Biomedical subjects

P A Offit

Publications and source records attributed to P A Offit.

At least 55 records · Page 3Linked to original sources

PubMed1991

Neutralizing antibodies to heterologous animal rotavirus serotypes 5, 6, 7, and 10 in sera from Ecuadorian children.

Serum samples from 870 Ecuadorian children who underwent natural rotavirus exposure were tested for neutralizing serum antibody to heterologous animal rotavirus (RV) serotypes. Six percent of the sera neutralized porcine RV OSU (serotype 5), 10% neutralized bovine RV NCDV (serotype 6), 4% neutralized avian RV Ch-2 (serotype 7), and 8% neutralized bovine RV V1005 (serotype 10). Neutralization was defined as a 90% reduction in infectious virus at a 1:100 serum dilution. The prevalence of antibody to all four heterotypic viruses increased with the age of the children and the number of human RV serotypes neutralized, but prevalences did not differ significantly between children from rural and urban areas of Ecuador. No serum sample that specifically neutralized bovine RV NCDV was identified. We inferred from the seroepidemiological analysis that human RVs contain immunorecessive neutralization epitopes that can stimulate cross-neutralizing antibody to heterotypic animal RVs. This occurs increasingly with age and with the number of human serotypes recognized by a child's neutralizing antibody. Thus, it appears that a broadened immune response to the heterotypic strains occurs with repetitive RV infections.

Animals↗

PubMed1991

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.

Animals↗

PubMed1990

Presence of three P types (VP4 serotypes) and two G types (VP7 serotypes) among bovine rotavirus strains.

Cross neutralization tests with a panel of rotavirus strains representing previously described nine VP7 (G) serotypes revealed that bovine rotavirus strain KK-3, a prototype Japanese bovine serotype 2, belonged to a new serotype (G10), confirming and extending the recent report of Snodgrass et al. [J. Clin. Microbiol. 28: 504-507 (1990)] which showed that hyperimmune serum to the KK-3 strain neutralized the B223 strain, a proposed type strain of G10. Further antigenic characteristics of the KK-3 strain, as well as the 0510 strain (a G6 strain isolated in Japan), were examined in terms of their VP4 (P) specificity. For the characterization of P types, we employed genetic reassortants that possess VP4 gene for UK and VP7 gene for D (G1), VP4 gene for NCDV and VP7 gene for SA11 (G3), or VP4 gene for SA11 and VP7 gene for NCDV (G6) in the plaque reduction neutralization assay with hyperimmune sera against these two Japanese strains and the prototype bovine rotavirus NCDV strain. While the 0510 strain had UK-like P and NCDV-like G types, the KK-3 strain had a distinct set of P and G types. Thus, at least three P types (NCDV-, UK-, and KK-3-like) and two G types (G6 and G10) are present among bovine rotavirus strains.

Animals↗

PubMed1990

Rotavirus-specific cytotoxic T lymphocytes passively protect against gastroenteritis in suckling mice.

Suckling mice are protected against murine rotavirus-induced gastroenteritis after adoptive transfer of splenic lymphocytes from immunized animals. Adoptive transfer of Thy1(+)-depleted or CD8(+)-depleted lymphocytes abrogated protection against challenge. (We previously found that depletion of Thy1+ or CD8+ lymphocytes from rotavirus-immunized mice decreased rotavirus-specific cytotoxic activity in vitro.) Protection against disease occurred in the absence of rotavirus-specific neutralizing antibodies in the sera of suckling mice. Rotavirus-specific cytotoxic T lymphocytes may be important in either amelioration of acute infection or protection against reinfection.

Animals↗

PubMed1990

Polypeptide specificity of antiviral serum antibodies in children naturally infected with human rotavirus.

