Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus

Respiratory symptoms with rotavirus shedding in nasopharyngeal secretions have been reported in children with and without gastrointestinal symptoms (Zheng et al., 1991, J. Med. Virol. 34:29-37). To investigate if attenuated and virulent human rotavirus (HRV) strains cause upper respiratory tract infections or viremia in gnotobiotic pigs, we inoculated them with attenuated or virulent HRV intranasally, intravenously, or orally or via feeding tube (gavage) and assayed virus shedding. After oral or intranasal inoculation with attenuated HRV, the pigs remained asymptomatic, but 79 to 95% shed virus nasally and 5 to 17% shed virus rectally. After inoculation by gavage, no pigs shed virus nasally or rectally, but all pigs seroconverted with antibodies to HRV. No viremia was detected through postinoculation day 10. Controls inoculated intranasally with nonreplicating rotavirus-like particles or mock inoculated did not shed virus. In contrast, 100% of pigs inoculated with virulent HRV (oral, intranasal, or gavage) developed diarrhea, shed virus nasally and rectally, and had viremia. The infectivity of sera from the viremic virulent HRV-inoculated pigs was confirmed by inoculating gnotobiotic pigs orally with pooled HRV-positive serum. Serum-inoculated pigs developed diarrhea and fecal and nasal virus shedding and seroconverted with serum and intestinal HRV antibodies. Pigs inoculated intravenously with serum or intestinal contents from the viremic virulent HRV-inoculated pigs developed diarrhea, virus shedding, and viremia, similar to the orally inoculated pigs. This study provides new evidence that virulent HRV causes transient viremia and upper respiratory tract infection in addition to gastrointestinal infection in gnotobiotic pigs, confirming previous reports of rotavirus antigenemia (Blutt et al., Lancet 362:1445-1449, 2003). Our data also suggest that intestinal infection might be initiated from the basolateral side of the epithelial cells via viremia. Additionally, virus shedding patterns indicate a different pathogenesis for attenuated versus virulent HRV.     

IgY Antibodies Protect against Human Rotavirus Induced Diarrhea in the Neonatal Gnotobiotic Piglet Disease Model

Group A Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The aim of the present work was to evaluate protection against rotavirus (RV) diarrhea conferred by the prophylactic administration of specific IgY antibodies (Ab) to gnotobiotic piglets experimentally inoculated with virulent Wa G1P[8] human rotavirus (HRV). Chicken egg yolk IgY Ab generated from Wa HRV hyperimmunized hens specifically recognized (ELISA) and neutralized Wa HRV in vitro. Supplementation of the RV Ab free cow milk diet with Wa HRV-specific egg yolk IgY Ab at a final ELISA Ab titer of 4096 (virus neutralization –VN- titer = 256) for 9 days conferred full protection against Wa HRV associated diarrhea and significantly reduced virus shedding. This protection was dose-dependent. The oral administration of semi-purified passive IgY Abs from chickens did not affect the isotype profile of the pig Ab secreting cell (ASC) responses to Wa HRV infection, but it was associated with significantly fewer numbers of HRV–specific IgA ASC in the duodenum. We further analyzed the pigś immune responses to the passive IgY treatment. The oral administration of IgY Abs induced IgG Ab responses to chicken IgY in serum and local IgA and IgG Ab responses to IgY in the intestinal contents of neonatal piglets in a dose dependent manner. To our knowledge, this is the first study to show that IgY Abs administered orally as a milk supplement passively protect neonatal pigs against an enteric viral pathogen (HRV). Piglets are an animal model with a gastrointestinal physiology and an immune system that closely mimic human infants. This strategy can be scaled-up to inexpensively produce large amounts of polyclonal IgY Abs from egg yolks to be applied as a preventive and therapeutic passive Ab treatment to control RV diarrhea.     

