Application of a Novel Proteomic Microarray Reveals High Exposure to Diarrhoeagenic Escherichia coli among Children in Zambia Participating in a Phase I Clinical Trial.

Diarrhoeagenic E. coli (DEC) significantly contributes to the burden of diarrhoea among children. Currently, there is no approved vaccine against DEC, but several vaccines against the enterotoxigenic E. coli (ETEC) pathotype are in advanced clinical trial stages, including the ETVAX® vaccine, undergoing evaluation in Zambia. This study reports on the reactivity of antibodies from ETVAX® vaccine and placebo recipients in a phase I clinical trial to proteins derived from (DEC) other than ETEC. Plasma samples collected at two time points (prior to any vaccination and post-third dose vaccination) from 16 vaccinated and 4 placebo participants in a phase 1 clinical trial examining the safety, tolerability, and immunogenicity of ETVAX® with dmLT adjuvant were evaluated for IgG response to E. coli antigens other than ETEC using the Pan-DEC protein microarray. This was the first field application of the novel pan-DEC array as a new tool in assessing the antigenic breadth of antibody responses induced by the ETVAX vaccine, as well as to assess early life exposure to DEC pathotypes and other bacterial enteric pathogens. We observed that plasma obtained from ETVAX® and placebo recipients had high antibody reactivity to Ipa, SseC and EspB proteins. These findings suggest that there is high exposure early in life to DEC pathogens, like EPEC, EHEC, EAEC and EIEC in addition to ETEC, in the Zambian population. These immunological observations are consistent with the results of recent epidemiological studies assessing the etiology of diarrheal disease among infants and young children in Zambia.

Association of biomarkers of enteric dysfunction, systemic inflammation, and growth hormone resistance with seroconversion to oral rotavirus vaccine: A lasso for inference approach.

BACKGROUND: Rotavirus gastroenteritis remains a leading cause of morbidity and mortality despite the introduction of vaccines. Research shows there are several factors contributing to the reduced efficacy of rotavirus vaccines in low- and middle-income settings. Proposed factors include environmental enteric dysfunction (EED), malnutrition, and immune dysfunction. This study aimed to assess the effect of these factors on vaccine responses using a machine learning lasso approach. METHODS: Serum samples from two rotavirus clinical trials (CVIA 066 n = 99 and CVIA 061 n = 124) were assessed for 11 analytes using the novel Micronutrient and EED Assessment Tool (MEEDAT) multiplex ELISA. Immune responses to oral rotavirus vaccines (Rotarix, Rotavac, and Rotavac 5D) as well as a parenteral rotavirus vaccine (trivalent P2-VP8) were also measured and machine learning using the lasso approach was then applied to investigate any associations between immune responses and environmental enteric dysfunction, systemic inflammation, and growth hormone resistance biomarkers. RESULTS: Both oral and parenteral rotavirus vaccine responses were negatively associated with retinol binding protein 4 (RBP4), albeit only weakly for oral vaccines. The parenteral vaccine responses were positively associated with thyroglobulin (Tg) and histidine-rich protein 2 (HRP2) for all three serotypes (P8, P6 and P4), whilst intestinal fatty acid binding protein (I-FABP) was negatively associated with P6 and P4, but not P8, and soluble transferrin receptor (sTfR) was positively associated with P6 only. CONCLUSION: MEEDAT successfully measured biomarkers of growth, systemic inflammation, and EED in infants undergoing vaccination, with RBP4 being the only analyte associated with both oral and parenteral rotavirus vaccine responses. Tg and HRP2 were associated with responses to all three serotypes in the parenteral vaccine, while I-FABP and sTfR results indicated possible strain specific immune responses to parenteral immunization.

Characterizing Epstein-Barr virus infection of the central nervous system in Zambian adults living with HIV.