Reassortants between serotype 3 SA11 and serotype 6 NCDV rotaviruses were used to determine the relative amounts of serum-neutralizing antibody to VP4 and VP7 of serotype 3 SA11 rotavirus in children after natural rotavirus exposure. Sera from Ecuadorian children of a population-based study and sera from children of a hospital-based study in Germany (excluding diarrhea patients) demonstrated high titers of VP7-specific but only low titers of VP4-specific antibodies. In contrast, paired sera from German children hospitalized with a symptomatic primary rotavirus gastroenteritis demonstrated a titer increase to VP4 more frequently than to VP7 protein by neutralization test and immunoblotting. For these rotavirus patients, we provided, previously, direct evidence for the development of cross-neutralizing antibodies.

Antibodies, Viral↗

PubMed1989

Rotavirus-specific cytotoxic T lymphocyte response of mice after oral inoculation with candidate rotavirus vaccine strains RRV or WC3.

The immunologic basis of homotypic and heterotypic protection by immunization with two candidate rotavirus vaccine strains (simian strain RRV and bovine strain WC3, respectively) was investigated. Mice were orally inoculated with RRV, and 6 d later splenic lymphocytes lysed target cells infected with RRV (serotype 3) but not with human rotavirus serotypes 1 or 2; 4 w after inoculation, cytotoxic T lymphocytes (CTLs) generated in vitro from CTL precursors were also strain-specific. Also, the frequency of RRV-specific CTLs from CTLp after RRV immunization was 20-fold greater than that of cross-reactive CTLs. Inoculation of mice with WC3 (serotype 6), on the other hand, produced cross-reactive CTLs from CTLp at 4 w; CTLs lysed target cells infected with WC3 or human serotypes 1, 2, or 3 to the same extent. The frequency of cross-reactive CTLs after WC3 immunization was greater than or equal to 20-fold greater than that of WC3-specific CTLs. Cross-reactive, rotavirus-specific CTLs induced after WC3 immunization may in part explain the immunologic basis of protection against heterotypic challenge.

Administration, Oral↗

PubMed1989

Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to the heterotypic neutralization domain of VP7 and the VP8 fragment of VP4.

A murine model was used to determine whether neutralizing monoclonal antibodies (MAbs) with heterotypic specificity directed to VP7 (MAb 57-8) or to the VP8 fragment of VP4 (MAb M14) passively protect mice against challenge with various strains of rotavirus. (The gene 4 product, an outer capsid protein, has traditionally been called VP3. It has been proposed, however, that the rotavirus gene 4 product be named VP4. The gene 3 product, a core protein, has been identified recently and named VP3 [M. Liu, P. A. Offit, and M. K. Estes, Virology 163:28-32, 1988]). Suckling mice orally inoculated with MAb 57-8 did not develop diarrhea when challenged with virulent serotype 3, 4, or 6 rotaviruses, while those inoculated with MAb M14 were passively protected from challenge with serotype 3 or 6 rotaviruses, as predicted by in vitro neutralization tests. These MAbs, however, did not protect mice from infection when the mice were challenged with rotaviruses of other serotypes. We conclude that specific neutralization epitopes on each surface protein are capable of mediating protection against one or several rotavirus serotypes.

Animals↗

PubMed1989

Noninfectious rotavirus (strain RRV) induces an immune response in mice which protects against rotavirus challenge.

We found that female adult mice parenterally inoculated with noninfectious rotavirus (simian strain RRV) developed virus-specific neutralizing antibodies in the serum; newborn mice from these dams were protected against RRV-induced gastroenteritis. In addition, mice parenterally inoculated with noninfectious RRV developed virus-specific cytotoxic T-lymphocyte precursors in the spleen. Replication of rotavirus in intestinal epithelial cells was apparently not required to induce rotavirus-neutralizing antibodies or rotavirus-specific cytotoxic T lymphocytes. Parenteral immunization of infants and young children with noninfectious rotaviruses may induce an immune response which protects against rotavirus challenge.

Animals↗

PubMed1989

Rotavirus-specific protein synthesis is not necessary for recognition of infected cells by virus-specific cytotoxic T lymphocytes.

