Histo-blood group antigen profile of Australian Aboriginal children and seropositivity following oral rotavirus vaccination.

BACKGROUND: Histo-blood group antigens (HBGAs) may influence immune responses to rotavirus vaccination. METHODS: HBGA phenotyping was determined by detection of antigens A, B, H and Lewis a and b in saliva using enzyme-linked immunosorbent assay. Secretor status was confirmed by lectin antigen assay if A, B and H antigens were negative or borderline (OD ± 0.1 of threshold of detection). PCR-RFLP analysis was used to identify the FUT2 'G428A' mutation in a subset. Rotavirus seropositivity was defined as serum anti-rotavirus IgA ≥ 20 AU/mL. RESULTS: Of 156 children, 119 (76 %) were secretors, 129 (83 %) were Lewis antigen positive, and 105 (67 %) were rotavirus IgA seropositive. Eighty-seven of 119 (73 %) secretors were rotavirus seropositive, versus 4/9 (44 %) weak secretors and 13/27 (48 %) non-secretors. CONCLUSIONS: Most Australian Aboriginal children were secretor and Lewis antigen positive. Non-secretor children were less likely to be seropositive to rotavirus antibodies following vaccination, but this phenotype was less common. HBGA status is unlikely to fully explain underperformance of rotavirus vaccines among Australian Aboriginal children.

Respiratory Virus Surveillance Among Children with Acute Respiratory Illnesses - New Vaccine Surveillance Network, United States, 2016-2021.

The New Vaccine Surveillance Network (NVSN) is a prospective, active, population-based surveillance platform that enrolls children with acute respiratory illnesses (ARIs) at seven pediatric medical centers. ARIs are caused by respiratory viruses including influenza virus, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), human parainfluenza viruses (HPIVs), and most recently SARS-CoV-2 (the virus that causes COVID-19), which result in morbidity among infants and young children (1-6). NVSN estimates the incidence of pathogen-specific pediatric ARIs and collects clinical data (e.g., underlying medical conditions and vaccination status) to assess risk factors for severe disease and calculate influenza and COVID-19 vaccine effectiveness. Current NVSN inpatient (i.e., hospital) surveillance began in 2015, expanded to emergency departments (EDs) in 2016, and to outpatient clinics in 2018. This report describes demographic characteristics of enrolled children who received care in these settings, and yearly circulation of influenza, RSV, HMPV, HPIV1-3, adenovirus, human rhinovirus and enterovirus (RV/EV),* and SARS-CoV-2 during December 2016-August 2021. Among 90,085 eligible infants, children, and adolescents (children) aged <18 years† with ARI, 51,441 (57%) were enrolled, nearly 75% of whom were aged <5 years; 43% were hospitalized. Infants aged <1 year accounted for the largest proportion (38%) of those hospitalized. The most common pathogens detected were RV/EV and RSV. Before the emergence of SARS-CoV-2, detected respiratory viruses followed previously described seasonal trends, with annual peaks of influenza and RSV in late fall and winter (7,8). After the emergence of SARS-CoV-2 and implementation of associated pandemic nonpharmaceutical interventions and community mitigation measures, many respiratory viruses circulated at lower-than-expected levels during April 2020-May 2021. Beginning in summer 2021, NVSN detected higher than anticipated enrollment of hospitalized children as well as atypical interseasonal circulation of RSV. Further analyses of NVSN data and continued surveillance are vital in highlighting risk factors for severe disease and health disparities, measuring the effectiveness of vaccines and monoclonal antibody-based prophylactics, and guiding policies to protect young children from pathogens such as SARS-CoV-2, influenza, and RSV.

Clinical Presentation and Severity of Adenovirus Detection Alone vs Adenovirus Co-detection With Other Respiratory Viruses in US Children With Acute Respiratory Illness from 2016 to 2018.

