubin and oxygen saturation of neonates simultaneously using whole optical spectrum analysis at point of care

The study was aimed to evaluate the performance of a newly developed spectroscopy-based non-invasive and noncontact device (SAMIRA) for the simultaneous measurement of hemoglobin, bilirubin and oxygen saturation as an alternative to the invasive biochemical method of blood sampling. The accuracy of the device was assessed in 4318 neonates having incidences of either anemia, jaundice, or hypoxia. Transcutaneous bilirubin, hemoglobin and blood saturation values were obtained by the newly developed instrument which was corroborated with the biochemical blood tests by expert clinicians. The instrument is trained using Artificial Neural Network Analysis to increase the acceptability of the data. The artificial intelligence incorporated within the instrument determines the disease condition of the neonate. The Pearson's correlation coefficient, r was found to be 0.987 for hemoglobin estimation and 0.988 for bilirubin and blood gas saturation respectively. The bias and the limits of agreement for the measurement of all the three parameters were within the clinically acceptance limit

Analytical Treatment Interruption after Short-Term Antiretroviral Therapy in a Postnatally Simian-Human Immunodeficiency Virus-Infected Infant Rhesus Macaque Model

Novel interventions that do not rely on daily adherence to ART are needed to achieve sustained viral remission for perinatally infected children, who currently rely on lifelong ART. Considering the risks and expense associated with ART interruption trials, the identification of biomarkers of viral rebound will prioritize promising therapeutic intervention strategies, including anti-HIV Env protein therapeutics. However, comprehensive studies to identify those biomarkers are logistically challenging in human infants, demanding the need for relevant nonhuman primate models of HIV rebound. In this study, we developed an infant RM model of oral infection with simian-human immunodeficiency virus expressing clade C HIV Env and short-term ART followed by ATI, longitudinally characterizing the immune responses to viral infection during ART and after ATI. Additionally, we compared this infant RM model to an analogous adult RM rebound model and identified virologic and immunologic correlates of the time to viral rebound after ATI.To achieve long-term viral remission in human immunodeficiency virus (HIV)-infected children, novel strategies beyond early antiretroviral therapy (ART) will be necessary. Identifying clinical predictors of the time to viral rebound upon ART interruption will streamline the development of novel therapeutic strategies and accelerate their evaluation in clinical trials. However, identification of these biomarkers is logistically challenging in infants, due to sampling limitations and the potential risks of treatment interruption. To facilitate the identification of biomarkers predicting viral rebound, we have developed an infant rhesus macaque (RM) model of oral simian-human immunodeficiency virus (SHIV) SHIV.CH505.375H.dCT challenge and analytical treatment interruption (ATI) after short-term ART. We used this model to characterize SHIV replication kinetics and virus-specific immune responses during short-term ART or after ATI and demonstrated plasma viral rebound in 5 out of 6 (83%) infants. We observed a decline in humoral immune responses and partial dampening of systemic immune activation upon initiation of ART in these infants. Furthermore, we monitored SHIV replication and rebound kinetics in infant and adult RMs and found that both infants and adults demonstrated equally potent virus-specific humoral immune responses. Finally, we validated our models by confirming a well-established correlate of the time to viral rebound, namely, the pre-ART plasma viral load, as well as identified additional potential humoral immune correlates. Thus, this model of infant ART and viral rebound can be used and further optimized to define biomarkers of viral rebound following long-term ART as well as to preclinically assess novel therapies to achieve a pediatric HIV functional cure

Maternal Broadly Neutralizing Antibodies Can Select for Neutralization-Resistant, Infant-Transmitted/Founder HIV Variants

Efforts to eliminate MTCT of HIV with antiretroviral therapy (ART) have met little success, with >180,000 infant infections each year worldwide. It is therefore likely that additional immunologic strategies that can synergize with ART will be required to eliminate MTCT of HIV. To this end, understanding the role of maternal HIV Env-specific IgG antibodies in the setting of MTCT is crucial. In this study, we found that maternal-plasma broadly neutralizing antibody (bNAb) responses can select for T/F viruses that initiate infection in infants. We propose that clinical trials testing the efficacy of single bNAb specificities should not include HIV-infected pregnant women, as a single bNAb might select for neutralization-resistant infant-T/F viruses.Each year, >180,000 infants become infected via mother-to-child transmission (MTCT) of HIV despite the availability of effective maternal antiretroviral treatments, underlining the need for a maternal HIV vaccine. We characterized 224 maternal HIV envelope (Env)-specific IgG monoclonal antibodies (MAbs) from seven nontransmitting and transmitting HIV-infected U.S. and Malawian mothers and examined their neutralization activities against nontransmitted autologous circulating viruses and infant-transmitted founder (infant-T/F) viruses. Only a small subset of maternal viruses, 3 of 72 (4%), were weakly neutralized by maternal linear V3 epitope-specific IgG MAbs, whereas 6 out of 6 (100%) infant-T/F viruses were neutralization resistant to these V3-specific IgG MAbs. We also show that maternal-plasma broadly neutralizing antibody (bNAb) responses targeting the V3 glycan supersite in a transmitting woman may have selected for an N332 V3 glycan neutralization-resistant infant-T/F virus. These data have important implications for bNAb-eliciting vaccines and passively administered bNAbs in the setting of MTCT