We found that rotavirus-specific protein synthesis was not necessary for recognition by virus-specific cytotoxic T lymphocytes (CTLs). In addition, CTLs lysed rotavirus-infected target cells prior to production of infectious virus. Target cell processing of rotavirus antigens for presentation to CTLs was enhanced by treatment of rotavirus with trypsin prior to infection; trypsin-induced cleavage of the viral hemagglutinin (vp4) has previously been found to facilitate rotavirus entry into target cells by direct penetration of virions through the plasma membrane. We conclude that sufficient quantities of exogenous viral proteins may be introduced into the cytoplasm for processing by target cells. The mechanism by which rotavirus proteins are processed for presentation to the target cell surface remains to be determined.

Animals↗

PubMed1989

Rotavirus-specific cytotoxic T lymphocytes appear at the intestinal mucosal surface after rotavirus infection.

The gastrointestinal tract is constantly exposed to a variety of potentially invasive bacteria and viruses. The first line of defense of the host against these pathogens is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IELs), mucus, and secretory immunoglobulins. Little is known about the function, memory, or trafficking of IELs after intestinal infection. We found that IELs obtained 6 days after oral inoculation of mice with the intestinal pathogen rotavirus (simian strain RRV) lysed rotavirus-infected target cells; cytotoxic T lymphocytes (CTLs) were responsible for rotavirus-specific cytotoxic activity. Rotavirus-specific cytotoxic activity by IELs was (i) eliminated by treatment with Thy 1.2-specific immunoglobulin M plus complement, (ii) restricted by proteins encoded at the major histocompatibility complex, and (iii) absent in mock-infected animals. Oral inoculation of mice with RRV also induced rotavirus-specific CTLs in splenic and intestinal lymphocytes (mesenteric lymph nodes, Peyer's patch). Parenteral inoculation induced rotavirus-specific CTLs in splenic, intestinal (IELs, mesenteric lymph nodes, Peyer's patch), and nonintestinal lymphocytes (inguinal nodes). Therefore, presentation of rotavirus to the intestinal mucosal surface was not necessary to induce IELs with virus-specific cytotoxic activity. At 4 weeks after oral or parenteral inoculation of mice with RRV, rotavirus-specific CTL precursors appeared among splenic, Peyer's patch, inguinal, and mesenteric node lymphocytes, but not among IELs. IELs with rotavirus-specific cytotoxic activity may be generated from precursors at a site other than the intestinal mucosal surface. Part of the response of the host to enteric infection may include surveillance and lysis of virus-infected villus epithelial cells by IELs.

Animals↗

PubMed1988

Identification of the simian rotavirus SA11 genome segment 3 product.

Previous studies on rotavirus gene-coding assignments failed to clearly identify the simian rotavirus SA11 genome segment 3 protein product. This question was reexamined by using new conditions of electrophoresis with improved resolution of proteins in the high-molecular-weight range. Our results showed that the SA11 genome segment 3 codes for a protein with an apparent mol wt of 88,000. This protein normally comigrates with the protein product of genome segment 4. The gene 3 protein was located in viral core particles by comparing the electrophoretic patterns of [35S]methionine-labeled viral polypeptides from infected cells and from purified double-shelled, single-shelled, and core particles. To confirm the identity of the gene 3 product, we also studied two reassortant viruses in which genome segment 3 was reassorted from each of two parental virus strains (SA11 and NCDV). The gene 3 and gene 4 products of these viruses were identified by (i) their separation by two different polyacrylamide gel systems, (ii) their location in distinct particle types, (iii) their differential sensitivity to trypsin digestion, and (iv) their distinctive protease peptide maps. We propose that the genome segment 3 product be called VP3 and that the gene 4 product be named VP4 from now on.

Animals↗

PubMed1988

Rotavirus-specific cytotoxic T lymphocytes cross-react with target cells infected with different rotavirus serotypes.