BACKGROUND: Human adenovirus (HAdV) is commonly associated with acute respiratory illnesses (ARI) in children and is also frequently co-detected with other viral pathogens. We compared clinical presentation and outcomes in young children with HAdV detected alone vs co-detected with other respiratory viruses. METHODS: We used data from a multicenter, prospective, viral surveillance study of children seen in the emergency department and inpatient pediatric settings at seven US sites. Children less than 18 years old with fever and/or respiratory symptoms were enrolled between 12/1/16 and 10/31/18 and tested by molecular methods for HAdV, human rhinovirus/enterovirus (HRV/EV), respiratory syncytial virus (RSV), parainfluenza (PIV, types 1-4), influenza (flu, types A-C), and human metapneumovirus (HMPV). Our primary measure of illness severity was hospitalization; among hospitalized children, secondary severity outcomes included oxygen support and length of stay (LOS). RESULTS: Of the 18,603 children enrolled, HAdV was detected in 1,136 (6.1%), among whom 646 (56.9%) had co-detection with at least one other respiratory virus. HRV/EV (n = 293, 45.3%) and RSV (n = 123, 19.0%) were the most frequent co-detections. Children with HRV/EV (aOR = 1.61; 95% CI = [1.11-2.34]), RSV (aOR = 4.48; 95% CI = [2.81-7.14]), HMPV (aOR = 3.39; 95% CI = [1.69-6.77]), or ≥ 2 co-detections (aOR = 1.95; 95% CI = [1.14-3.36]) had higher odds of hospitalization compared to children with HAdV alone. Among hospitalized children, HAdV co-detection with RSV or HMPV was each associated with higher odds of oxygen support, while co-detection with PIV or influenza viruses was each associated with higher mean LOS. CONCLUSIONS: HAdV co-detection with other respiratory viruses was associated with greater disease severity among children with ARI compared to HAdV detection alone.

Lethal Pediatric Cerebral Vasculitis Triggered by Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: We report the clinical, radiological, laboratory, and neuropathological findings in support of the first diagnosis of lethal, small-vessel cerebral vasculitis triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a pediatric patient. PATIENT DESCRIPTION: A previously healthy, eight-year-old Hispanic girl presented with subacute left-sided weakness two weeks after a mild febrile illness. SARS-CoV-2 nasopharyngeal swab was positive. Magnetic resonance imaging revealed an enhancing right frontal lobe lesion with significant vasogenic edema. Two brain biopsies of the lesion showed perivascular and intraluminal lymphohistiocytic inflammatory infiltrate consistent with vasculitis. Despite extensive treatment with immunomodulatory therapies targeting primary angiitis of the central nervous system, she experienced neurological decline and died 93 days after presentation. SARS-CoV-2 testing revealed positive serum IgG and positive cerebrospinal fluid IgM. Comprehensive infectious, rheumatologic, hematologic/oncologic, and genetic evaluation did not identify an alternative etiology. Postmortem brain autopsy remained consistent with vasculitis. CONCLUSION: This is the first pediatric presentation to suggest that SARS-CoV-2 can lead to a fatal, postinfectious, inflammatory small-vessel cerebral vasculitis. Our patient uniquely included supportive cerebrospinal fluid and postmortem tissue analysis. While most children recover from the neurological complications of SARS-CoV-2, we emphasize the potential mortality in a child with no risk factors for severe disease.

High Mobility Group Box 1 Release by Cholangiocytes Governs Biliary Atresia Pathogenesis and Correlates With Increases in Afflicted Infants.

BACKGROUND AND AIMS: Biliary atresia (BA) is a devastating cholangiopathy of infancy. Upon diagnosis, surgical reconstruction by Kasai hepatoportoenterostomy (HPE) restores biliary drainage in a subset of patients, but most patients develop fibrosis and progress to end-stage liver disease requiring liver transplantation for survival. In the murine model of BA, rhesus rotavirus (RRV) infection of newborn pups results in a cholangiopathy paralleling that of human BA. High-mobility group box 1 (HMGB1) is an important member of the danger-associated molecular patterns capable of mediating inflammation during infection-associated responses. In this study, we investigated the role of HMGB1 in BA pathogenesis. APPROACH AND RESULTS: In cholangiocytes, RRV induced the expression and release of HMGB1 through the p38 mitogen-activated protein kinase signaling pathway, and inhibition of p38 blocked HMGB1 release. Treatment of cholangiocytes with ethyl pyruvate suppressed the release of HMGB1. Administration of glycyrrhizin in vivo decreased symptoms and increased survival in the murine model of BA. HMGB1 levels were measured in serum obtained from infants with BA enrolled in the PROBE and START studies conducted by the Childhood Liver Disease Research Network. High HMGB1 levels were found in a subset of patients at the time of HPE. These patients had higher bilirubin levels 3 months post-HPE and a lower survival of their native liver at 2 years. CONCLUSIONS: These results suggest that HMGB1 plays a role in virus induced BA pathogenesis and could be a target for therapeutic interventions in a subset of patients with BA and high HMGB1.