Protective immunity and antibody-secreting cell responses elicited by combined oral attenuated Wa human rotavirus and intranasal Wa 2/6-VLPs with mutant Escherichia coli heat-labile toxin in gnotobiotic pigs

Two combined rotavirus vaccination regimens were evaluated in a gnotobiotic pig model of rotavirus infection and disease and were compared to previously tested rotavirus vaccination regimens. The first (AttHRV/VLP2x) involved oral inoculation with one dose of attenuated (Att) Wa human rotavirus (HRV), followed by two intranasal (i.n.) doses of a rotavirus-like particle (2/6-VLPs) vaccine derived from Wa (VP6) and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were coadministered with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT) adjuvant. For the second regimen (VLP2x/AttHRV), two i.n. doses of 2/6-VLPs+mLT were given, followed by one oral dose of attenuated Wa HRV. To compare the protective efficacy and immune responses induced by the combined vaccine regimens with individual rotavirus vaccine regimens, we included in the experiments the following vaccine groups: one oral dose of attenuated Wa HRV (AttHRV1x and Mock2x/AttHRV, respectively), three oral doses of attenuated Wa HRV (AttHRV3x), three i.n. doses of 2/6-VLPs plus mLT (VLP3x), three i.n. doses of purified double-layered inactivated Wa HRV plus mLT (InactHRV3x), mLT alone, and mock-inoculated pigs. The isotype, magnitude, and tissue distribution of antibody-secreting cells (ASCs) in the intestinal and systemic lymphoid tissues were evaluated using an enzyme-linked immunospot assay. The AttHRV/VLP2x regimen stimulated the highest mean numbers of intestinal immunoglobulin A (IgA) ASCs prechallenge among all vaccine groups. This regimen induced partial protection against virus shedding (58%) and diarrhea (44%) upon challenge of pigs with virulent Wa HRV. The reverse VLP2x/AttHRV regimen was less efficacious than the AttHRV/VLP2x regimen in inducing IgA ASC responses and protection against diarrhea (25% protection rate) but was more efficacious than VLP3x or InactHRV3x (no protection). In conclusion, the AttHRV/VLP2x vaccination regimen stimulated the strongest B-cell responses in the intestinal mucosal immune system at challenge and conferred a moderately high protection rate against rotavirus disease, indicating that priming of the mucosal inductive site at the portal of natural infection with a replicating vaccine, followed by boosting with a nonreplicating vaccine at a second mucosal inductive site, may be a highly effective approach to stimulate the mucosal immune system and induce protective immunity against various mucosal pathogens.     

Efficacy,safety and immunogenicity of hexavalent rotavirus vaccine in Chinese infants

A randomized, double-blind, placebo-controlled multicenter trial was conducted in healthy Chinese infants to assess the efficacy and safety of a hexavalent live human-bovine reassortant rotavirus vaccine (HRV) against rotavirus gastroenteritis (RVGE). A total of 6400 participants aged 6–12 weeks were enrolled and randomly assigned to either HRV (n = 3200) or placebo (n = 3200) group. All the subjects received three oral doses of vaccine four weeks apart. The vaccine efficacy (VE) against RVGE caused by rotavirus serotypes contained in HRV was evaluated from 14 days after three doses of administration up until the end of the second rotavirus season. VE against severe RVGE, VE against RVGE hospitalization caused by serotypes contained in HRV, and VE against RVGE, severe RVGE, and RVGE hospitalization caused by natural infection of any serotype of rotavirus were also investigated. All adverse events (AEs) were collected for 30 days after each dose. Serious AEs (SAEs) and intussusception cases were collected during the entire study. Our data showed that VE against RVGE caused by serotypes contained in HRV was 69.21% (95%CI: 53.31–79.69). VE against severe RVGE and RVGE hospitalization caused by serotypes contained in HRV were 91.36% (95%CI: 78.45–96.53) and 89.21% (95%CI: 64.51–96.72) respectively. VE against RVGE, severe RVGE, and RVGE hospitalization caused by natural infection of any serotype of rotavirus were 62.88% (95%CI: 49.11–72.92), 85.51% (95%CI: 72.74–92.30) and 83.68% (95%CI: 61.34–93.11). Incidences of AEs from the first dose to one month post the third dose in HRV and placebo groups were comparable. There was no significant difference in incidences of SAEs in HRV and placebo groups. This study shows that this hexavalent reassortant rotavirus vaccine is an effective, well-tolerated, and safe vaccine for Chinese infants.     

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.     

