Sung-Sil Moon

Inhibitory Effect of Breast Milk on Infectivity of Live Oral Rotavirus Vaccines

Background: Live oral rotavirus vaccines have been less immunogenic and efficacious among children in poor developing countries compared with middle income and industrialized countries for reasons that are not yet completely understood. We assessed whether the neutralizing activity of breast milk could lower the titer of vaccine virus and explain this difference in vitro. Methods: Breast milk samples were collected from mothers who were breast-feeding infants 4 to 29 weeks of age (ie, vaccine eligible age) in India (N = 40), Vietnam (N = 77), South Korea (N = 34), and the United States (N = 51). We examined breast milk for rotavirus-specific IgA and neutralizing activity against 3 rotavirus vaccine strains—RV1, RV5 G1, and 116E using enzyme immunoassays. The inhibitory effect of breast milk on RV1 was further examined by a plaque reduction assay. Findings: Breast milk from Indian women had the highest IgA and neutralizing titers against all 3 vaccine strains, while lower but comparable median IgA and neutralizing titers were detected in breast milk from Korean and Vietnamese women, and the lowest titers were seen in American women. Neutralizing activity was greatest against the 2 vaccine strains of human origin, RV1 and 116E. This neutralizing activity in one half of the breast milk specimens from Indian women could reduce the effective titer of RV1 by ∼2 logs, of 116E by 1.5 logs, and RV5 G1 strain by ∼1 log more than that of breast milk from American women. Interpretation: The lower immunogenicity and efficacy of rotavirus vaccines in poor developing countries could be explained, in part, by higher titers of IgA and neutralizing activity in breast milk consumed by their infants at the time of immunization that could effectively reduce the potency of the vaccine. Strategies to overcome this negative effect, such as delaying breast-feeding at the time of immunization, should be evaluated.

Dose sparing and enhanced immunogenicity of inactivated rotavirus vaccine administered by skin vaccination using a microneedle patch

Skin immunization is effective against a number of infectious diseases, including smallpox and tuberculosis, but is difficult to administer. Here, we assessed the use of an easy-to-administer microneedle (MN) patch for skin vaccination using an inactivated rotavirus vaccine (IRV) in mice. Female inbred BALB/c mice in groups of six were immunized once in the skin using MN coated with 5 μg or 0.5 μg of inactivated rotavirus antigen or by intramuscular (IM) injection with 5 μg or 0.5 μg of the same antigen, bled at 0 and 10 days, and exsanguinated at 28 days. Rotavirus-specific IgG titers increased over time in sera of mice immunized with IRV using MN or IM injection. However, titers of IgG and neutralizing activity were generally higher in MN immunized mice than in IM immunized mice; the titers in mice that received 0.5 μg of antigen with MN were comparable or higher than those that received 5 μg of antigen IM, indicating dose sparing. None of the mice receiving negative-control, antigen-free MN had any IgG titers. In addition, MN immunization was at least as effective as IM administration in inducing a memory response of dendritic cells in the spleen. Our findings demonstrate that MN delivery can reduce the IRV dose needed to mount a robust immune response compared to IM injection and holds promise as a strategy for developing a safer and more effective rotavirus vaccine for use among children throughout the world.

Differential Profiles and Inhibitory Effect on Rotavirus Vaccines of Nonantibody Components in Breast Milk From Mothers in Developing and Developed Countries

Background: Live oral rotavirus vaccines have been less immunogenic and efficacious for children of developing countries than for those in middle income and industrialized countries, and the basis for these differences is not fully understood. Recently, we demonstrated that breastmilk from mothers in India had significantly higher IgA and neutralizing activity against rotavirus that could reduce the effective titer of rotavirus vaccines reaching the gut when compared with that from mothers in the United States. We extended our study to understand the specific contribution of those nonantibody components in breastmilk to the neutralizing activity against rotavirus vaccine we observed. Methods: Breastmilk samples were collected from mothers of breast-feeding infants aged between 4 and 29 weeks (ie, vaccine eligible age) in India (N = 40), South Africa (N = 50) and the United States (N = 51). We examined breastmilk for lactoferrin, lactadherin, rotavirus-specific IgA and neutralizing activity against 3 rotavirus vaccine strains (Rotarix, RotaTeq G1 and 116E) using enzyme immunoassays, a plaque reduction assay or a microneutralization assay. Results: We observed higher levels of lactoferrin, lactadherin, IgA and neutralizing activity in breastmilk specimens from Indian and South African women than those from American women. We demonstrated positive associations between levels of lactoferrin or IgA and neutralizing activity in Indian and South African specimens, but not in American specimens. We demonstrated that the inhibitory effect of lactoferrin was dose- or species-dependent, as evidenced by greater reduction in titer of Rotarix and 116E by human lactoferrin. Lactadherin also exhibited inhibitory activity to rotavirus vaccines but appeared to be less effective. Conclusions: The lower immunogenicity and efficacy of rotavirus vaccines in developing countries could be explained, in part, by synergistic inhibitory effect of high levels of antibody and nonantibody components in breastmilk consumed by infants at the time of immunization. Therefore, there is a need for alternative rotavirus vaccine strategies in breast-feeding populations.