Safety and immunogenicity of an intranasal sendai virus-based vaccine for human parainfluenza virus type I and respiratory syncytial virus (SeVRSV) in adults.

SeVRSV is a replication-competent Sendai virus (SeV)-based vaccine carrying the respiratory syncytial virus (RSV) fusion protein (F) gene. Unmanipulated, non-recombinant SeV is a murine parainfluenza virus type 1 (PIV-1) and serves as a Jennerian vaccine for human PIV-1 (hPIV-1). SeV protects African green monkeys (AGM) from infection after hPIV-1 challenge. The recombinant SeVRSV additionally targets RSV and protects AGM from lower respiratory infections after RSV challenge. The present study is the first to report on the safety, viral genome detection, and immunogenicity following SeVRSV vaccination of healthy adults. Seventeen and four healthy adults received intranasal SeVRSV and PBS, respectively, followed by six months of safety monitoring. Virus genome (in nasal wash) and vaccine-specific antibodies (in sera) were monitored for two and four weeks, respectively, post-vaccination. The vaccine was well-tolerated with only mild to moderate reactions that were also present in the placebo group. No severe reactions occurred. As expected, due to preexisting immunity toward hPIV-1 and RSV in adults, vaccine genome detection was transient. There were minimal antibody responses to SeV and negligible responses to RSV F. Results encourage further studies of SeVRSV with progression toward a clinical trial in seronegative children. Abbreviations: AE-adverse event; SAE-serious adverse event; SeV-Sendai virus; RSV-respiratory syncytial virus; PIV-1-parainfluenza virus-type 1; hPIV-1-human parainfluenza virus-type 1; F-RSV fusion protein; SeVRSV-recombinant SeV carrying the RSV F gene; Ab-antibody; MSW-medically significant wheezing; NOCMC-new onset chronic medical condition, mITT-modified Intent to Treat; ALRI-acute lower respiratory tract infection.

Cytomegalovirus Genetic Diversity Following Primary Infection.

BACKGROUND: Infection with multiple cytomegalovirus (CMV) strains (mixed infection) was reported in a variety of hosts. As the virus genetic diversity in primary CMV infection and the changes over time remain incompletely defined, we examined CMV diversity and changes in diversity over time in healthy adolescent females who participated in a phase 2 CMV gB/MF59 vaccine trial. METHODS: CMV genetic diversity was determined by genotyping of 5 genes-gB (UL55), gH (UL75), gN (UL73), US28, and UL144-in urine, saliva, and plasma samples from 15 study subjects. RESULTS: At the time of primary infection, 5 of 12 (42%) urine samples had multiple virus strains, and 50% of vaccine recipients were infected with gB1 genotype (vaccine strain). Mixed infection was documented in all 15 subjects within 3 months after primary infection, and the majority had different CMV genotypes in different compartments. Changes in genotypes over time were observed in all subjects. CONCLUSIONS: Infection with multiple CMV genotypes was common during primary infection and further diversification occurred over time. Infection with gB1 genotype in vaccine recipients suggests a lack of strain-specific protection from the vaccine. As only 5 polymorphic genes were assessed, this study likely underestimated the true genetic diversity in primary CMV infection.

Human VP8* mAbs neutralize rotavirus selectively in human intestinal epithelial cells.

We previously generated 32 rotavirus-specific (RV-specific) recombinant monoclonal antibodies (mAbs) derived from B cells isolated from human intestinal resections. Twenty-four of these mAbs were specific for the VP8* fragment of RV VP4, and most (20 of 24) were non-neutralizing when tested in the conventional MA104 cell-based assay. We reexamined the ability of these mAbs to neutralize RVs in human intestinal epithelial cells including ileal enteroids and HT-29 cells. Most (18 of 20) of the "non-neutralizing" VP8* mAbs efficiently neutralized human RV in HT-29 cells or enteroids. Serum RV neutralization titers in adults and infants were significantly higher in HT-29 than MA104 cells and adsorption of these sera with recombinant VP8* lowered the neutralization titers in HT-29 but not MA104 cells. VP8* mAbs also protected suckling mice from diarrhea in an in vivo challenge model. X-ray crystallographic analysis of one VP8* mAb (mAb9) in complex with human RV VP8* revealed that the mAb interaction site was distinct from the human histo-blood group antigen binding site. Since MA104 cells are the most commonly used cell line to detect anti-RV neutralization activity, these findings suggest that prior vaccine and other studies of human RV neutralization responses may have underestimated the contribution of VP8* antibodies to the overall neutralization titer.

