Immune response in silico (IRIS): immune-specific genes identified from a compendium of microarray expression data.

Immune cell-specific expression is one indication of the importance of a gene's role in the immune response. We have compiled a compendium of microarray expression data for virtually all human genes from six key immune cell types and their activated and differentiated states. Immune Response In Silico (IRIS) is a collection of genes that have been selected for specific expression in immune cells. The expression pattern of IRIS genes recapitulates the phylogeny of immune cells in terms of the lineages of their differentiation. Gene Ontology assignments for IRIS genes reveal significant involvement in inflammation and immunity. Genes encoding CD antigens, cytokines, integrins and many other gene families playing key roles in the immune response are highly represented. IRIS also includes proteins of unknown function and expressed sequence tags that may not represent genes. The predicted cellular localization of IRIS proteins is evenly distributed between cell surface and intracellular compartments, indicating that immune specificity is important at many points in the signaling pathways of the immune response. IRIS provides a resource for further investigation into the function of the immune system and immune diseases.

Hypertrophy, hyperplasia, and infectious virus in gut-associated lymphoid tissue of mice after oral inoculation with simian-human or bovine-human reassortant rotaviruses.

Oral inoculation of infants with a vaccine that contains simian-human reassortant rotaviruses has been found to be a rare cause of intussusception. Because intussusception can be associated with enlargement of gut-associated lymphoid tissue, we studied the capacity of simian-human and bovine-human reassortant rotaviruses to cause lymphoid hypertrophy and hyperplasia of Peyer's patches (PP) of adult BALB/c mice. Neither hypertrophy nor hyperplasia was detected in PP after oral inoculation with simian-human or bovine-human reassortant rotaviruses. However, infectious virus was detected in PP and mesenteric lymph nodes after oral inoculation with simian, but not bovine, reassortant rotaviruses. Implications of these findings on the pathogenesis of intussusception are discussed.

Molecular characterization of serotype G9 rotavirus strains from a global collection.

Between 1992 and 1998, serotype G9 human rotavirus (RV) strains have been detected in 10 countries, including Thailand, India, Brazil, Bangladesh, Malawi, Italy, France, the United States, the United Kingdom, and Australia, suggesting the possible emergence of the fifth common serotype worldwide. Unlike the previously characterized reference G9 strains (i.e., WI61 and F45), the recent G9 isolates had a variety of gene combinations, raising questions concerning their origin and evolution. To identify the progenitor strain and examine the on-going evolution of the recent G9 strains, we characterized by genetic and antigenic analyses 16 isolates obtained from children with diarrhea in India, Bangladesh, the United States, and Malawi. Specifically, we sequenced their VP7 and NSP4 genes and compared the nucleotide (nt) and deduced amino acid sequences with the reference G9 strains. To identify reassortment, we examined the products of five gene segments; VP4, VP7, and NSP4 genotypes (genes 4, 9, and 10); subgroups (gene 6); electropherotypes (gene 11); and the genogroup profiles of all of the recent G9 isolates. Sequence analysis of the VP7 gene indicated that the recent U.S. P[6],G9 strains were closely related to the Malawian G9 strains (>99% nt identity) but distinct from G9 strains of India ( approximately 97% nt identity), Bangladesh ( approximately 98% nt identity), and the reference strains ( approximately 97% nt identity). Phylogenetic analysis identified a single cluster for the U.S. P[6],G9 strains that may have common progenitors with Malawian P[6],G9 strains whereas separate lineages were defined for the Indian, Bangladeshi, and reference G9 strains. Northern hybridization results indicated that all 11 gene segments of the Malawian P[6],G9 strains hybridized with a probe derived from a U.S. strain of the same genotype and may have the same progenitor, different from the Indian G9 strains, whereas the Bangladesh strains may have evolved from the U.S. G9 progenitors. Overall, our findings suggest that much greater diversity among the newly identified G9 strains has been generated by reassortment between gene segments than through the accumulation of mutations in a single gene.

