Rotavirus infection and intussusception: a view from retrospect

A live orally-administrable rhesus rotavirus (RRV) tetravalent (TV) vaccine, licensed in the U.S.A. and the European Union, was recalled from the market because it was suspected to increase the risk of intussusception during the week following immunization. In contrast, natural rotavirus infection is generally believed not to cause intussusception. Because my experience contributed to the first paper that linked intussusception with rotavirus infection, I have re-examined our own data published 22 years ago and other studies on this issue. I also made a case study of adenovirus and intussusception as a paradigm to establish an etiological association of viral infection and intussusception. My hypothesis postulated in this review is that natural infection of susceptible (or predisposed) infants with some rotavirus strains, probably serotype G3 rotaviruses, will result in an appreciable fraction of idiopathic intussusception. Thus, the number of rotavirus-induced intussusception cases may change reflecting the relative frequency of G3 strains, which I believe was much higher in the 70s than during the last two decades. The epidemiological data indicate that the RRV-TV vaccine triggers intussusception at a rate significantly higher than the background incidence rate following the week of vaccination, particularly after the first dose. In contrast, the data do not suggest that the cumulative incidence among the vaccine recipients increases accordingly, implicating that the risk of intussusception attributable to the RRV-TV vaccine may be minimal.     

The epithelial cell response to rotavirus infection.

Rotavirus is the most important worldwide cause of severe gastroenteritis in infants and young children. Intestinal epithelial cells are the principal targets of rotavirus infection, but the response of enterocytes to rotavirus infection is largely unknown. We determined that rotavirus infection of HT-29 intestinal epithelial cells results in prompt activation of NF-kappaB (<2 h), STAT1, and ISG F3 (3 h). Genetically inactivated rotavirus and virus-like particles assembled from baculovirus-expressed viral proteins also activated NF-kappaB. Rotavirus infection of HT-29 cells induced mRNA for several C-C and C-X-C chemokines as well as IFNs and GM-CSF. Mice infected with simian rotavirus or murine rotavirus responded similarly with the enhanced expression of a profile of C-C and C-X-C chemokines. The rotavirus-stimulated increase in chemokine mRNA was undiminished in mice lacking mast cells or lymphocytes. Rotavirus induced chemokines only in mice <15 days of age despite documented infection in older mice. Macrophage inflammatory protein-1beta and IFN-stimulated protein 10 mRNA responses occurred, but were reduced in p50-/- mice. Macrophage inflammatory protein-1beta expression during rotavirus infection localized to the intestinal epithelial cell in murine intestine. These results show that the intestinal epithelial cell is an active component of the host response to rotavirus infection.     

Lymphotoxin Alpha-Deficient Mice Clear Persistent Rotavirus Infection after Local Generation of Mucosal IgA

Rotavirus is a major cause of pediatric diarrheal illness worldwide. To explore the role of organized intestinal lymphoid tissues in infection by and immunity to rotavirus, lymphotoxin alpha-deficient (LTα^−/−) mice that lack Peyer''s patches and mesenteric lymph nodes were orally infected with murine rotavirus. Systemic rotavirus was cleared within 10 days in both LTα^−/− and wild-type mice, and both strains developed early and sustained serum antirotavirus antibody responses. However, unlike wild-type mice, which resolved the intestinal infection within 10 days, LTα^−/− mice shed fecal virus for approximately 50 days after inoculation. The resolution of fecal virus shedding occurred concurrently with induction of intestinal rotavirus-specific IgA in both mouse strains. Induction of intestinal rotavirus-specific IgA in LTα^−/− mice correlated with the (late) appearance of IgA-producing plasma cells in the small intestine. This, together with the absence of rotavirus-specific serum IgA, implies that secretory rotavirus-specific IgA was produced locally. These findings indicate that serum IgG responses are insufficient and imply that local intestinal IgA responses are important for the clearance of rotavirus from intestinal tissues. Furthermore, they show that while LTα-dependent lymphoid tissues are important for the generation of IgA-producing B cells in the intestine, they are not absolutely required in the setting of rotavirus infection. Moreover, the induction of local IgA-producing B cell responses can occur late after infection and in an LTα-independent manner.     