Splenocytes from adult C57BL6 (H-2b) mice orally inoculated with nonmurine rotaviruses lysed syngeneic rotavirus-infected target cells. Cytotoxic T lymphocytes (CTLs) were responsible for this cytotoxic activity. Cytotoxic activity was (i) detected 7 days after primary oral inoculation; (ii) not detected in uninoculated animals; (iii) specific for rotavirus-infected target cells; (iv) eliminated by treatment with Thy 1.2-specific immunoglobulin M and complement; and (v) restricted at H-2Db. In addition, rotavirus-specific CTLs cross-reacted with target cells infected with different human or animal rotavirus serotypes. Heterotypic protection against rotavirus challenge may be mediated by cross-reactive CTLs.

Animals↗

PubMed1988

Rotavirus serotype-specific neutralizing activity in human milk.

A plaque reduction neutralization assay was used to determine rotavirus serotype-specific neutralizing activity in human breast milk from 25 mothers of upper socioeconomic background and 20 mothers of a lower socioeconomic status. Levels of neutralizing activity, as well as those of rotavirus-specific antibodies detected by enzyme-linked immunosorbent assay (ELISA), were comparable for each socioeconomic group. Overall, neutralizing activity in human milk was detected in the majority of samples and can be increased many months postpartum. The prevalence of neutralization antibodies (titers, greater than or equal to 1:10) was 77% against Wa (serotype 1), 86% against SA-11 (serotype 3), and 75% against NCDV (bovine) rotavirus. Rotavirus-specific IgA and IgG antibodies detected by ELISA (titers, greater than or equal to 1:10) were present in 35% and 55% of breast milks, respectively. Sequential analysis of repeated breast milk samples from five individual mothers revealed that rotavirus neutralizing activity fluctuated over time, with high activity observed in one mother's milk at 18 months postpartum. Mothers who breast-fed for six months or more tended to have higher milk neutralizing titers against rotavirus.

Adolescent↗

PubMed1987

Rotavirus isolate WI61 representing a presumptive new human serotype.

A virus (strain WI61) representing a presumptive new human serotype was isolated from an 18-month-old child with gastroenteritis admitted to Children's Hospital of Philadelphia in February 1983. The WI61 virus was clearly distinguished by cross-neutralization tests from human rotaviruses of serotypes 1, 2, 3, and 4, human 69M, and representative bovine (NCDV), porcine (OSU), and chicken (Ch2) rotaviruses. Antisera generated in guinea pigs hyperimmunized to WI61 virus displayed a partial cross-reactivity with rotaviruses of human serotypes 1, 2, 3, and 4. By means of studies with reassortant rotaviruses, it was presumptively determined that the WI61 virus cross-reactive antigenic determinants are localized on the vp3 surface polypeptide coded by gene segment 4. The characteristic RNA genome electropherotype of WI61 virus was observed in 5 of 59 cases of infant gastroenteritis detected in 1983 and 1984 but has not been observed in a subsequently at Children's Hospital. Serotype WI61-specific neutralizing antibodies were observed in a majority of sera of normal adults and infants of less than 4 or greater than 12 months of age collected in the Philadelphia area. Median antibody titers to WI61 equaled or exceeded those to rotaviruses of serotype 1 or 3. Each of seven samples of commercial cow's milk exhibited neutralizing antibodies to WI61 virus at a titer greater than or equal to that to serotype 1 or 3 or bovine (strain NCDV) rotavirus. However, WI61 rotavirus did not induce disease or a specific serum-neutralizing antibody response when fed to a caesarean-derived colostrum-deprived newborn calf. WI61 rotavirus caused diarrhea in newborn mice with a 50% diarrhea-inducing dose of 10(7.0) PFU.

Animals↗

PubMed1987

Gastroenteritis caused by human rotaviruses (serotype three) in a suckling mouse model.