The rotavirus vaccine.

A rotavirus vaccine was recently licensed by the Food and Drug Administration and is likely to be recommended for use in all infants by both the Advisory Committee on Immunization Practices and the American Academy of Pediatrics. If used as recommended, the vaccine is likely to prevent much of the 500,000 physician visits, 50,000 hospitalizations, and 20 to 40 deaths caused by rotavirus infections every year in the United States. An understanding of the biology, immunology, and pathogenesis of rotavirus infection will help to explain the strengths and limitations of the rotavirus vaccine.

Genetic and antigenic characterization of a serotype P[6]G9 human rotavirus strain isolated in the United States.

During an epidemiologic survey of rotavirus infections established to monitor the prevalent G serotypes circulating in the United States, human P[6]G9, subgroup I rotavirus strains causing symptomatic infections were identified as the fourth most common serotype. In this report we describe the molecular and antigenic characterization of one of these P[6]G9 isolates (US1205). Neutralization and sequencing studies have demonstrated that both outer capsid proteins, VP7 and VP4, of US1205 are closely related to but genetically and antigenically distinguishable from those of standard G9 strains (e.g., F45, WI61) and standard P2A[6] strains (e. g., ST3, M37). Thus the complete antigenic type of US1205 is P2A[6]G9, subgroup I. Sequence analysis of the VP6 and NSP4 genes of US1205 indicates that strain US1205 possessed VP6 subgroup I and NSP4A genotype specificities. Finally, Northern hybridization studies suggest that the P[6]G9 strains are closely related to members of the DS-1 genogroup except for their P[6] VP4 gene, which has been commonly identified in strains of both major human genogroups, and their G9 VP7 gene, which may have been derived by reassortment with a Wa genogroup strain. Examination of historic collections and prospective surveillance of strains will be needed to determine whether this strain has been present for some time or if it is emerging to compete with the other common serotypes of rotavirus.

Identification of two lineages (WA-like and F45-like) within the major rotavirus genotype P[8].

The fourth gene of a porcine (S8) and eight human rotavirus isolates possessing the major human VP4 specificity (P1A serotype and/or P[8] genotype) were partially sequenced and compared to other available P[8] sequences from rotaviruses types G1, G3, G5 and G9 specificities which had been originally recovered from children with diarrhea in Japan, Brazil and the USA. Brazilian rotavirus S8 represented the single known porcine rotavirus with this P specificity. Phylogenetic analysis revealed two lineages or subgenotypes within P[8] strains: the F45-like P subgenotype comprised most of the strains, including all the human G5 isolates analyzed, whereas the Wa- or S8-like subgenotype consisted of only a human isolate obtained in the same geographic region as S8 and an American strain with atypical RNA profile besides the prototypes Wa and S8 viruses. A conserved basic amino acid residue at position 131 in VP4 seemed characteristic of the F45-like P[8] subgenotype.

Relationship among serotype G3P5A rotavirus strains isolated from different host species.

The relationship among G3P5A rotavirus strains was analysed by restriction endonuclease assay of the VP4, VP6 and VP7 encoding genes, neutralization assay and phylogenetic analysis. The restriction patterns of the capsid encoding genes were species specific allowing the differentiation among the strains of different origin. The VP7 profiles differentiated human from animal strains more efficiently. The phylogenetic analysis of the VP4 gene demonstrated that HCR3A and K9 are closer related to each other than to other P5A strains. The same occurs to strains Ro1845 and Cat 97. The CU-1 virus appears to be an ancestor of the P5A strains by neutralization and phylogenetic analysis. The results placed the RRV strain definitely in a separate VP4 serotype and genotype from that of P5A strains. Restriction endonuclease assay of the capsid encoding genes seems to be a useful tool to identify the host species of rotavirus strains belonging to the same serotype and/or genotype.

Relative importance of rotavirus-specific effector and memory B cells in protection against challenge.