Association of gamma interferon and interleukin-17 production in intestinal CD4+ T cells with protection against rotavirus shedding in mice intranasally immunized with VP6 and the adjuvant LT(R192G)

Mucosal immunization of mice with chimeric, Escherichia coli-expressed VP6, the protein that comprises the intermediate capsid layer of the rotavirus particle, together with attenuated E. coli heat-labile toxin LT(R192G) as an adjuvant, reduces fecal shedding of rotavirus antigen by >95% after murine rotavirus challenge, and the only lymphocytes required for protection are CD4+ T cells. Because these cells produce cytokines with antiviral properties, the cytokines whose expression is upregulated in intestinal memory CD4+ T cells immediately after rotavirus challenge of VP6/LT(R192G)-immunized mice may be directly or indirectly responsible for the rapid suppression of rotavirus shedding. This study was designed to identify which cytokines are significantly upregulated in intestinal effector sites and secondary lymphoid tissues of intranasally immunized BALB/c mice after challenge with murine rotavirus strain EDIM. Initially, this was done by using microarray analysis to quantify mRNAs for 96 murine common cytokines. With this procedure, the synthesis of mRNAs for gamma interferon (IFN-gamma) and interleukin-17 (IL-17) was found to be temporarily upregulated in intestinal lymphoid cells of VP6/LT(R192G)-immunized mice at 12 h after rotavirus challenge. These cytokines were also produced in CD4+ T cells obtained from intestinal sites specific to VP6/LT(R192G)-immunized mice after in vitro exposure to VP6 as determined by intracellular cytokine staining and secretion of cytokines. Although genetically modified mice that lack receptors for either IFN-gamma or IL-17 remained protected after immunization, these results provide suggestive evidence that these cytokines may play direct or indirect roles in protection against rotavirus after mucosal immunization of mice with VP6/LT(R192G).     

Migration of antigen-presenting B cells from peripheral to mucosal lymphoid tissues may induce intestinal antigen-specific IgA following parenteral immunization.

Parenterally administered immunizations have long been used to induce protection from mucosal pathogens such as Bordetella pertussis and influenza virus. We previously found that i.m. inoculation of mice with the intestinal pathogen, rotavirus, induced virus-specific Ab production by intestinal lymphocytes. We have now used adoptive transfer studies to identify the cell types responsible for the generation of virus-specific Ab production by gut-associated lymphoid tissue (GALT) after i.m. immunization. Three days after i.m. immunization with rotavirus, cells obtained from the draining peripheral lymph nodes of donor mice were transferred into naive recipient mice. We found that intestinal lymphocytes produced rotavirus-specific Igs (IgM, IgA, and IgG) 2 wk after transfer of either unfractionated cells, or unfractionated cells rendered incapable of cellular division by mitomycin C treatment. Additional studies demonstrated that rotavirus-specific IgA, but not IgG, was produced by intestinal lymphocytes after transfer of purified B cells. Ig allotype analysis revealed that rotavirus-specific IgA was produced by intestinal B cells of recipient origin, suggesting that migration of Ag-presenting B cells from peripheral lymphoid tissues to GALT may contribute to the generation of mucosal IgA responses after parenteral immunization. Strategies that promote Ag uptake and presentation by B cells may enhance mucosal IgA production following parenteral immunization.     

Elevated lipopolysaccharide in the colon evokes intestinal inflammation, aggravated in immune modulator-impaired mice

Frequency of gram-negative bacteria is markedly enhanced in inflamed gut, leading to augmented LPS in the intestine. Although LPS in the intestine is considered harmless and, rather, provides protective effects against epithelial injury, it has been suggested that LPS causes intestinal inflammation, such as necrotizing enterocolitis. Therefore, direct effects of LPS in the intestine remain to be studied. In this study, we examine the effect of LPS in the colon of mice instilled with LPS by rectal enema. We found that augmented LPS on the luminal side of the colon elicited inflammation in the small intestine remotely, not in the colon; this inflammation was characterized by body weight loss, increased fluid secretion, enhanced inflammatory cytokine production, and epithelial damage. In contrast to the inflamed small intestine induced by colonic LPS, the colonic epithelium did not exhibit histological tissue damage or inflammatory lesions, although intracolonic LPS treatment elicited inflammatory cytokine gene expression in the colon tissues. Moreover, we found that intracolonic LPS treatment substantially decreased the frequency of immune-suppressive regulatory T cells (CD4(+)/CD25(+) and CD4(+)/Foxp3(+)). We were intrigued to find that LPS-promoted intestinal inflammation is exacerbated in immune modulator-impaired IL-10(-/-) and Rag-1(-/-) mice. In conclusion, our results provide evidence that elevated LPS in the colon is able to cause intestinal inflammation and, therefore, suggest a physiological explanation for the importance of maintaining the balance between gram-negative and gram-positive bacteria in the intestine to maintain homeostasis in the gut.     