Study of Environmental Enteropathy and Malnutrition (SEEM) in Pakistan: protocols for biopsy based biomarker discovery and validation.

BACKGROUND: Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority. METHODS: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn's disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community. DISCUSSION: Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals. TRIAL REGISTRATION: Retrospectively registered; clinicaltrials.gov ID NCT03588013 .

Safety and efficacy of a cytomegalovirus glycoprotein B (gB) vaccine in adolescent girls: A randomized clinical trial.

BACKGROUND: Cytomegalovirus (CMV) is a leading cause of congenital infection and an important target for vaccine development. METHODS: CMV seronegative girls between 12 and 17 years of age received CMV glycoprotein B (gB) vaccine with MF59 or saline placebo at 0, 1 and 6 months. Blood and urine were collected throughout the study for evidence of CMV infection based on PCR and/or seroconversion to non-vaccine CMV antigens. RESULTS: 402 CMV seronegative subjects were vaccinated (195 vaccine, 207 placebo). The vaccine was generally well tolerated, although local and systemic adverse events were significantly more common in the vaccine group. The vaccine induced gB antibody in all vaccine recipients with a gB geometric mean titer of 13,400 EU; 95%CI 11,436, 15,700, after 3 doses. Overall, 48 CMV infections were detected (21 vaccine, 27 placebo). In the per protocol population (124 vaccine, 125 placebo) vaccine efficacy was 43%; 95%CI: -36; 76, p=0.20. The most significant difference was after 2 doses, administered as per protocol; vaccine efficacy 45%, 95%CI: -9; 72, p=0.08. CONCLUSION: The vaccine was safe and immunogenic. Although the efficacy did not reach conventional levels of significance, the results are consistent with a previous study in adult women (Pass et al. N Engl J Med 2009;360:1191) using the same formulation.

The Safety and Immunogenicity of Rotavirus Vaccination in Infants With Intestinal Failure.

BACKGROUND: Young children with intestinal failure are at risk for complications from rotavirus gastroenteritis. To date, the safety and immunogenicity of rotavirus vaccines in these children are not known. We hypothesized that rotavirus vaccination would be safe and confer immunity to infants with intestinal failure and a history of abdominal surgery. METHODS: The study population consisted of infants with early intestinal failure who required abdominal surgery and parenteral nutrition for necrotizing enterocolitis, gastroschisis, jejunoileal atresia, or meconium peritonitis. Subjects received a rotavirus vaccine series at the appropriate age. Safety assessments were performed for the first 5 days after each vaccine dose. Viral stool shedding and serum rotavirus antigen were measured at multiple time points after each dose of the vaccine. A 3-fold increase in rotavirus immunoglobulin A titer at study completion defined seroconversion. RESULTS: Fifteen infants were enrolled and 14 infants completed the study protocol. Mild diarrhea, emesis, and fever were noted after vaccination in 33%, 40%, and 7% of subjects, respectively. No subject experienced postvaccine intussusception, viremia, dehydration, or required alterations in nutritional care. Viral stool shedding was noted in 47%, but only 1 child shed virus beyond 2 weeks postvaccination. All subjects who were not considered immune at baseline developed seroconversion to rotavirus after vaccination. CONCLUSIONS: In infants with intestinal failure, rotavirus vaccination appears to be safe and immunogenic. We found no evidence for altered viral shedding in this population. Given the profound consequences associated with rotavirus infection and potential benefits of rotavirus vaccine in this cohort, multicenter studies focusing on vaccine efficacy are warranted.

Role of myeloid differentiation factor 88 in Rhesus rotavirus-induced biliary atresia.