Adult BALB/c mice were orally inoculated with murine (strain EDIM), simian (strain RRV), or bovine (strain WC3) rotavirus. Six or 16 weeks after inoculation, mice were challenged with EDIM. At the time of challenge and in the days immediately following challenge, production of rotavirus-specific immunoglobulin A (IgA), IgG, and IgM by small intestinal lamina propria lymphocytes (LPL) was determined by fragment culture, and quantities of virus-specific antibodies at the intestinal mucosal surface were determined by intestinal lavage. Mice immunized with EDIM were completely protected against EDIM challenge both 6 and 16 weeks after immunization. Protection was associated with production of high levels of IgA by LPL and detection of virus-specific IgA at the intestinal mucosal surface. In addition, animals immunized and later challenged with EDIM did not develop a boost in antibody responses, suggesting that they were also not reinfected. We also found that in mice immunized with nonmurine rotaviruses, (i) quantities of virus-specific IgA generated following challenge were greater 16 weeks than 6 weeks after immunization, (ii) immunization enhanced the magnitude but did not hasten the onset of production of high quantities of virus-specific IgA by LPL after challenge, and (iii) immunization induced partial protection against challenge; however, protection was not associated with either production of virus-specific antibodies by LPL or detection of virus-specific antibodies at the intestinal mucosal surface.

Immunologic correlates of protection against rotavirus challenge after intramuscular immunization of mice.

The capacity of intramuscular (i.m.) inoculation of mice with homologous or heterologous host rotaviruses to induce protection from challenge was evaluated. i.m. inoculation with live, wild-type rotavirus (murine strain EDIM) induced complete protection from viral shedding after challenge for at least 6 weeks after inoculation; protection was correlated with production of virus-specific immunoglobulin A (IgA) by lamina propria (LP) lymphocytes. i.m. inoculation with inactivated EDIM, cell culture-adapted EDIM, or simian strain RRV was associated with partial protection, characterized by reduced viral shedding after challenge. Partial protection after challenge was not associated with production of virus-specific IgA by LP lymphocytes. The mechanisms by which i.m. inoculation induces virus-specific humoral immune responses in the small intestinal LP were examined.

The development of multivalent bovine rotavirus (strain WC3) reassortant vaccine for infants.

Laboratory and clinical studies have been directed toward development of a vaccine against rotavirus gastroenteritis in infants. First, bovine rotavirus strain WC3, which did not induce neutralizing antibodies to predominant human rotavirus (HRV) serotypes, was determined to be safe and immunogenic; however, it was not protective in all efficacy trials. HRVs adapted to cell culture retained some virulence for infants, but when further attenuated by cold adaptation, they were poorly immunogenic. Reassortant rotaviruses were designed to express HRV surface proteins VP7 (G) or VP4 (P) while retaining a bovine WC3 genome background. Reassortants containing either HRV surface protein and as few as four bovine rotavirus genes were safe in infants. A monovalent WC3 reassortant of serotype G1 specificity was 64%-100% protective in placebo-controlled trials. A quadrivalent WC3 reassortant vaccine with components of HRV G1, G2, G3, and P[8] specificity induced 67% protection against all rotavirus disease in a multicenter efficacy trial.

WC3 reassortant vaccines in children.

Bovine rotavirus strain WC3 (P7[5], G6) administered at the 12th passage level was well tolerated clinically in infants and efficiently induced serum virus neutralizing antibody (VNA) with bovine rotavirus G6 specificity. The protective efficacy of WC3 vaccine against all rotavirus disease was inconsistent, varying in four separate trials from 76% to 0%; some selective protection against severe disease was seen in all trials. WC3 reassortants containing the gene for an individual human rotavirus VP7 (G) or VP4 (P) surface antigen were also well tolerated, but preferentially induced VNA to the WC3 parent. Efficacy trials of human G1 VP7 reassortant WI79-9 (P7[5], G1) consistently led to > 60% protection against all rotavirus disease. A quadrivalent WC3 reassortant vaccine was developed to contain four separate monovalent reassortants expressing human rotaviruses surface proteins G1, G2, G3, and P1A [8] respectively. In a multicenter trial including 439 infants, this vaccine induced 67.1% protection against all rotavirus disease (defined as positive for rotavirus antigen by ELISA only [p = < 0.001]) and 72.6% protection when the standard for rotavirus diagnosis was a positive test of stool for both rotavirus antigen by ELISA and rotavirus RNA by electropherotype analysis (p = < 0.001). In this trial, episodes of the most severe rotavirus disease (clinical severity score > 16.0 eight cases) occurred only in placebo recipients.