Lactobacilli and Bifidobacteria Promote Immune Homeostasis by Modulating Innate Immune Responses to Human Rotavirus in Neonatal Gnotobiotic Pigs

The effects of co-colonization with Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on 3-dose vaccination with attenuated HRV and challenge with virulent human rotavirus (VirHRV) were assessed in 4 groups of gnotobiotic (Gn) pigs: Pro+Vac (probiotic-colonized/vaccinated), Vac (vaccinated), Pro (probiotic-colonized, non-vaccinated) and Control (non-colonized, non-vaccinated). Subsets of pigs were euthanized pre- [post-challenge day (PCD) 0] and post (PCD7)-VirHRV challenge to assess diarrhea, fecal HRV shedding and dendritic cell/innate immune responses. Post-challenge, Pro+Vac and Vac groups were completely protected from diarrhea; protection rates against HRV shedding were 100% and 83%, respectively. Diarrhea and HRV shedding were reduced in Pro compared to Control pigs following VirHRV challenge. Diarrhea scores and virus shedding were significantly higher in Controls, compared to all other groups, coincident with significantly higher serum interferon-alpha levels post-challenge. LGG+Bb12 colonization ±vaccine promoted immunomaturation as reflected by increased frequencies of CD4, SWC3a, CD11R1, MHCII expressing mononuclear cells (MNCs) and conventional dendritic cells in intestinal tissues and blood post-challenge. Colonization decreased frequencies of toll-like receptors (TLR) 2 and TLR4 expressing MNCs from vaccinated pigs (Pro+Vac) pre-challenge and increased frequencies of TLR3 expressing MNCs from Pro pigs post-challenge, suggesting that probiotics likely exert anti-inflammatory (TLR2 and 4 down-regulation) and antiviral (TLR3 up-regulation by HRV dsRNA) actions via TLR signaling. Probiotic colonization alone (Pro) increased frequencies of intestinal and systemic apoptotic MNCs pre-challenge, thereby regulating immune hyperreactivity and tolerance. However, these frequencies were decreased in intestinal and systemic tissues post-challenge, moderating HRV-induced apoptosis. Additionally, post-challenge, Pro+Vac and Pro groups had significantly decreased MNC proliferation, suggesting that probiotics control excessive lymphoproliferative reactions upon VirHRV challenge. We conclude that in the neonatal Gn pig disease model, selected probiotics contribute to immunomaturation, regulate immune homeostasis and modulate vaccine and virulent HRV effects, thereby moderating HRV diarrhea.     

Vitamin A Deficiency Impairs Adaptive B and T Cell Responses to a Prototype Monovalent Attenuated Human Rotavirus Vaccine and Virulent Human Rotavirus Challenge in a Gnotobiotic Piglet Model

Rotaviruses (RV) are a major cause of gastroenteritis in children. Widespread vitamin A deficiency is associated with reduced efficacy of vaccines and higher incidence of diarrheal infections in children in developing countries. We established a vitamin A deficient (VAD) gnotobiotic piglet model that mimics subclinical vitamin A deficiency in children to study its effects on an oral human rotavirus (HRV) vaccine and virulent HRV challenge. Piglets derived from VAD and vitamin A sufficient (VAS) sows were orally vaccinated with attenuated HRV or mock, with/without supplemental vitamin A and challenged with virulent HRV. Unvaccinated VAD control piglets had significantly lower hepatic vitamin A, higher severity and duration of diarrhea and HRV fecal shedding post-challenge as compared to VAS control pigs. Reduced protection coincided with significantly higher innate (IFNα) cytokine and CD8 T cell frequencies in the blood and intestinal tissues, higher pro-inflammatory (IL12) and 2-3 fold lower anti-inflammatory (IL10) cytokines, in VAD compared to VAS control pigs. Vaccinated VAD pigs had higher diarrhea severity scores compared to vaccinated VAS pigs, which coincided with lower serum IgA HRV antibody titers and significantly lower intestinal IgA antibody secreting cells post-challenge in the former groups suggesting lower anamnestic responses. A trend for higher serum HRV IgG antibodies was observed in VAD vs VAS vaccinated groups post-challenge. The vaccinated VAD (non-vitamin A supplemented) pigs had significantly higher serum IL12 (PID2) and IFNγ (PID6) compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions. Furthermore, regulatory T-cell responses were compromised in VAD pigs. Supplemental vitamin A in VAD pigs did not fully restore the dysregulated immune responses to AttHRV vaccine or moderate virulent HRV diarrhea. Our findings suggest that that VAD in children in developing countries may partially contribute to more severe rotavirus infection and lower HRV vaccine efficacy.     