BACKGROUND: Biliary atresia (BA) is a unique neonatal disease resulting from inflammatory and fibrosing obstruction of the extrahepatic biliary tree. Previous studies have demonstrated the critical role of innate immunity and the Th1 response to activated inflammatory cells and overexpressed cytokines in the pathogenesis of BA. Myeloid differentiation factor 88 (MyD88) is a critical adaptor molecule that has been shown to play a crucial role in immunity. We investigated the role of MyD88 in the inflammatory response and development of cholangiopathy in murine BA. METHODS: MyD88 knockout (MyD88(-/-)) and wild-type (WT) BALB/c pups were injected with Rhesus rotavirus or saline on day 1 of life. The mice were monitored for clinical symptoms of BA, including jaundice, acholic stools, bilirubinuria, and death. The liver and extrahepatic bile ducts were harvested for histologic evaluation and the quantification of viral content, determination of cytokine expression, and detection of inflammatory cells. RESULTS: Rhesus rotavirus infection produced symptoms in 100% of both MyD88(-/-) and WT pups, with survival of 18% of WT and 0% of MyD88(-/-) mice. Histologic analysis demonstrated bile duct obstruction in both MyD88(-/-) and WT mice. Viral titers obtained 7 d after infection and expression of interferon-γ and tumor necrosis factor-α at day 3, 5, 8, and 12 after infection revealed no significant differences between the WT and MyD88(-/-) mice. Flow cytometry demonstrated similar levels of activated CD8+ T cells and natural killer cells. CONCLUSIONS: The pathogenesis of murine BA is independent of the MyD88 signaling inflammatory pathway, suggesting alternative mechanisms are crucial in the induction of the model.

Rotavirus infection of human cholangiocytes parallels the murine model of biliary atresia.

INTRODUCTION: Biliary atresia (BA) is the leading indication for liver transplantation in the pediatric population. The murine model of BA supports a viral etiology, because infection of neonatal mice with rhesus rotavirus (RRV) results in biliary obstruction. Viral infection targets the biliary epithelium and development of the model is viral strain dependent. No study has yet determined whether human cholangiocytes are also susceptible to rotaviral infection. We established an in vitro human model using an immortalized human cholangiocyte cell line and primary human cholangiocytes obtained from explanted livers to determine human cholangiocyte susceptibility to rotavirus infection. METHODS: Replication and binding assays were performed on immortalized mouse (mCL) and human (H69) cells using six different strains of rotavirus. Primary human cholangiocytes were isolated from cadaveric livers, characterized in culture, and infected with RRV, which causes BA in mice, and another simian strain, TUCH, which does not cause BA in mice. RESULTS: Immortalized mouse and human cholangiocytes demonstrated similar patterns of infectivity and binding with different strains of rotavirus. Both cell lines produced a significantly higher viral yield with RRV infection than with the other strains tested. In primary human cholangiocytes, which maintained their epithelial characteristics, as demonstrated by cytokeratin staining, RRV replicated to a yield 1000-fold higher than TUCH. CONCLUSIONS: Both immortalized and primary human cholangiocytes are susceptible to RRV infection in a fashion similar to murine cholangiocytes. These novel findings suggest rotavirus infection could have a potential role in the pathogenesis of human BA.

Simian rotaviruses possess divergent gene constellations that originated from interspecies transmission and reassortment.

Although few simian rotaviruses (RVs) have been isolated, such strains have been important for basic research and vaccine development. To explore the origins of simian RVs, the complete genome sequences of strains PTRV (G8P[1]), RRV (G3P[3]), and TUCH (G3P[24]) were determined. These data allowed the genotype constellations of each virus to be determined and the phylogenetic relationships of the simian strains with each other and with nonsimian RVs to be elucidated. The results indicate that PTRV was likely transmitted from a bovine or other ruminant into pig-tailed macaques (its host of origin), since its genes have genotypes and encode outer-capsid proteins similar to those of bovine RVs. In contrast, most of the genes of rhesus-macaque strains, RRV and TUCH, have genotypes more typical of canine-feline RVs. However, the sequences of the canine and/or feline (canine/feline)-like genes of RRV and TUCH are only distantly related to those of modern canine/feline RVs, indicating that any potential transmission of a progenitor of these viruses from a canine/feline host to a simian host was not recent. The remaining genes of RRV and TUCH appear to have originated through reassortment with bovine, human, or other RV strains. Finally, comparison of PTRV, RRV, and TUCH genes with those of the vervet-monkey RV SA11-H96 (G3P[2]) indicates that SA11-H96 shares little genetic similarity to other simian strains and likely has evolved independently. Collectively, our data indicate that simian RVs are of diverse ancestry with genome constellations that originated largely by interspecies transmission and reassortment with nonhuman animal RVs.

The adjuvant CLDC increases protection of a herpes simplex type 2 glycoprotein D vaccine in guinea pigs.