Multiple virus infection alters rotavirus replication and expression of cytokines and Toll-like receptors in intestinal epithelial cells

Two live oral rotavirus vaccines have shown to be effective in protecting young children from severe illness in developed and middle income countries, but their efficacy is significantly lower in low income countries. One of the reasons for this lower efficacy may be mixed virus infection in the gut that is commonly encountered among infants in the developing world. We investigated whether multiple virus infection interferes with rotavirus replication and alters host response by comparing single and mixed enteric virus infections in Caco-2 cells. We observed a dramatic reduction in rotavirus replication and growth in mixed rotavirus, astrovirus and enterovirus infection compared to single rotavirus infection. By contrast, the levels of astrovirus and enterovirus RNA in mixed infection remained unchanged when compared to those of the corresponding single virus infection. We then examined cells with single or multiple virus infections for the expression of 10 cytokine genes and demonstrated elevated expressions for 7 (IFN-α, IFN-β, IFN-γ, TNF-α, IL-6, IL-8, and IL-17) in dual rotavirus and enterovirus or triple rotavirus, enterovirus and astrovirus-infected cells but only 3 (IFN-β, TNF-α, and IL-8) in dual rotavirus and astrovirus-infected cells. We further observed elevated levels of TLR4, TLR5, TLR7 and TLR9 mRNAs in cells with rotavirus and enterovirus or rotavirus, enterovirus and astrovirus infections when compared to single rotavirus infections. Our data suggest that rotavirus infection is susceptible to interference by other enteric viruses in the gut, which could result in reduced virus replication and contribute to lower immunogenicity and efficacy of oral rotavirus vaccines in low income countries.

Antigenemia, RNAemia, and innate immunity in children with acute rotavirus diarrhea

Antigenemia is commonly detected in children with acute rotavirus diarrhea, but the prevalence of viremia has not been clearly defined. We examined antigenemia in plasma and RNAemia in peripheral blood mononuclear cells (PBMC) of children with acute diarrhea by EIA, RT-PCR, and Southern hybridization, using primers and a probe specific to rotavirus NSP4 gene. We detected the presence of rotavirus antigen in 33.3% and almost full-length NSP4 gene in 70.8% of the acute-phase plasma and PBMC, respectively. In contrast, antigenemia and RNAemia were detected in 0% and 4.2% of the convalescent-phase plasma and PBMC, respectively, which were similar to antigenemia (0%) and RNAemia (7.7%) in healthy controls. We demonstrated an increase in the proportions of activated myeloid dendritic cells (mDC) and activated plasmacytoid DC (pDC) in acute-phase PBMC of patients when compared to those in convalescent phase of patients and in PBMC of healthy controls. The activation of mDC peaked on days 2-4 after illness onset, and the activation of acute-phase pDC appeared to correlate with levels of antigenemia. High prevalence of NSP4 gene in acute-phase PBMC indicates possible rotavirus replication in white blood cells, and extraintestinal spread and the activation of DC may have implications for the prevention of rotavirus disease in children.

Comparison of innate immune responses to pathogenic and putative non-pathogenic hantaviruses in vitro

Hantaviruses are human pathogens that cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. The mechanisms accounting for the differences in virulence between pathogenic and non-pathogenic hantaviruses are not well known. We have examined the pathogenesis of different hantavirus groups by comparing the innate immune responses induced in the host cell following infection by pathogenic (Sin Nombre, Hantaan, and Seoul virus) and putative non-pathogenic (Prospect Hill, Tula, and Thottapalayam virus) hantaviruses. Pathogenic hantaviruses were found to replicate more efficiently in interferon-competent A549 cells than putative non-pathogenic hantaviruses. The former also suppressed the expression of the interferon-β and myxovirus resistance protein genes, while the transcription level of both genes increased rapidly within 24 h post-infection in the latter. In addition, the induction level of interferon correlated with the activation level of interferon regulatory factor-3. Taken together, these results suggest that the observed differences are correlated with viral pathogenesis and further indicate that pathogenic and putative non-pathogenic hantaviruses differ in terms of early interferon induction via activation of the interferon regulatory factor-3 in infected host cells.

First detection of group C rotavirus in children with acute gastroenteritis in South Korea

Group C rotavirus (GpC RV) causes sporadic cases and outbreaks of acute diarrhoea in humans worldwide, but has not been detected among children in South Korea. The present study aimed to detect GpC RV among children hospitalized with gastroenteritis in South Korea and to perform a molecular characterization of GpC RV strains. From November 2003 to January 2006, 434 faecal samples were collected from children <10 years of age who were hospitalized for treatment of acute diarrhoea and screened for group C and A rotaviruses by enzyme immunoassay. GpC RV strains were characterized by sequence and phylogenetic analysis.Of the 434 samples screened, two were positive for GpC RV and one had a mixed GpC and GpA RV infection. One of the strains, Icheon, shared high sequence conservation in VP4, VP6 and VP7 genes with other published GpC RV. This is the first report describing the molecular characteristics of GpC RV among children in South Korea. Additional surveillance is needed to determine the burden of GpC RV gastroenteritis.