Gastroenteritis in suckling mice caused by human rotavirus can be prevented with egg yolk immunoglobulin (IgY) and treated with a protein-bound polysaccharide preparation (PSK)

Oral inoculation of human rotavirus MO strain (serotype 3) into 5-day-old BALB/c mice cause gastroenteritis characterized by diarrhea. Clinical symptoms, histopathological changes in the small intestine, and the detection of rotavirus antigen in enterocytes were all characteristic of rotavirus-induced gastroenteritis. Using this small animal model, passive protection of suckling mice against human rotavirus infection was achieved with the use of immunoglobulin (IgY) from the yolks of eggs of rotavirus-immunized hens. When IgY against a rotavirus strain homotypic to the challenge virus (MO strain) was administered in the mice, complete protection against rotavirus infection was achieved. On the other hand, with oral administration of IgY against a heterotypic strain (serotype 1, Wa strain), a lower protective effect was nevertheless obtained. The four different strains of human rotavirus (Wa, KUN, MO, and ST3) were inactivated in vitro by treatment with PSK, a protein-bound polysaccharide preparation, in a dose-dependent manner. Oral administration of 2.5 mg of PSK caused a therapeutic effect on experimentally MO-infected suckling mice. The antiviral effect of PSK was indicated by the reduction of the duration of diarrhea.     

Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen.

A single-gene substitution reassortant 11-1 was generated from two porcine rotaviruses, OSU (serotype 5) and Gottfried (serotype 4). This reassortant derived 10 genes, including gene 4 encoding VP3, from the OSU strain and only gene 9, encoding a major neutralization glycoprotein (VP7), from the Gottfried strain and was thus designated VP3:5; VP7:4. Oral administration of this reassortant to colostrum-deprived gnotobiotic newborn pigs induced a high level of neutralizing antibodies not only to Gottfried VP7 but also to OSU VP3, thus demonstrating that VP3 is as potent an immunogen as VP7 in inducing neutralizing antibodies during experimental oral infection. Gnotobiotic piglets infected previously with the reassortant were completely resistant to oral challenge with the virulent Gottfried strain (VP3:4; VP7:4), as indicated by failure of symptoms to develop and lack of virus shedding. Similarly, prior infection with the reassortant induced almost complete protection against diarrhea and significant restriction of virus replication after oral challenge with the virulent OSU strain (VP3:5; VP7:5). Thus, it appears that (i) the immune system of the piglet responds equally well to two rotavirus outer capsid proteins, VP3 and VP7, during primary enteric rotavirus infection; (ii) antibody to VP3 and antibody to VP7 are each associated with resistance to diarrhea; and (iii) infection with a reassortant rotavirus bearing VP3 and VP7 neutralization antigens derived from two viruses of different serotype induces immunity to both parental viruses. The relevance of these findings to the development of effective reassortant rotavirus vaccines is discussed.     

Cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus

To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma interferon (IFN-gamma) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor beta (Th3) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-gamma, IL-4, and IL-10 cytokine-secreting cell (CSC) responses of mononuclear cells from ileum, spleen, and blood by ELISPOT. Pigs received the virulent Wa P1A[8]G1 strain of human rotavirus (HRV) (VirHRV), attenuated Wa HRV (AttHRV), or mock (controls). The TNF-alpha levels peaked earlier and remained elevated in serum of the VirHRV group but peaked later in the AttHRV group. In serum, IL-6 was significantly elevated at postinoculation day (PID) 1 in the VirHRV group and at PID 3 in both HRV groups. The IL-12 was detected in serum of all pigs including controls with significantly elevated peaks in both HRV-infected groups, indicating a role for IL-12 in the induction of immune responses to rotavirus infection. Only low and transient IFN-gamma responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-gamma responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-gamma-secreting cells was significantly higher in the ileum of the VirHRV group than in that of the controls. The number of IL-4 CSCs was significantly higher in ileum of both HRV groups than in that of the controls. Significantly higher levels of IL-10 in the serum occurred early in the VirHRV group, compared to lower levels in the AttHRV group. However, the number of IL-10 CSCs was significantly higher later in ileum and spleen of the AttHRV than in the VirHRV group, suggesting a delayed initiation of a Th2 response induced by AttHRV. A significantly higher percentage of pigs had IFN-gamma and IL-10 responses in serum after VirHRV infection than after AttHRV infection or in controls. These data indicate a balanced Th1/Th2 response during rotavirus infection, with higher cytokine levels early after infection with VirHRV compared to that with AttHRV. Mapping the kinetics and patterns of cytokine responses after rotavirus infection has important implications for induction of protective immunity by HRV vaccines. Higher protection rates may be associated with more balanced Th1- and Th2-type responses, but induction of higher earlier IFN-gamma (Th1) and proinflammatory cytokines triggered by VirHRV may also play an important role in the higher intestinal immunoglobulin A responses and protection rates induced by VirHRV.     

Human rotavirus K8 strain represents a new VP4 serotype.

The complete VP4 gene of the human rotavirus (HRV) K8 strain (G1 serotype) was cloned and inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. A K8VP4 recombinant baculovirus was obtained by cotransfection of Spodoptera frugiperda (Sf9) cells with transfer vector DNA containing the K8VP4 gene and wild-type baculovirus DNA. Infection of Sf9 cells with this VP4 recombinant baculovirus resulted in the production of a protein that is similar in size and antigenic activity to the authentic VP4 of the K8 strain. Guinea pigs immunized with the expressed VP4 developed antibodies that neutralized the infectivity of the K8 strain. This antiserum neutralized HRV strains belonging to VP4 serotypes 1A, 1B, and 2 with efficiency eightfold or lower than that of the homologous virus, indicating that the human rotavirus K8 strain represents a distinct VP4 serotype (P3). In addition, low levels of cross-immunoprecipitation of the K8VP4 and its VP5 and VP8 subunits with hyperimmune antisera to HRV strains representing different VP4 serotype specificities also suggested that the K8 strain possesses a unique VP4 with few epitopes in common with other P-serotype strains.     

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.     

Sequential rotavirus infections: characterization of serotypes and electrophoretypes

Few studies have characterized the rotaviruses observed in repeated infections in the same patient. Rotavirus serotypes and electrophoretypes were determined from faecal samples of 24 children hospitalized between 1984 and 1989 in a Clermont-Ferrand hospital and who had had repeated rotavirus shedding.The patients were aged between 3 days and 4 years. Fifty infections were recorded. Of the 48 serotypes obtained, 18 were serotype 1 (37.5 %), 10 serotype 2 (20.8 %) and 20 serotype 4 (41.7 %). Serotype 3 was not found. Seven serotype 2 infections were characterized during rehospitalization. Six patients had two subsequent infections with the same serotype.     

Rotavirus neutralizing protein VP7: antigenic determinants investigated by sequence analysis and peptide synthesis.

The rotavirus neutralizing antigen, VP7, is a 37,000-molecular-weight glycoprotein which is a major component of the outer shell of the virion. The amino acid sequence of VP7 for strain S2 (human serotype 2) and Nebraska calf diarrhea virus (bovine serotype) has been inferred from the nucleic acid sequence of cloned copies of genomic segment nine. Comparison of the amino acid sequences of these two VP7 proteins with those already determined for other rotavirus strains reveals extensive sequence conservation between serotypes with clusters of amino acid differences sited predominantly in hydrophilic domains of the protein. Six peptides have been synthesized that span the hydrophilic regions of the molecule. Antisera to these peptides both recognize the respective homologous peptides in a solid-phase radioimmunoassay and bind to denatured VP7 in a Western blot. However, none of the antisera either recognize virus or exhibit significant neutralizing activity, indicating that these peptide sequences are not available on the surface of the virus.     