Herpes simplex virus (HSV) infections are common but there is no vaccine available. We evaluated cationic liposome-DNA complexes (CLDC) as an adjuvant for an HSV gD2 vaccine and compared it to an MPL/Alum adjuvant in a guinea pig model of genital herpes. The addition of CLDC to the gD2 vaccine significantly decreased acute and recurrent disease and most importantly the number of days with recurrent virus shedding compared to gD2 alone. Reductions in these outcomes were also detected when gD2+CLDC was compared to gD2+MPL/Alum. When the vaccine and adjuvants were evaluated as therapeutic vaccines, they were ineffective. CLDC enhanced protection compared to MPL/Alum and is the first vaccine to reduce recurrent virus shedding, a key to decreasing the spread of HSV-2.

Prevention of the murine model of biliary atresia after live rotavirus vaccination of dams.

PURPOSE: Biliary atresia (BA) is a neonatal disease that results in the obliteration of the biliary tree. The murine model of BA has been established where rhesus rotavirus (RRV) infection of newborn mice leads to an obstructive cholangiopathy. We determined whether maternal postconception rotavirus vaccination could prevent the murine model of BA. MATERIALS AND METHODS: Female mice were mated and injected intraperitoneally with one of the following materials: purified rotavirus strains RRV or Wa, high or low-dose Rotateq (Merck and Co Inc, Whitehouse Station, NJ) (a pentavalent rotavirus vaccine [PRV]), purified recombinant viral antigens of rotavirus (VP6) or influenza (NP), or saline. B-cell-deficient females also underwent postconception PRV injection. RESULTS: Maternal vaccination with PRV improves survival of pups infected with RRV. Serum rotavirus IgG, but not IgA, levels were increased in pups delivered from dams who received RRV, Wa, PRV, or VP6, but in the case of the Wa, PRV, and VP6 groups, these antibodies were not neutralizing. Postconception injection of high-dose PRV did not improve survival of pups born to B-cell-deficient dams. CONCLUSION: Maternal vaccination against RRV can prevent the rotavirus-induced murine model of BA in newborn mouse pups.

Immune response to influenza vaccine in children with inflammatory bowel disease.

OBJECTIVES: Patients with inflammatory bowel disease (IBD) frequently receive immunosuppressive therapy. The immune response in these patients to vaccines has not been well studied. We conducted a prospective, open label study to evaluate the serologic response to influenza vaccine in children with IBD. METHODS: Serum was obtained from 146 children and young adults with IBD (96 Crohn's disease, 47 ulcerative colitis, and 3 indeterminate colitis) for baseline influenza titer, immediately followed by immunization with trivalent (A/Solomon Islands/3/2006 (H1N1), A/Wisconsin/67/2005 (H3N2), and B/Malaysia/2506/2004 (B)) inactivated influenza vaccine. Patients returned for repeat titers 3-9 weeks later. Seroprotection against each influenza strain was defined as hemagglutination inhibition titer > or = 40. Patients were categorized as nonimmunosuppressed (NIS; aminosalicylates only, antibiotics only, or no therapy) or immunosuppressed (IS; any immunosuppressive agent). IS patients were further subcategorized as: (i) tacrolimus, (ii) tumor necrosis factor-alpha (TNF-alpha) inhibitor, (3) immunomodulator, and (4) corticosteroids only. RESULTS: More patients were seroprotected against strains A/H1N1 and A/H3N2 than B strain (P<0.02), regardless of immunosuppression status. The proportion of seroprotected patients and geometric mean titers at post-vaccination were similar between NIS and IS groups for all three strains. Subanalysis of patients not seroprotected at baseline showed that those receiving anti-TNF therapy were less likely to be seroprotected against strain B (14%) compared to patients in the NIS group (39%, P=0.025). There were no serious vaccine-associated adverse events. CONCLUSIONS: Influenza vaccination produces a high prevalence of seroprotection in IBD patients, particularly against A strains. The vaccine is well tolerated. Routine influenza vaccination in IBD patients is recommended, irrespective of whether patients receive immunosuppressive medications.

IFN-gamma is the only anti-rotavirus cytokine found after in vitro stimulation of memory CD4+ T cells from mice immunized with a chimeric VP6 protein.