The significance of Epstein-Barr virus (EBV) detection in the cerebrospinal spinal fluid (CSF) in people living with HIV (PLWH) is not entirely understood. The detection of EBV DNA may represent active central nervous system (CNS) infection, reactivation in the setting of another CNS pathogen or due to impaired immunity, or detection of quiescent virus. We screened 470 adult PLWH in Zambia with neurological symptoms for the presence of EBV DNA in the CSF. We performed quantitative EBV PCR on the CSF and blood. We then performed quantitative EBV DNA PCR on the blood of controls with documented HIV viral suppression without CNS symptoms. The prevalence of EBV DNA in the CSF of patients with CNS symptoms was 28.9% (136/470). EBV DNA positivity was associated with younger age, shorter duration of HIV diagnosis, lower CSF glucose levels, higher CSF protein and white blood cell levels, and a positive CSF Mycobacterium tuberculosis result. The median EBV DNA load was 8000 cps/mL in both the CSF and blood with a range of 2000-2,753,000 cps/mL in the CSF and 1000 to 1,871,000 cps/mL in the blood. Molecular screening of CSF for other possible causes of infection identified Mycobacterium tuberculosis in 30.1% and cytomegalovirus (CMV) in 10.5% of samples. EBV DNA load in the blood and CSF was not associated with mortality. Our results suggest that even though EBV DNA was commonly detected in the CSF of our population, it appears to have limited clinical significance regardless of EBV DNA load.

Maternal and Infant Histo-Blood Group Antigen (HBGA) Profiles and Their Influence on Oral Rotavirus Vaccine (RotarixTM) Immunogenicity among Infants in Zambia.

Live-attenuated, oral rotavirus vaccines have significantly reduced rotavirus-associated diarrhoea morbidity and infant mortality. However, vaccine immunogenicity is diminished in low-income countries. We investigated whether maternal and infant intrinsic susceptibility to rotavirus infection via histo-blood group antigen (HBGA) profiles influenced rotavirus (ROTARIX®) vaccine-induced responses in Zambia. We studied 135 mother-infant pairs under a rotavirus vaccine clinical trial, with infants aged 6 to 12 weeks at pre-vaccination up to 12 months old. We determined maternal and infant ABO/H, Lewis, and secretor HBGA phenotypes, and infant FUT2 HBGA genotypes. Vaccine immunogenicity was measured as anti-rotavirus IgA antibody titres. Overall, 34 (31.3%) children were seroconverted at 14 weeks, and no statistically significant difference in seroconversion was observed across the various HBGA profiles in early infant life. We also observed a statistically significant difference in rotavirus-IgA titres across infant HBGA profiles at 12 months, though no statistically significant difference was observed between the study arms. There was no association between maternal HBGA profiles and infant vaccine immunogenicity. Overall, infant HBGAs were associated with RV vaccine immunogenicity at 12 months as opposed to in early infant life. Further investigation into the low efficacy of ROTARIX® and appropriate intervention is key to unlocking the full vaccine benefits for U5 children.

Use of an ETEC Proteome Microarray to Evaluate Cross-Reactivity of ETVAX® Vaccine-Induced IgG Antibodies in Zambian Children.

Developing a broadly protective vaccine covering most ETEC variants has been elusive. The most clinically advanced candidate yet is an oral inactivated ETEC vaccine (ETVAX®). We report on the use of a proteome microarray for the assessment of cross-reactivity of anti-ETVAX® IgG antibodies against over 4000 ETEC antigens and proteins. We evaluated 40 (pre-and post-vaccination) plasma samples from 20 Zambian children aged 10-23 months that participated in a phase 1 trial investigating the safety, tolerability, and immunogenicity of ETVAX® adjuvanted with dmLT. Pre-vaccination samples revealed high IgG responses to a variety of ETEC proteins including classical ETEC antigens (CFs and LT) and non-classical antigens. Post-vaccination reactivity to CFA/I, CS3, CS6, and LTB was stronger than baseline among the vaccinated compared to the placebo group. Interestingly, we noted significantly high post-vaccination responses to three non-vaccine ETEC proteins: CS4, CS14, and PCF071 (p = 0.043, p = 0.028, and p = 0.00039, respectively), suggestive of cross-reactive responses to CFA/I. However, similar responses were observed in the placebo group, indicating the need for larger studies. We conclude that the ETEC microarray is a useful tool for investigating antibody responses to numerous antigens, especially because it may not be practicable to include all antigens in a single vaccine.

Epstein-Barr Virus Detection in the Central Nervous System of HIV-Infected Patients.