The pathogenic potential of human rotaviruses of serotypes 1 through 4 was evaluated in suckling mice. Oral inoculation of three different human rotaviruses of serotype 3 into 5-6 day old CD-1 mice caused disease characterized by diarrhea and dehydration. The mean 50% diarrhea inducing dose (DD50) was 5 X 10(5) pfu. Histopathological examination of small intestines revealed villus epithelial cell vacuolization localized to the distal one-third of the villus. Only Serotype 3 rotaviruses exhibited a rapid phase of viral growth in the intestine between 7 and 12 hours post-inoculation. Larger inocula of rotavirus serotypes 1, 2, and 4 did not cause disease or typical histopathologic changes. However, immunoperoxidase staining for rotavirus antigen was positive in all serotypes tested indicating that infection can occur without apparent disease and is not serotype specific. This convenient in-vivo model can be used to evaluate attenuation of human origin vaccine candidates of serotype 3.

Animals↗

PubMed1986

Immune response of infants and children to low-passage bovine rotavirus (strain WC3).

A bovine rotavirus (strain WC3) was isolated from a calf in Pennsylvania and adapted to growth in continuous Cercopithecus cell line CV1. A pool for human vaccine trials was produced at the 12th cell culture passage level. After preliminary testing in adults and older children, a dose of 3 X 10(7) plaque-forming units was given by mouth to 52 infants and children aged 5 months to 6 years. No clinical sequelae were detected, and shedding in feces was detected in only 30% of tested infants. A serum-neutralizing antibody response was induced in 95% of 21 infants aged 5 to 11 months; response rates were slightly reduced in older infants. The antibody response was primarily directed toward bovine rotavirus, but a response to human serotype 3 rotavirus was also observed in approximately 50% of vaccinees. After vaccination with WC3, infants with preexisting antibody to rotaviruses of human serotype 1 or 3 frequently exhibited a booster response to those serotypes. WC3 is a candidate rotaviral vaccine deserving larger trials in children.

Adult↗

PubMed1986

Antigenic mapping of the surface proteins of rhesus rotavirus.

Monoclonal antibodies have been produced and used to map the functional topography of the surface proteins of rhesus rotavirus (RRV) that mediate viral neutralization. Ten monoclonal antibodies directed to VP7 were studied in neutralization assays and competitive binding studies. A large neutralization domain with several interrelated epitopes on VP7 was apparent. Twelve monoclonal antibodies directed to VP3 were used in similar studies and delineated at least 2 distinct neutralization domains on that protein. Neutralizing monoclonal antibodies directed at both VP3 and VP7 were used to isolate viral antigenic variants, which were than studied in neutralization and hemagglutination inhibition assays. The viral variant studies, while confirming the general conclusions obtained from the competitive binding studies, allowed the apparent distinction of two separate neutralization domains on VP7 and three on VP3. All VP7-specific monoclonal antibodies (mAb) mediated serotype-specific neutralization, but a VP3-specific mAb was identified that neutralized rotaviruses of three distinct serotypes. No alteration of viral virulence was apparent in studies of suckling mice orally inoculated with antigenic variant viruses selected with our panel of neutralizing VP3 or VP7-specific mAbs.

Antibodies, Monoclonal↗

PubMed1986

Response of adult human volunteers to oral administration of bovine and bovine/human reassortant rotaviruses.

Small groups of adult volunteers, in sequence, were inoculated orally with inactivated purified bovine rotavirus of strain NCDV, with live NCDV purified or unpurified and with two different NCDV X human rotavirus reassortant viruses. One of five volunteers given 200 micrograms of ultravioletinactivated NCDV developed a virus-neutralizing (VN) and a binding antibody response detected by enzyme-linked immunosorbent assay (ELISA). Four of 10 volunteers given from 1 X 10(6) to 1 X 10(8) p.f.u. of live NCDV developed VN antibody, but nine of 10 responded when ELISA, HAI and radioimmuno-precipitation tests for serum antibody were also considered. Two different NCDV X human serotype 1 Wa strain virus reassortants, each containing Wa gene segment 9 and the serotype 1 neutralization phenotype, were administered orally in doses up to 10(6) p.f.u. The reassortants were relatively ineffective in eliciting a serum antibody response at the dosage level employed.

Administration, Oral↗