CD4+ T cells are the only lymphocytes required for protection of mice against rotavirus shedding after mucosal immunization with chimeric VP6 (MBP::VP6) and the adjuvant LT(R192G). One possible effector of protection is CD4+ T-cell cytokines. To determine if memory CD4+ T cells of immunized mice produce cytokines with direct anti-rotavirus activity, an in vitro infection model was developed using mouse CMT-93 cells and rhesus rotavirus (RRV). Spleen and lamina propria (LP) cells, as well as purified splenic CD4T cells obtained after intranasal immunization of BALB/c mice with MBP::VP6/LT(R192G) released large quantities of two cytokines (IL-17 and IFN-gamma) into cell supernatants when stimulated with MBP::VP6. Production of these same cytokines is rapidly upregulated in intestinal lymphocytes after rotavirus inoculation of immunized mice. IL-17 pretreatment of CMT-93 cells had no effect on subsequent RRV replication, but IFN-gamma was the most potent inhibitor within a panel of nine cytokines tested. Supernatants obtained after in vitro stimulation of splenic CD4+ T cells of immunized mice had high levels of anti-RRV activity and their pretreatment with mAb against IFN-gamma caused essentially complete loss of activity. Thus, IFN-gamma was the only cytokine identified in stimulated CD4+ T cells from immunized mice that directly inhibited rotavirus replication.

MAPK signaling contributes to rotaviral-induced cholangiocyte injury and viral replication.

BACKGROUND: Biliary atresia is a disease of newborns that results in obliteration of the biliary tree. Infection of mice with rhesus rotavirus (RRV) results in a cholangiopathy mirroring human disease. The Mitogen Associated Protein Kinase (MAPK) signaling pathway can be activated by viral binding to cell-surface receptors. We hypothesized that RRV infection of cholangiocytes results in activation of MAPK signaling. METHODS: Extrahepatic bile ducts from BALB/c pups or immortalized cholangiocytes subjected to RRV infection or control were analyzed, using Western blots, for phosphorylated members of the MAPK family: p38, ERK 1/2, JNK 1/2, and downstream transcription factors. Inhibitors of the MAPK were used to downregulate activity. Viral replication and cytolysis in cholangiocytes were evaluated post-MAPK inhibition. RESULTS: Phosphorylation of all MAPK increased in RRV-infected mice and cholangiocytes. Several downstream transcription factors had increased activity in vitro. Inhibition of p38 and ERK 1/2 resulted in decreased viral replication. ERK 1/2 inhibition decreased cytolysis without affecting viral entry or binding. CONCLUSIONS: RRV infection of cholangiocytes resulted in increased MAPK signaling. Inhibition of p38 and ERK 1/2 influenced the ability of rotavirus to replicate. These novel findings provide insight into the signaling cascade involved in RRV-induced cholangiocyte injury.

Effector role of neonatal hepatic CD8+ lymphocytes in epithelial injury and autoimmunity in experimental biliary atresia.

BACKGROUND & AIMS: Lymphocytes populate the livers of infants with biliary atresia, but it is unknown whether neonatal lymphocytes regulate pathogenesis of disease. Here, we investigate this question by examining the role of T lymphocytes in the destruction of extrahepatic bile ducts of neonatal mice using an experimental model of biliary atresia. METHODS: Inoculation of neonatal mice with rhesus rotavirus followed by multistaining flow cytometry to quantify expression of interferon-gamma by hepatic lymphocytes, and real-time polymerase chain reaction for mRNA expression of pro-inflammatory cytokines. This was followed by determining the consequences of antibody-mediated depletion of lymphocyte subtypes on the development of biliary obstruction, and coculture and cell transfer experiments to investigate the effector role of lymphocyte subtypes on neonatal biliary disease. RESULTS: Rotavirus infection results in overexpression of interferon-gamma by neonatal hepatic T cells. Among these cells, depletion of CD4(+) cells did not change the course of inflammatory injury and obstruction of neonatal bile ducts. In contrast, loss of CD8(+) cells remarkably suppressed duct injury, prevented luminal obstruction, and restored bile flow. Coculture experiments showed that rotavirus-primed, but not naïve, CD8(+) cells were cytotoxic to cholangiocytes. In adoptive transfer experiments, we found that primed CD8(+) cells preferentially homed to extrahepatic bile ducts of neonatal mice and invaded their epithelial lining. CONCLUSIONS: Primed neonatal CD8(+) cells can activate a pro-inflammatory program, target diseased and healthy duct epithelium, and drive the phenotypic expression of biliary atresia, thus constituting a potential therapeutic target to halt disease progression.