Simply detecting Epstein-Barr virus deoxyribonucleic acid (EBV-DNA) is insufficient to diagnose EBV-associated diseases. The current literature around EBV-DNA detection from cerebrospinal fluid (CSF) in human immunodeficiency virus (HIV)-positive non-lymphoma patients was systematically reviewed and a meta-analysis reporting the estimated pooled prevalence in this population when PCR methods are employed, targeting different sequence segments within the EBV genome, was conducted. Using a combination of three key concepts-Epstein-Barr virus detection, central nervous system disease, and human cerebrospinal fluid-and their MeSH terms, the PubMed database was searched. A total of 273 papers reporting the detection of EBV in CNS were screened, of which 13 met the inclusion criteria. The meta-analysis revealed a pooled prevalence of EBV-DNA in CSF of 20% (CI: 12-31%). The highest pooled prevalence was from studies conducted on the African population at 39% (CI: 27-51%). The investigation of the presence of EBV-DNA in the CSF was also very varied, with several gene targets used. While most patients from the articles included in this review and meta-analysis were symptomatic of CNS disorders, the pathogenicity of EBV in non-lymphoma HIV patients when detected in CSF has still not been determined. The presence of EBV-DNA in the CNS remains a concern, and further research is warranted to understand its significance in causing CNS disorders.

Performance of Machine Learning Classifiers in Classifying Stunting among Under-Five Children in Zambia.

Stunting is a global public health issue. We sought to train and evaluate machine learning (ML) classification algorithms on the Zambia Demographic Health Survey (ZDHS) dataset to predict stunting among children under the age of five in Zambia. We applied Logistic regression (LR), Random Forest (RF), SV classification (SVC), XG Boost (XgB) and Naïve Bayes (NB) algorithms to predict the probability of stunting among children under five years of age, on the 2018 ZDHS dataset. We calibrated predicted probabilities and plotted the calibration curves to compare model performance. We computed accuracy, recall, precision and F1 for each machine learning algorithm. About 2327 (34.2%) children were stunted. Thirteen of fifty-eight features were selected for inclusion in the model using random forest. Calibrating the predicted probabilities improved the performance of machine learning algorithms when evaluated using calibration curves. RF was the most accurate algorithm, with an accuracy score of 79% in the testing and 61.6% in the training data while Naïve Bayesian was the worst performing algorithm for predicting stunting among children under five in Zambia using the 2018 ZDHS dataset. ML models aids quick diagnosis of stunting and the timely development of interventions aimed at preventing stunting.

T-Cell Responses after Rotavirus Infection or Vaccination in Children: A Systematic Review.

Cellular immunity against rotavirus in children is incompletely understood. This review describes the current understanding of T-cell immunity to rotavirus in children. A systematic literature search was conducted in Embase, MEDLINE, Web of Science, and Global Health databases using a combination of "t-cell", "rotavirus" and "child" keywords to extract data from relevant articles published from January 1973 to March 2020. Only seventeen articles were identified. Rotavirus-specific T-cell immunity in children develops and broadens reactivity with increasing age. Whilst occurring in close association with antibody responses, T-cell responses are more transient but can occur in absence of detectable antibody responses. Rotavirus-induced T-cell immunity is largely of the gut homing phenotype and predominantly involves Th1 and cytotoxic subsets that may be influenced by IL-10 Tregs. However, rotavirus-specific T-cell responses in children are generally of low frequencies in peripheral blood and are limited in comparison to other infecting pathogens and in adults. The available research reviewed here characterizes the T-cell immune response in children. There is a need for further research investigating the protective associations of rotavirus-specific T-cell responses against infection or vaccination and the standardization of rotavirus-specific T-cells assays in children.

Immunogenicity and waning immunity from the oral cholera vaccine (Shanchol™) in adults residing in Lukanga Swamps of Zambia.