Removing the Age Restrictions for Rotavirus Vaccination: A Benefit-Risk Modeling Analysis

Background

To minimize potential risk of intussusception, the World Health Organization (WHO) recommended in 2009 that rotavirus immunization should be initiated by age 15 weeks and completed before 32 weeks. These restrictions could adversely impact vaccination coverage and thereby its health impact, particularly in developing countries where delays in vaccination often occur.

Methods and Findings

We conducted a modeling study to estimate the number of rotavirus deaths prevented and the number of intussusception deaths caused by vaccination when administered on the restricted schedule versus an unrestricted schedule whereby rotavirus vaccine would be administered with DTP vaccine up to age 3 years. Countries were grouped on the basis of child mortality rates, using WHO data. Inputs were estimates of WHO rotavirus mortality by week of age from a recent study, intussusception mortality based on a literature review, predicted vaccination rates by week of age from USAID Demographic and Health Surveys, the United Nations Children''s Fund (UNICEF) Multiple Indicator Cluster Surveys (MICS), and WHO-UNICEF 2010 country-specific coverage estimates, and published estimates of vaccine efficacy and vaccine-associated intussusception risk. On the basis of the error estimates and distributions for model inputs, we conducted 2,000 simulations to obtain median estimates of deaths averted and caused as well as the uncertainty ranges, defined as the 5th–95th percentile, to provide an indication of the uncertainty in the estimates.We estimated that in low and low-middle income countries a restricted schedule would prevent 155,800 rotavirus deaths (5th–95th centiles, 83,300–217,700) while causing potentially 253 intussusception deaths (76–689). In contrast, vaccination without age restrictions would prevent 203,000 rotavirus deaths (102,000–281,500) while potentially causing 547 intussusception deaths (237–1,160). Thus, removing the age restrictions would avert an additional 47,200 rotavirus deaths (18,700–63,700) and cause an additional 294 (161–471) intussusception deaths, for an incremental benefit-risk ratio of 154 deaths averted for every death caused by vaccine. These extra deaths prevented under an unrestricted schedule reflect vaccination of an additional 21%–25% children, beyond the 63%–73% of the children who would be vaccinated under the restricted schedule. Importantly, these estimates err on the side of safety in that they assume high vaccine-associated risk of intussusception and do not account for potential herd immunity or non-fatal outcomes.

Conclusions

Our analysis suggests that in low- and middle-income countries the additional lives saved by removing age restrictions for rotavirus vaccination would far outnumber the potential excess vaccine-associated intussusception deaths. Please see later in the article for the Editors'' Summary     

Role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice

Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-α/β and IFN-γ receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B- or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-α/β receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-γ receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.     

Protective Immunity Induced by Oral Immunization with a Rotavirus DNA Vaccine Encapsulated in Microparticles

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.     

Enhanced disease and pulmonary eosinophilia associated with formalin-inactivated respiratory syncytial virus vaccination are linked to G glycoprotein CX3C-CX3CR1 interaction and expression of substance P

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine or RSV G glycoprotein results in enhanced pulmonary disease after live RSV infection. Enhanced pulmonary disease is characterized by pulmonary eosinophilia and is associated with a substantial inflammatory response. We show that the absence of the G glycoprotein or G glycoprotein CX3C motif during FI-RSV vaccination or RSV challenge of FI-RSV-vaccinated mice, or treatment with anti-substance P or anti-CX3CR1 antibodies, reduces or eliminates enhanced pulmonary disease, modifies T-cell receptor Vbeta usage, and alters CC and CXC chemokine expression. These data suggest that the G glycoprotein, and in particular the G glycoprotein CX3C motif, is key in the enhanced inflammatory response to FI-RSV vaccination, possibly through the induction of substance P.     

