Biomedical subjects

M E Conner

Publications and source records attributed to M E Conner.

35 records · Page 2Linked to original sources

PubMed1997

Comparative amino acid sequence analysis of the outer capsid protein VP4 from four lapine rotavirus strains reveals identity with genotype P[14] human rotaviruses.

The genes encoding the outer capsid VP4 proteins of four lapine rotavirus strains, three isolated in the US (ALA, C-11 and BAP-2) and one isolated in Japan (R-2) were sequenced, and the predicted amino acid (aa) sequence was compared to all known rotavirus genotypes. A high degree of aa identity (96.8-98.9%) was found among the American lapine strains, while the Japanese rotavirus strain R-2 shared less aa identity (89.5-90.0%) with the American strains. The four lapine rotaviruses shared the closest aa identity (90.6-94.9%) with the P[14] genotype, consisting of viruses isolated from humans in Italy, Finland and Thailand. These results indicate that the VP4 protein of the four lapine strains are genotype P14, and that among lapine strains there are possibly two subtypes, one represented by the American lapine strains and the other by the Japanese R-2 strain.

Amino Acid Sequence↗

PubMed1997

Determination of the duration of a primary immune response and the ID50 of ALA rabbit rotavirus in rabbits.

The rabbit model of rotavirus infection has been used to examine the immune response to rotavirus infection and to evaluate strategies for rotavirus vaccine development. To determine the 50% infectious does (ID50) of tissue culture adapted ALA virus, rabbit were orally inoculated with 10(1)-10(3) PFU of ALA rotavirus. The ID50 of ALA virus was determined to be 1.7 x 10(2) PFU. The immune response induced in rabbits infected at low virus doses (10(2)-10(3) PFU) was of similar magnitude to the immune responses induced with a high dose (10(6) PFU) inoculum, indicating that the immune response to ALA rotavirus in rabbits is not dose dependent. To determine if a single rotavirus inoculation would induce a long lasting immune response, four rabbits were inoculated once with ALA virus (3.5 x 10(5) PFU) and their serologic and mucosal antirotavirus titers were monitored at intervals for 1.5-2 years. The infected rabbits maintained serologic and mucosal rotavirus antibody titers until the final time point more than 700 days post inoculation. These data are important because they indicate that the antigenic load achieved following a single oral inoculation is sufficient to achieve long lasting immunity, the goal of any potential vaccine.

Animals↗

PubMed1997

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.

Adjuvants, Immunologic↗

PubMed1996

Rotavirus subunit vaccines.

We evaluated rotavirus subunit vaccines for use in humans and animals. Insect cells were co-infected with combinations of individual baculovirus recombinants expressing human, bovine or simian rotavirus VP2, VP4, VP6 or VP7 to produce virus-like particles (VLPs). To determine whether immunization with VLPs could induce active protective immunity, VLPs were administered parenterally to rabbits, and the immune response and protection from rabbit ALA rotavirus challenge were evaluated. Complete or partial protection was attained, showing that parenteral immunization with VLPs induces active protective immunity. We also examined whether heterotypic immune responses could be induced with a limited number of broadly reactive VP7 proteins or with chimeric particles (multiple VP7 types on individual particles). The feasibility of this approach was determined by immunizing mice with VLPs containing a G3 VP7 or G1 VP7 and chimeric G1/G3 VLPs. Broadly reactive neutralizing antibody was induced by the G1 VLPs. VLPs also have been successfully used to boost lactogenic (colostral and milk) immunity in dairy cows. Taken together, these results show that VLPs can be effective immunogens in rabbits, mice and dairy cattle when administered parenterally, a limited number of VLPs may be sufficient to produce a broadly protective vaccine, and G3 VLPs may serve as an effective subunit vaccine for use in bovines.

Animals↗

PubMed1996

Recombinant Norwalk virus-like particles as an oral vaccine.