Dachsous encodes a member of the cadherin superfamily that controls imaginal disc morphogenesis in Drosophila.

Mutations in the dachsous gene of Drosophila lead to striking defects in the morphogenesis of the thorax, legs, and wings. The dachsous gene has been cloned and shown to encode a huge transmembrane protein that is a member of the cadherin superfamily, similar to the fat gene reported previously. Both the Dachsous and Fat proteins contain large tandem arrays of cadherin domains--27 and 34, respectively--as compared with 4 cadherin domains in classic vertebrate cadherins. In addition, Dachsous and Fat each has a cytoplasmic domain with sequence similarity to the cytoplasmic beta-catenin-binding domain of classic vertebrate cadherins. The dachsous gene is expressed in the ectoderm of embryos, whereas its expression in larvae is restricted to imaginal discs and specific regions of the brain. The phenotypes of, and genetic interactions between dachsous and fat are consistent with a model in which cell proliferation and morphogenesis of imaginal structures depends on the coupled equilibria between homo- and heterophilic interactions of the Dachsous and Fat cadherin proteins.

Evaluation of the protective efficacy of a serotype 1 bovine-human rotavirus reassortant vaccine in infants.

The objective of this study was to evaluate the efficacy of a live, attenuated bovine (strain WC3) x human (strain WI79, serotype G1) rotavirus reassortant (WI79-9) virus vaccine for prevention of symptomatic rotavirus gastroenteritis in infants. The study was a prospective, randomized, double-blind, placebo-controlled trial, conducted over a single rotavirus season in 325 infants who were 2 to 8 months old at enrollment. Subjects were randomized to receive either placebo or WI79-9 virus vaccine at 10(7.3) plaque-forming units in three oral doses each separated by 2 months. Subjects were followed for 7 days after each dose for occurrence of adverse events and during the subsequent winter for development of rotavirus gastroenteritis. Administration of WI79-9 virus vaccine was well-tolerated, and the rates of low grade fever after each dose were no higher in vaccine recipients (8 to 21%) than in placebo recipients (14 to 19%). The protective efficacy of the WI79-9 vaccine during a subsequent epidemic of predominantly serotype G1 rotavirus was 87.0% (95% confidence limits, 62.6 to 95.5%) against relatively severe rotavirus gastroenteritis (rotavirus gastroenteritis with a clinical severity score of > 8) and was 64.1% (95% confidence limits 35.9 to 79.9%) against all symptomatic rotavirus episodes. The WI79-9 vaccine was safe and effective in prevention of homotypic human rotavirus infection in infants. Further studies of reassortant vaccines based on the bovine WC3 rotavirus should be performed.

Enhancement of rotavirus immunogenicity by microencapsulation.

It was determined whether microencapsulation of rotavirus enhanced virus-specific immunity in mice. Combinations of several water-soluble anionic polymers and amines were tested for their capacity to form microcapsules which were stable in the presence of simulated gastric acid. Using the combinations of sodium alginate and spermine hydrochloride or sodium chondroitin sulfate and spermine hydrochloride we found that microcapsules (1) captured infectious rotavirus, (2) penetrated into the persisted in gut-associated lymphoid tissue (GALT) after oral inoculation, (3) delivered rotavirus antigen to GALT at levels greater than those detected after oral inoculation with free virus, and (4) enhanced the virus-specific humoral immune response after oral or parenteral immunization.