INTRODUCTION: In cholera endemic areas, the periodicity of cholera outbreaks remains unpredictable, making it difficult to organize preventive efforts. Lack of data on duration of protection conferred by oral cholera vaccines further makes it difficult to determine when to deploy preemptive vaccination. We report on the immunogenicity and waning of immunity to Shanchol™ in Lukanga Swamps. METHODS: We enrolled a cohort of 223 participants aged between 18 and 65 years old from whom serum samples were collected at baseline, day 28 before administration of the second dose, and consecutively at 6, 12, 24, 30, 36, and 48 months. Vibriocidal antibody titres were measured and expressed as geometric mean titres. Box plots and 95% CI were computed at each visit for both Inaba and Ogawa. Seroconversion was defined as a four fold or greater increase in antibody titres compared to baseline titres. RESULTS: Overall, seroconversion against V. cholerae Inaba and Ogawa after 1st dose was 35/134 (26%) and 34/134 (25%) respectively. We observed a statistical difference in seroconversion between the two subgroups of baseline titres (low <80 and high ≥80) for both Inaba (p = 0.02) and Ogawa (p<0.0001). From a baseline of 13.58, anti-Ogawa GMT increased to 21.95 after the first dose, but rapidly waned to 14.52, 13.13, and 12.78 at months 6, 12 and 24 respectively, and then increased to 13.21, 18.67 and 23.65 at months 30, 36 and 48 respectively. A similar trend was observed for anti-Inaba GMT across the same time points. CONCLUSION: We found that Shanchol™ was immunogenic in our study population and that vibriocidal antibodies may not be a good marker for long-term immunity. The observed rise in titres after 36 months suggests natural exposure, and this may be a critical time window opening for natural transmission in an endemic areas. We recommend re-vaccination at this time point in high risk areas.

Comparing growth velocity of HIV exposed and non-exposed infants: An observational study of infants enrolled in a randomized control trial in Zambia.

BACKGROUND: Impaired growth among infants remains one of the leading nutrition problems globally. In this study, we aimed to compare the growth trajectory rate and evaluate growth trajectory characteristics among children, who are HIV exposed uninfected (HEU) and HIV unexposed uninfected (HUU), under two years in Zambia. METHOD: Our study used data from the ROVAS II study (PACTR201804003096919), an open-label randomized control trial of two verses three doses of live, attenuated, oral RotarixTM administered 6 &10 weeks or at 6 &10 weeks plus an additional dose at 9 months of age, conducted at George clinic in Lusaka, Zambia. Anthropometric measurements (height and weight) were collected on all scheduled and unscheduled visits. We defined linear growth velocity as the rate of change in height and estimated linear growth velocity as the first derivative of the mixed effect model with fractional polynomial transformations and, thereafter, used the second derivative test to determine the peak height and age at peak heigh. RESULTS: We included 212 infants in this study with median age 6 (IQR: 6-6) weeks of age. Of these 97 (45.3%) were female, 35 (16.4%) were stunted, and 59 (27.6%) were exposed to HIV at baseline. Growth velocity was consistently below the 3rd percentile of the WHO linear growth standard for HEU and HUU children. The peak height and age at peak height among HEU children were 74.7 cm (95% CI = 73.9-75.5) and 15.5 months (95% CI = 14.7-16.3) respectively and those for HUU were 73 cm (95% CI = 72.1-74.0) and 15.6 months (95% CI = 14.5-16.6) respectively. CONCLUSION: We found no difference in growth trajectories between infants who are HEU and HUU. However, the data suggests that poor linear growth is universal and profound in this cohort and may have already occurred in utero.

Mosquito-Borne Viral Pathogens Detected in Zambia: A Systematic Review.

Emerging and re-emerging mosquito-borne viral diseases are a threat to global health. This systematic review aimed to investigate the available evidence of mosquito-borne viral pathogens reported in Zambia. A search of literature was conducted in PubMed and Google Scholar for articles published from 1 January 1930 to 30 June 2020 using a combination of keywords. Eight mosquito-borne viruses belonging to three families, Togaviridae, Flaviviridae and Phenuiviridae were reported. Three viruses (Chikungunya virus, Mayaro virus, Mwinilunga virus) were reported among the togaviruses whilst four (dengue virus, West Nile virus, yellow fever virus, Zika virus) were among the flavivirus and only one virus, Rift Valley fever virus, was reported in the Phenuiviridae family. The majority of these mosquito-borne viruses were reported in Western and North-Western provinces. Aedes and Culex species were the main mosquito-borne viral vectors reported. Farming, fishing, movement of people and rain patterns were among factors associated with mosquito-borne viral infection in Zambia. Better diagnostic methods, such as the use of molecular tools, to detect the viruses in potential vectors, humans, and animals, including the recognition of arboviral risk zones and how the viruses circulate, are important for improved surveillance and design of effective prevention and control measures.