Viruses that infect cells in the gastrointestinal tract are well suited for examining the immune response to oral delivery of antigen and for exploring the advantages and pitfalls of oral vaccines. Norwalk virus (NV) (family Caliciviridae, genus Calicivirus) causes acute gastroenteritis in all age groups. The NV capsid is composed of 180 copies of a single 58000 molecular weight protein which spontaneously forms virus-like particles (VLPs) that can be purified in extremely high yields (22 mg per 300 ml culture) when produced using the baculovirus expression system. We are testing the potential of these recombinant NV (rNV) particles for use as an oral vaccine by administering them to mice and volunteers. Mice were orally inoculated four times with rNV particles in concentrations ranging from 5 to 500 micrograms in the absence of adjuvant or from 5 to 200 micrograms with 10 micrograms of cholera toxin. Serum IgG and fecal IgA immune responses were monitored. rNV particles were found to be immunogenic when orally given to mice with or without adjuvant. These particles also were safe and immunogenic when orally given to volunteers. These studies show that rNV particles are an excellent model to test the oral delivery of mucosal immunogens in general, and that rNV particles are ideal candidates for vaccine development in particular.

Administration, Oral↗

PubMed1996

Isotype-specific antibody responses to rotavirus and virus proteins in cows inoculated with subunit vaccines composed of recombinant SA11 rotavirus core-like particles (CLP) or virus-like particles (VLP).

The isotype antibody responses to bovine IND P5, G6 and simian SA11 P2, G3 rotavirus and SA11 rotavirus proteins (VP4, VP6 and VP7) in serum, colostrum and milk were analysed by ELISA in three groups of vaccinated cows and nonvaccinated controls. Pregnant cows were vaccinated intramuscularly and intramammarily with recombinant baculovirus-expressed SA11 rotavirus VLP (triple-layered virus-like particles containing rotavirus VP2, VP4, VP6 and VP7); CLP (double-layered core-like particles containing rotavirus VP2 and VP6); or inactivated SA11 rotavirus, respectively. Rotavirus antigen titers were highest (30-200-fold) in ELISA in the VLP vaccine compared to the inactivated SA11 vaccine. The IgG1, IgG2 and IgM geometric mean antibody titers (GMT) to rotavirus (titers to bovine rotavirus vs SA11 rotavirus did not differ significantly for any isotype or group) and the IgG2 GMT to VP6 in serum at calving in the vaccinated groups were significantly (P < 0.05) higher than in the control group. In colostrum, IgG1 and IgA rotavirus antibody titers were significantly elevated for VLP (IgG1 GMT 832225; IgA GMT 16384), CLP (IgG1 GMT 660561; IgA GMT 10321) and SA11 (IgG1 GMT 131072; IgA GMT 1448) vaccinated cows compared to control cows (IgG1 GMT 11585; IgA GMT 45). The IgG1 and IgA GMT to rotavirus were significantly elevated (6-100-fold) in milk of VLP and CLP vaccinated cows compared to SA11 vaccinated or control cows. The isotype antibody responses to VP6 in serum, colostrum and milk paralleled the responses to rotavirus, but titers were approximately 2-10-fold lower. Only cows vaccinated with VLP had significantly enhanced serum, colostral and milk antibody titers to rotavirus VP4 and VP7. These results demonstrate that rotavirus antibody titers in serum, colostrum and milk are significantly enhanced by use of non-infectious VLP, CLP and inactivated SA11 rotavirus vaccines, but the VLP or CLP vaccines induced the highest antibody responses, corresponding to their higher rotavirus antigen titers measured by ELISA.

Animals↗

PubMed1996

Virus-like particles as a rotavirus subunit vaccine.

Rotavirus subunit vaccines are being evaluated for use in humans. The virus-like particles (VLPs) for these vaccines are produced in insect cells coinfected with combinations of baculovirus recombinants expressing bovine RIF VP2 and simian SA11, VP4, VP6, or VP7 rotavirus proteins. VLPs were administered parenterally to mice and rabbits, and the immunogenicity and protective efficacy of the vaccines were evaluated. Rabbits vaccinated with VP2/4/6/7 or VP2/6/7 VLP combinations developed high levels of rotavirus-specific serum antibody and fecal IgG but not fecal IgA. The induction of fecal IgG was associated with total or partial protection from oral challenge with ALA rotavirus. Heterotypic serum and fecal neutralizing antibody was induced in mice vaccinated parenterally with G1 VP2/6/7 or VP2/4/6n VLPs. VLPs were highly immunogenic when administered in QS21 adjuvant, inducing serum neutralizing antibody titers comparable to those induced by SA11 virus. VLPs are effective immunogens when administered parenterally and may be an effective subunit vaccine.

Animals↗

PubMed1994

Rotavirus vaccines and vaccination potential.