Hantaan virus surveillance targeting small mammals at Dagmar North Training Area, Gyeonggi Province, Republic of Korea, 2001-2005

ABSTRACT: In response to a hemorrhagic fever with renal syndrome case in November 2000, a seasonal rodent-borne disease surveillance program was initiated at Dagmar North Training Area (DNTA), Gyeonggi Province, Republic of Korea. From April 2001-December 2005, 1,848 small mammals were captured. Apodemus agrarius accounted for 92.5%, followed by Mus musculus (3.6%), Crocidura lasiura (2.1%), and Microtus fortis (1.1%). Three species of rodents were found to be antibody-positive (Ab+) for Hantaan virus (HTNV): A. agrarius (22.3%), M. musculus (9.1%), and M. fortis (5.0%). Ab+ rates for A. agrarius increased with increasing weight (age), except for those weighing <10 g. The peak HTNV transmission period in Korea coincided with the peak reproductive potential of A. agrarius during the fall (August/September) surveys. HTNV strains from DNTA were distinct from HTNV strains from the Peoples Republic of China. From these studies, more accurate risk assessments can be developed to better protect personnel from rodent-borne diseases.

Rotavirus-specific IgG antibodies from mothers' serum may inhibit infant immune responses to the pentavalent rotavirus vaccine.

© 2014 Lippincott Williams & Wilkins www.pidj.com | 115 Rotavirus-specific IgG Antibodies From Mothers’ Serum May Inhibit Infant Immune Responses to the Pentavalent Rotavirus Vaccine in Exon 2 of the IL2RG gene (c.252C>A, p.Asn84Lys; MIM# 300400) and curative therapy with stem cell transplantation was initiated at our specialized medical center. We identified rotavirus vaccine strain in stool samples by sequencing of 8 different gene segments (VP1, VP2, VP4, VP6, VP7, NSP2, NSP4 and NSP5). In all segments vaccine-associated sequences harboring few new mutations were found, leading to the conclusion that the child’s chronic gastroenteritis was caused by persisting infection with the vaccine rotavirus strain (see Table, Supplemental Digital Content 1, http://links.lww.com/INF/B978). No other pathogen could be identified in multiple stool samples, including uncommon parasites, coccidia and helminths. Notably, all stool, blood and respiratory specimens were also tested by Luminex xTAG respiratory and xTAG gastrointestinal pathogen panel (Abbott Molecular, Wiesbaden, Germany). In samples from the respiratory tract, we detected high copy numbers of human bocavirus (HBoV) and rhinovirus, which most likely subsequently caused respiratory failure in our patient. Despite all intensive supportive care and treatment, the boy’s condition deteriorated and he died on day 1 after stem cell transplantation. In addition to the association with intussusception, shedding of rotavirus for a period of time after oral vaccination and vaccineinduced severe gastroenteritis including infection of healthy siblings have been reported. The observation of acute and chronic infections by vaccine strain rotavirus in immunocompromised children led to the addition of SCID as contraindication for administration of rotavirus vaccine. Our SCID patient presented already with advanced disease and severely reduced general condition. We believe that viral shedding of rotavirus for such a prolonged period of time has not been previously reported and might be explained by the lack of timely adequate medical management. We cannot completely exclude that other gastrointestinal infections and other comorbidities might have been involved, but we propose that vaccine-acquired rotavirus-infection had probably contributed significantly to the fatal outcome. The clinical manifestation and diagnosis of SCID patients might be delayed well into the life period after the recommended vaccination schedule. Therefore, we emphasize the negative effect that rotavirus vaccination might have in these endangered patients and we strongly support efforts of mandatory newborn screening for SCID.

Hantaan virus surveillance in small mammals at firing points 10 and 60, Yeoncheon, Gyeonggi Province, Republic of Korea.

We used epidemiological data and indirect fluorescent antibody tests to determine the Hantaan virus (HTNV) antibody-positive (Ab+) prevalence in small mammals captured at firing point 10 (FP-10) and firing point 60 (FP-60), Gyeonggi Province, near the demilitarized zone, Republic of Korea (ROK), from 2001 to 2005. We used these data, combined with the partial M segment amplified from HTNV recovered from lung tissues of Apodemus agrarius, to clarify the genetic diversity and phylogenetic relationships among HTNV strains in the ROK. Of the eight species of rodents and one insectivore species captured, A. agrarius accounted for 93.4% and 88.5% at FP-10 and FP-60, respectively. Only two species of rodents, A. agrarius and Micromys minutus, were HTNV Ab+. The overall HTNV Ab+ prevalence for A. agrarius captured at FP-10 and FP-60 was 23.3% (121/520) and 14.5% (94/647), respectively. The hantaviral reverse transcription-polymerase chain reaction-positive rate of Ab+ A. agrarius was 74.2% (167/215), and the phylogenetic trees, based on the 269-nucleotide G2-encoding M segment, demonstrated that HTNV strains from FP-10 and FP-60 were distantly segregated from HTNV of other geographic regions in Korea and China. These data are useful in the development of risk reduction strategies for the prevention of hantavirus infections among military personnel, especially during training or the event of hostilities, and civilian populations.