Comparative analysis of VP8* sequences from rotaviruses possessing M37-like VP4 recovered from children with and without diarrhoea.

Rotavirus strains belonging to G types 1 to 4 and having a P3 genotype (M37-like VP4) were recovered from children with symptomatic and asymptomatic infections. Partial sequences of their VP4 genes were determined in an attempt to characterize these strains further. The genomic regions encoding VP8*, the connecting and putative fusion peptides and three other regions in VP5* were sequenced. The deduced amino acid sequences were compared with rotavirus strains belonging to different P genotypes that had been previously reported. High degrees of identity were found between the VP8* fragment of all human P3 strains (92.7 to 99.7%) suggesting that they belong to the same genotype, regardless of differences in their virulence. Furthermore, based on comparative sequence analysis, we did not identify any amino acid(s) that differ appreciably between symptomatic and asymptomatic strains and could therefore be associated with virulence. The results suggest that the P3 genotype, although frequently associated with asymptomatic infections, may not be the single determining factor in attenuation of symptoms.

Amino acid sequence similarity of the VP7 protein of human rotavirus HCR3 to that of canine and feline rotaviruses.

The sequence of the VP7 gene of human rotavirus strain HCR3 was determined and its predicted amino acid sequence was compared with that of other rotavirus strains. The VP7 gene is 1062 nucleotides long and contains a single open reading frame of 981 nucleotides capable of encoding a protein of 326 amino acids. The VP7 amino acid sequence similarity of strain HCR3 to those of various human and animal G3 serotypes ranged from 88.7 to 99.4%, and from 60.4 to 88.3% to strains representing each of the other 13 G serotypes. Alignment of four variable regions [VR4, VR5(A), VR7(B) and VR8(C)] of HCR3 with those of G3 strains of different host species showed that HCR3 possesses a sequence almost identical to that of canine rotaviruses and feline rotavirus strain CAT97 in all four regions. A considerable divergence in regions VR4, VR7(B) and VR8(C) was found with strains of human, mouse, monkey, horse and rabbit rotaviruses. This observation together with results of our previous study on VP4 indicated that human rotavirus HCR3 is genetically more closely related to animal rotaviruses than to other human rotaviruses.

VP4 genotyping of human rotavirus in the United States.

The VP4 (P) and VP7 (G) types of 171 rotavirus isolates obtained from children with diarrhea in the United States were characterized by PCR typing assays. Strains P1G1 predominated (71%); this was followed by strains P1G3 (20%) and P2G2 and P1G4 (2% each). Mixed types were identified in five (3%) specimens. Two (1%) strains bearing the P3 genotype (P3G1 and P3G2) were found in children with severe dehydrating diarrhea, although the P3 genotype has been regarded as a possible marker for virus attenuation.

Nucleotide sequence of the VP4-encoding gene of an unusual human rotavirus (HCR3).

Human rotavirus strain HCR3 was isolated from the stool of a clinically normal infant and identified as a serotype G3 rotavirus; however, it could not be grouped into any known human VP4 genetic groups by a polymerase chain reaction assay. The fourth gene of strain HCR3, which encodes the outer capsid protein VP4, was sequenced. This gene is 2362 nucleotides in length and contains one open reading frame capable of encoding a protein of 776 amino acids. The VP4 protein of strain HCR3 shared 67.5-73.5% amino acid identity with those of strains KU, RV-5, 1076, and K8, representing four human genetic groups, and relatively high homology (84.7%) with a fifth genetic group represented by strain 69M, whose VP4 shows more similarity to animal than to human strains. Strain HCR3 shared higher VP4 amino acid homology with various animal rotaviruses, ranging from 74.5 to 89.4%. These observations suggest that the VP4 outer capsid protein of strain HCR3 represents a new VP4 genetic group that is more closely related to animal rotaviruses than to human rotaviruses.