The development of a successful rotavirus vaccine is a complex problem. Our review of rotavirus vaccine development shows that many challenges remain, and priorities for future studies need to be established. For example, the evaluation of administration of a vaccine with OPV or breast milk might receive less emphasis until a vaccine is made that shows clear efficacy against all virus serotypes. Samples remaining from previous trials should be analyzed to determine epitope-specific serum and coproantibody responses to clarify why only some trials were successful. Detailed evaluation of the antigenic properties of the viruses circulating and causing illness in vaccinated children also should be performed for comparisons with the vaccine strains. In future trials, sample collection should include monitoring for asymptomatic infections and cellular immune responses should be analyzed. The diversity of rotavirus serotype distribution must be monitored before, during, and after a trial in the study population and placebo recipients must be matched carefully to vaccine recipients. Epidemiologic and molecular studies should be expanded to document, or disprove, the possibility of animal to human rotavirus transmission, because, if this occurs, vaccine protection may be more difficult in those areas of the world where cohabitation with animals occurs. We also need to have an accurate assessment of the rate of protection that follows natural infections. Is it realistic to try to achieve 90% protective efficacy with a vaccine if natural infections with these enteric pathogens only provide 60% or 70% protection? Subunit vaccines should be considered to be part of vaccine strategies, especially if maternal antibody interferes with the take of live vaccines. The constraints on development of new vaccines are not likely to come from molecular biology. The challenge remains whether the biology and immunology of rotavirus infections can be understood and exploited to permit effective vaccination. Recent advances in developing small animal models for evaluation of vaccine efficacy should facilitate future vaccine development and understanding of the protective immune response(s) (Ward et al. 1990b; Conner et al. 1993).

Animals↗

PubMed1993

Rotavirus vaccine administered parenterally induces protective immunity.

We performed experiments to determine whether parenteral immunization with SA11 rotavirus can induce active protective immunity in a rabbit model of rotavirus infection. After one or two intramuscular injections of 1 ml of live or formalin-inactivated SA11 virus, we evaluated the mucosal and serologic immune response and protection from challenge with a high dose of live, virulent rabbit (Ala) rotavirus. Inactivated SA11 virus preparations, evaluated by enzyme-linked immunosorbent assay (ELISA) with a panel of VP4- and VP7-specific neutralizing and nonneutralizing monoclonal antibodies, did not show a loss of epitopes from the inactivation procedure compared with live virus. Administration of two doses of vaccine, one at zero days postvaccination (DPV) and a booster shot at 49 DPV, followed by challenge at 71 DPV with 3.5 x 10(5) PFU of Ala virus resulted in protection from challenge. None of the two-dose virus-vaccinated rabbits shed virus after challenge, while virus shedding was detected in all control rabbits (P = 0.001, Fisher's exact two-tailed test). Differences in total serum immunoglobulin (Ig) antirotavirus ELISA titers (P < 0.05, Wilcoxon's rank sum test) were observed between groups vaccinated with virus in aluminum phosphate or Freund's adjuvant but not between groups vaccinated with live or inactivated virus in either adjuvant. All rabbits given two doses of vaccine had detectable antirotavirus intestinal antibody of the IgG, but not IgA, isotype. After challenge, fourfold or greater increases in intestinal IgG antibody responses were observed in three rabbits, whereas all controls and all but one virus-vaccinated rabbit had an intestinal IgA antibody response. In contrast, vaccination of rabbits with one dose of SA11 followed by challenge at 21 DPV did not protect from challenge; no difference in the mean number of days of virus shedding between any of the vaccinated groups and controls was observed. A serologic, but not a mucosal, antibody response was observed after the one-dose vaccination regimen. Differences in serologic antibody titers were not observed between any of the one-dose virus-vaccinated groups. These data indicate that parenteral vaccination with two, but not one, doses of rotavirus in either Freund's adjuvant or aluminum phosphate can induce active protection from challenge.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Extraintestinal rotavirus infections in children with immunodeficiency.