Rotavirus virus-like particles administered mucosally induce protective immunity.

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.     

Probiotic Lactobacillus rhamnosus GG Enhanced Th1 Cellular Immunity but Did Not Affect Antibody Responses in a Human Gut Microbiota Transplanted Neonatal Gnotobiotic Pig Model

This study aims to establish a human gut microbiota (HGM) transplanted gnotobiotic (Gn) pig model of human rotavirus (HRV) infection and diarrhea, and to verify the dose-effects of probiotics on HRV vaccine-induced immune responses. Our previous studies using the Gn pig model found that probiotics dose-dependently regulated both T cell and B cell immune responses induced by rotavirus vaccines. We generated the HGM transplanted neonatal Gn pigs through daily feeding of neonatal human fecal suspension to germ-free pigs for 3 days starting at 12 hours after birth. We found that attenuated HRV (AttHRV) vaccination conferred similar overall protection against rotavirus diarrhea and virus shedding in Gn pigs and HGM transplanted Gn pigs. HGM promoted the development of the neonatal immune system, as evidenced by the significantly enhanced IFN-γ producing T cell responses and reduction of regulatory T cells and their cytokine production in the AttHRV-vaccinated pigs. The higher dose Lactobacillus rhamnosus GG (LGG) feeding (14 doses, up to 10⁹ colony-forming-unit [CFU]/dose) effectively increased the LGG counts in the HGM Gn pig intestinal contents and significantly enhanced HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine. Lower dose LGG (9 doses, up to 10⁶ CFU/dose) was ineffective. Neither doses of LGG significantly improved the protection rate, HRV-specific IgA and IgG antibody titers in serum, or IgA antibody titers in intestinal contents compared to the AttHRV vaccine alone, suggesting that an even higher dose of LGG is needed to overcome the influence of the microbiota to achieve the immunostimulatory effect in the HGM pigs. This study demonstrated that HGM Gn pig is an applicable animal model for studying immune responses to rotavirus vaccines and can be used for studying interventions (i.e., probiotics and prebiotics) that may enhance the immunogenicity and protective efficacy of vaccines through improving the gut microbiota.     

Age-Dependent TLR3 Expression of the Intestinal Epithelium Contributes to Rotavirus Susceptibility

Rotavirus is a major cause of diarrhea worldwide and exhibits a pronounced small intestinal epithelial cell (IEC) tropism. Both human infants and neonatal mice are highly susceptible, whereas adult individuals remain asymptomatic and shed only low numbers of viral particles. Here we investigated age-dependent mechanisms of the intestinal epithelial innate immune response to rotavirus infection in an oral mouse infection model. Expression of the innate immune receptor for viral dsRNA, Toll-like receptor (Tlr) 3 was low in the epithelium of suckling mice but strongly increased during the postnatal period inversely correlating with rotavirus susceptibility, viral shedding and histological damage. Adult mice deficient in Tlr3 (Tlr3^−/−) or the adaptor molecule Trif (Trif^Lps2/Lps2) exerted significantly higher viral shedding and decreased epithelial expression of proinflammatory and antiviral genes as compared to wild-type animals. In contrast, neonatal mice deficient in Tlr3 or Trif did not display impaired cell stimulation or enhanced rotavirus susceptibility. Using chimeric mice, a major contribution of the non-hematopoietic cell compartment in the Trif-mediated antiviral host response was detected in adult animals. Finally, a significant age-dependent increase of TLR3 expression was also detected in human small intestinal biopsies. Thus, upregulation of epithelial TLR3 expression during infancy might contribute to the age-dependent susceptibility to rotavirus infection.     

Transient TLR activation restores inflammatory response and ability to control pulmonary bacterial infection in germfree mice

Mammals are colonized by an astronomical number of commensal microorganisms on their environmental exposed surfaces. These symbiotic species build up a complex community that aids their hosts in several physiological activities. We have shown that lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness. The present study investigated whether the germfree state and its hyporesponsive phenotype alter host resistance to an infectious bacterial insult. Experiments performed in germfree mice infected with Klebsiella pneumoniae showed that these animals are drastically susceptible to bacterial infection in an IL-10-dependent manner. In germfree mice, IL-10 restrains proinflammatory mediator production and neutrophil recruitment and favors pathogen growth and dissemination. Germfree mice were resistant to LPS treatment. However, priming of these animals with several TLR agonists recovered their inflammatory responsiveness to sterile injury. LPS pretreatment also rendered germfree mice resistant to pulmonary K. pneumoniae infection, abrogated IL-10 production, and restored TNF-α and CXCL1 production and neutrophil mobilization into lungs of infected germfree mice. This effective inflammatory response mounted by LPS-treated germfree mice resulted in bacterial clearance and enhanced survival upon infection. Therefore, host colonization by indigenous microbiota alters the way the host reacts to environmental infectious stimuli, probably through activation of TLR-dependent pathways. Symbiotic gut colonization enables proper inflammatory response to harmful insults to the host, and increases resilience of the entire mammal-microbiota consortium to environmental pressures.     

轮状病毒VP4亚单位疫苗研究进展

轮状病毒是全球范围内导致5岁以下婴幼儿严重腹泻的主要病原体,造成了巨大的经济负担和社会负担。疫苗预防接种是控制轮状病毒感染最为有效的手段,但在轮状病毒导致的死亡率较高的非洲和亚洲部分低收入国家,目前已经上市的轮状病毒疫苗的有效性较低,且会增加肠套叠的风险。更加安全、有效的轮状病毒疫苗对于降低轮状病毒感染导致的发病率和死亡率具有重要意义。目前,各国科研人员试图从多个方面提高轮状病毒疫苗的有效性,非复制型基因工程亚单位疫苗是目前轮状病毒疫苗研究的主要方向。文中就目前轮状病毒亚单位疫苗,特别是基于VP4蛋白的亚单位疫苗的研究进展进行了综述,以期对轮状病毒疫苗的发展提供借鉴意义。     

Memory and distribution of virus-specific cytotoxic T lymphocytes (CTLs) and CTL precursors after rotavirus infection.

The gastrointestinal tract is constantly exposed to a variety of potentially invasive bacteria, viruses, and parasites. The first line of defense against these pathogens is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IELs), mucus, and secretory immunoglobulins. In addition, the intestine is a rich source of lymphocytes located within Peyer's patches and the lamina propria. Little is known about the function, memory, trafficking, or origin of intestinal T lymphocytes after intestinal infection. We studied the murine cytotoxic T-lymphocyte (CTL) response to the intestinal pathogen rotavirus (simian strain RRV). Adult mice were inoculated orally or via the hind footpad with RRV; virus-specific cytotoxic activities in intestinal and nonintestinal lymphocyte populations were determined by 51Cr release assays. In addition, virus-specific CTL precursor (CTLp) frequencies were determined by limiting-dilution analysis. IELs containing rotavirus-specific cytotoxic activity were detected after oral but not footpad inoculation and expressed alpha/beta but not gamma/delta cell surface protein; virus-specific CTLs did not appear to arise from CTLp among IELs. In addition, the site at which RRV was presented to the immune system determined the site at which RRV-specific CTLp first appeared. Frequencies of rotavirus-specific CTLp detected in Peyer's patches were 25- to 30-fold greater after oral than after footpad inoculation. However, regardless of the route of inoculation, rotavirus-specific CTLp were distributed throughout the lymphoid system 21 days after infection. Implications of these findings for vaccine design are discussed.     

Childhood Intussusception: A Literature Review

Background

Postlicensure data has identified a causal link between rotavirus vaccines and intussusception in some settings. As rotavirus vaccines are introduced globally, monitoring intussusception will be crucial for ensuring safety of the vaccine programs.

Methods

To obtain updated information on background rates and clinical management of intussusception, we reviewed studies of intussusception in children <18 years of age published since 2002. We assessed the incidence of intussusception by month of life among children <1 year of age, seasonality, method of diagnosis, treatment, and case-fatality.