Some rotavirus strains, including vaccine candidates, have been demonstrated to cause hepatitis in immunodeficient and malnourished mice and to grow in human liver cells. To determine whether rotavirus spreads outside the intestine in naturally infected children, we examined tissues from four immunodeficient children affected with severe combined immunodeficiency disease, acquired immunodeficiency disease syndrome, or DiGeorge syndrome. Chronic rotavirus-related diarrhea, which persisted until death, had also developed in each child. Using indirect immunoperoxidase techniques, we identified rotavirus antigen in the liver and kidney with a hyperimmune guinea pig antiserum prepared to double-shelled rotavirus particles. Similar immunostaining with an antiserum to a rotavirus nonstructural protein (NS26) provided evidence of active virus replication. The observed reactivity was eliminated specifically when serial sections were immunostained with the same antiserum that had been absorbed with either double-shelled rotavirus particles or NS26. Immunostaining was not observed in the liver of children with other diseases (alpha 1-antitrypsin deficiency, inspissated bile syndrome, and acute rejection of a transplanted liver). These findings demonstrate that rotavirus infections in children can extend beyond the intestinal tract. Further studies are warranted to determine whether extraintestinal rotavirus replication occurs in children without severe immunodeficiency, such as malnourished children.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Analysis of serotypes and electropherotypes of equine rotaviruses isolated in the United States.

Equine group A rotaviruses isolated over a 10-year period in New York State, New Jersey, Kentucky, and Texas were compared serotypically and electropherotypically. All isolates were determined to be serotype 3 by reaction with hyperimmune antiserum to the serotype 3 H-2 strain of equine rotavirus. All displayed RNA electrophoretic migration patterns related to that of the H-2 strain but distinct from that of serotype 5 strain H-1. A serologic survey of 184 mares in Kentucky, which was done to determine the incidence of H-1 and H-2 infections, showed geometric mean serum neutralizing titers to the H-2 strain of equine rotavirus to be significantly higher than those to the H-1 strain. These data suggest that the serotype 3 H-2 strain is the dominant equine rotavirus in Kentucky and perhaps elsewhere in the United States. We were unable to produce confirmational evidence that the H-1 strain occurs as a natural infection in the United States.

Animals↗

PubMed1991

Serologic and mucosal immune response to rotavirus infection in the rabbit model.

We examined the humoral immune response to rotavirus infection in specific pathogen-free rabbits inoculated and challenged orally with rabbit Ala rotavirus (7.5 x 10(5) to 1 x 10(7) PFU). The humoral immune response in both serologic and mucosal samples was monitored by using total antibody enzyme-linked immunosorbent assays (ELISAs), isotype-specific ELISAs, and plaque reduction neutralization assays. Following a primary infection, all rabbits shed virus and serologic and mucosal antibody responses were initially detected by 1 week postinoculation. Intestinal immunoglobulin M was detected by 3 days postinoculation, and secretory immunoglobulin A was detected by 6 days postinoculation. Following challenge, rabbits were protected (no detectable virus shedding) from infection. An anamnestic immune response was observed only with mucosal neutralizing antibodies, and all serologic and mucosal immune responses persisted at high levels until at least 175 days postchallenge (204 days postinoculation). Detection of neutralization responses was influenced by the virus strain used in the neutralization assay; all inoculated rabbits developed detectable serum and intestinal neutralizing antibodies against the infecting (Ala) virus strain. Neutralization activity in both serum and mucosal samples was generally, but not exclusively, homotypic (VP7 serotype 3) after both primary and challenge inoculations with Ala virus. Heterotypic serum neutralization activity was observed with serotype 8 (9 of 12 rabbits) and 9 (12 of 12 rabbits) viruses and may be based on reactivity with the outer capsid protein VP4 or on a shared epitope in the C region of VP7. Comparisons of heterologous (serotype 3) and heterotypic neutralizing responses in mucosal and serologic samples revealed that 43% (21 of 49) of the responses were discordant. In 19 of 49 (39%) of these cases, a heterotypic serologic response was seen in the absence of a heterotypic mucosal response, but in 2 of 49 (4%) instances, a heterotypic mucosal response was seen in the absence of a concomitant serologic response. These results provide insight into factors which may affect detection of heterotypic responses.

Animals↗

PubMed1988

Molecular characterization of three rabbit rotavirus strains.

We report biochemical (RNA and protein patterns and gene-coding assignments) and serologic (serotype and subgroup) properties of three strains of rabbit rotaviruses--Ala C11 and R2. The RNA electropherotypes were a standard "short" pattern for R2, an unusual "short" pattern for Ala, and an unusual "long" pattern for C11. Serologic studies indicated that these viruses were all group A serotype 3 rotaviruses. In addition, the Ala and C11 viruses were found to possess subgroup I specificity, whereas the R2 virus possessed subgroup II specificity. In contrast to their distinctive RNA patterns, the polypeptide patterns of the rabbit viruses were similar to those of SA11. To identify cognate genes and determine gene-coding assignments for the rabbit rotaviruses, cDNA probes of individual SA11 genes were hybridized to rabbit rotaviral genomic RNA segments that had been separated by polyacrylamide gel electrophoresis and transferred to filters (Northern blots). The order of genome segments 7-11 for each of the rabbit rotaviruses was unique and differed from that of SA11 genes. These differences were possibly due to rearrangements of the RNA sequences within these individual genome segments. Sequence analysis of the individual RNA segments will confirm whether genome rearrangements are the molecular basis for these novel migration patterns.

Animals↗

PubMed1988

Rabbit model of rotavirus infection.

A new small animal model was developed to study parameters of rotavirus infections, including the active immune response. Seronegative New Zealand White rabbits (neonatal to 4 months old) were inoculated orally with cultivatable rabbit rotavirus strains Ala, C11, and R2 and with the heterologous simian strain SA11. The course of infection was evaluated by clinical findings, virus isolation (plaque assay and enzyme-linked immunosorbent assay), and serologic response. All four strains of virus were capable of infecting rabbits as determined by isolation of infectious virus from intestinal contents or fecal samples, by seroconversion, or by a combination of these methods. The responses differed depending on the virus strain used for inoculation. Rabbits remained susceptible to primary infection to at least 16 weeks of age (upper limit examined). Virus excretion in intestinal contents was detected from 6 h to 7 days postinoculation. RNA electropherotypes of inocula and viruses isolated from rabbits were the same in all samples tested. Transmission of Ala virus and R2 virus but not SA11 virus from inoculated animals to uninoculated controls also occurred. In a challenge experiment with Ala virus, 74- and 90-day-old rabbits were rechallenged with Ala 5 weeks after a primary infection with Ala. Virus was excreted in feces from 2 to 8 days after the primary infection but was not excreted after challenge. These results indicate that the rabbit provides an ideal model to investigate both the primary and secondary active immune responses to rotavirus infections and to evaluate candidate vaccines.

Age Factors↗

PubMed1983

Detection of rotavirus in horses with and without diarrhea by electron microscopy and Rotazyme test.

A total of 142 equine fecal samples (93 field fecal and 49 experimental fecal specimens) were examined for rotavirus using direct electron microscopy (EM) and the Rotazyme test. Eighty-six stool specimens were diarrhea samples. The Rotazyme test sensitivity and accuracy as compared to EM was determined by the visual (color reaction) and spectrophotometric methods. The overall agreement was 94.8% and 92.3% between EM and Rotazyme visual and spectrophotometric methods, respectively when suspect reactions (1 + color reaction or net absorbance between 0.05 and 0.1) were not included. The Rotazyme test is a quick, simple, and accurate diagnostic test for detection of rotavirus in equine fecal samples. It could be used by the equine practitioner with a minimum of laboratory facilities and by diagnostic and research laboratories.

Animals↗

PubMed1980

Rotavirus infection in foals.

Fecal samples from 86 foals with diarrhea were examined by electron microscopy during a 2.5 year period. Of these, 26 (30%) were positive for rotavirus. All of the cases were found in epizootic areas. The disease was produced in an experimental foal by inoculation via stomach tube of a bacteria-free fecal filtrate containing rotavirus. Examination of postmortem tissues from the duodenum and jejunum of 2 naturally infected foals and an experimentally infected foal revealed replicating virus in the intestinal epithelial cells. A limited survey of complement-fixing antibody to rotavirus in horses from Kentucky, Virginia, and France indicated that all horses had antibody to the virus. The sole exception was 1 foal from which blood samples were collected prior to suckling. These results were presumptive evidence that rotavirus is a major cause of diarrhea in foals, and the presence of antibody in horses from diverse areas is evidence for the ubiquitousness of this infection.

Animals↗

PubMedDate not supplied

Molecular biology and immunology of rotavirus infections.

Rotaviruses were first recognized about 15 years ago in association with diarrhea in children and animals. Since then, rotaviruses have been determined to be the most important viral agent that causes clinically significant diarrhea in children and a need for an effective vaccination program has been recognized. This article reviews the progress which has been made in understanding the molecular biology of rotaviruses and summarizes information on the immune responses to rotavirus infections obtained in a new animal model in rabbits. This model is useful to systematically evaluate active protective immunity following infection of seronegative animals.

Animals↗