Findings

We identified 82 studies from North America, Asia, Europe, Oceania, Africa, Eastern Mediterranean, and Central & South America that reported a total of 44,454 intussusception events. The mean incidence of intussusception was 74 per 100,000 (range: 9–328) among children <1 year of age, with peak incidence among infants 5–7 months of age. No seasonal patterns were observed. A radiographic modality was used to diagnose intussusception in over 95% of the cases in all regions except Africa where clinical findings or surgery were used in 65% of the cases. Surgical rates were substantially higher in Africa (77%) and Central and South America (86%) compared to other regions (13–29%). Case-fatality also was higher in Africa (9%) compared to other regions (<1%). The primary limitation of this review relates to the heterogeneity in intussusception surveillance across different regions.

Conclusion

This review of the intussusception literature from the past decade provides pertinent information that should facilitate implementation of intussusception surveillance for monitoring the postlicensure safety of rotavirus vaccines.     

Peroral immunization of microencapsulated human VP8 in combination with cholera toxin induces intestinal antibody responses

To develop an orally delivered subunit vaccine for rotavirus infection, a trypsin cleavage product of VP4, recombinant VP8*, was expressed in Escherichia coli. The recombinant VP8* (rVP8*), purified by affinity chromatography, was reactive against human rotavirus positive serum in Western-blot analysis. To further evaluate the immunogenicity of the oral-delivered rVP8*, it was encapsulated with alginate-microsphere and administered in combination with cholera toxin (CT) as a mucosal adjuvant perorally into mice. The ELISPOT assay showed that the number of rVP8*-specific IgG1 antibody secreting cells increased about 3-fold and about 2-fold in spleen and Peyer's patch, respectively as compared to non-immune mice. In addition, the number of rVP8*-specific IgA antibody secreting cells increased about 2-fold in Peyer's patch. Finally, rVP8*-specific IgA antibody response was significantly enhanced in the intestinal fluids from the mice immunized perorally with encapsulated rVP8* and CT. Taken together, these results indicate that rVP8* possessed proper immunogenicity and it would be potentially useful as a subunit vaccine against rotavirus-associated disease through peroral immunization.     

Production of Two Vaccinating Recombinant Rotavirus Proteins in the Milk of Transgenic Rabbits

Rotaviruses are the main cause of infantile viral gastroenteritis worldwide leading to approximately 500,000 deaths each year mostly in the developing world. For unknown reasons, live attenuated viruses used in classical vaccine strategies were shown to be responsible for intussusception (a bowel obstruction). New strategies allowing production of safe recombinant non-replicating rotavirus candidate vaccine are thus clearly needed. In this study we utilized transgenic rabbit milk as a source of rotavirus antigens. Individual transgenic rabbit lines were able to produce several hundreds of micrograms per ml of secreted recombinant VP2 and VP6 proteins in their milk. Viral proteins expressed in our model were immunogenic and were shown to induce a significant reduction in viral antigen shedding after challenge with virulent rotavirus in the adult mouse model. To our knowledge, this is the first report of transgenic mammal bioreactors allowing the rapid co-production of two recombinant viral proteins in milk to be used as a vaccine.     

Induction and function of lipocalin prostaglandin D synthase in host immunity

Although mainly expressed in neuronal cells, lipocalin-type PGD synthase (L-PGDS) is detected in the macrophages infiltrated to atherosclerotic plaques. However, the regulation and significance of L-PGDS expression in macrophages are unknown. Here, we found that treatment of macrophages with bacterial endotoxin (LPS) or Pseudomonas induced L-PGDS expression. Epigenetic suppression of L-PGDS expression in macrophages blunted a majority of PGD(2) produced after LPS treatment. Chromatin immunoprecipitation assays show that L-PGDS induction was regulated positively by AP-1, but negatively by p53. L-PGDS expression was detected in whole lung and alveolar macrophages treated with LPS or Pseudomonas. L-PGDS overexpressing transgenic mice improved clearance of Pseudomonas from the lung compared with nontransgenic mice. Similarly, intratracheal instillation of PGD(2) enhanced removal of Pseudomonas from the lung in mice. In contrast, L-PGDS knockout mice were impaired in their ability to remove Pseudomonas from the lung. Together, our results identify induction of L-PGDS expression by inflammatory stimuli or bacterial infection, the regulatory mechanism of L-PGDS induction, and the protective role of L-PGDS expression in host immune response. Our study suggests a potential therapeutic usage of L-PGDS or PGD(2) against Pseudomonas pneumonia.