Intranasal administration of 2/6-rotavirus-like particles with mutant Escherichia coli heat-labile toxin (LT-R192G) induces antibody-secreting cell responses but not protective immunity in gnotobiotic pigs

We investigated the immunogenicity of recombinant double-layered rotavirus-like particle (2/6-VLPs) vaccines derived from simian SA11 or human (VP6) Wa and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were administered to gnotobiotic pigs intranasally (i.n.) with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT), as mucosal adjuvant. Pigs were challenged with virulent Wa (P1A[8],G1) human rotavirus at postinoculation day (PID) 21 (two-dose VLP regimen) or 28 (three-dose VLP regimen). In vivo antigen-activated antibody-secreting cells (ASC) (effector B cells) and in vitro antigen-reactivated ASC (derived from memory B cells) from intestinal and systemic lymphoid tissues (duodenum, ileum, mesenteric lymph nodes [MLN], spleen, peripheral blood lymphocytes [PBL], and bone marrow lymphocytes) collected at selected times were quantitated by enzyme-linked immunospot assays. Rotavirus-specific immunoglobulin M (IgM), IgA, and IgG ASC and memory B-cell responses were detected by PID 21 or 28 in intestinal and systemic lymphoid tissues after i.n. inoculation with two or three doses of 2/6-VLPs with or without mLT. Greater mean numbers of virus-specific ASC and memory B cells in all tissues prechallenge were induced in pigs inoculated with two doses of SA11 2/6-VLPs plus mLT compared to SA11 2/6-VLPs without mLT. After challenge, anamnestic IgA and IgG ASC and memory B-cell responses were detected in intestinal lymphoid tissues of all VLP-inoculated groups, but serum virus-neutralizing antibody titers were not significantly enhanced compared to the challenged controls. Pigs inoculated with Wa-RF 2/6-VLPs (with or without mLT) developed higher anamnestic IgA and IgG ASC responses in ileum after challenge compared to pigs inoculated with SA11 2/6-VLPs (with or without mLT). Three doses of SA 11 2/6-VLP plus mLT induced the highest mean numbers of IgG memory B cells in MLN, spleen, and PBL among all groups postchallenge. However, no significant protection against diarrhea or virus shedding was evident in any of the 2/6-VLP (with or without mLT)-inoculated pigs after challenge with virulent Wa human rotavirus. These results indicate that 2/6-VLP vaccines are immunogenic in gnotobiotic pigs when inoculated i.n. and that the adjuvant mLT enhanced their immunogenicity. However, i.n. inoculation of gnotobiotic pigs with 2/6-VLPs did not confer protection against human rotavirus challenge.     

Effects of Maternal Antibodies on Protection and Development of Antibody Responses to Human Rotavirus in Gnotobiotic Pigs

Although maternal antibodies can protect against infectious disease in infancy, they can also suppress active immune responses. The effects of circulating maternal antibodies, with and without colostrum and milk antibodies, on passive protection and active immunity to human rotavirus (HRV) were examined in gnotobiotic pigs. Pigs received intraperitoneal injections of high-titer serum (immune pigs [groups 1 and 2]) from immunized sows, low-titer serum from naturally infected sows (control pigs [groups 3 and 4]), or no serum (group 5). Immune or control colostrum and milk were added to the diet of groups 2 and 4, respectively. After inoculation (3 to 5 days of age) and challenge (postinoculation day [PID] 21) with virulent HRV, the effects of maternal antibodies on protection (from diarrhea and virus shedding), and on active antibody responses (measured by quantitation of antibody-secreting cells [ASC] in intestinal and systemic lymphoid tissues by ELISPOT) were evaluated. Groups 1 and 2 had significantly less diarrhea and virus shedding after inoculation but higher rates of diarrhea and virus shedding after challenge than did groups 3 and 5. Group 1 and 2 pigs had significantly fewer immunoglobulin A (IgA) ASC in intestinal tissues at PID 21 and at postchallenge day (PCD) 7 compared to group 5. Significantly fewer IgG ASC were present in the intestines of group 2 pigs at PID 21 and PCD 7 compared to group 5. There was a trend towards fewer ASC in intestinal tissues of group 2 than group 1, from PID 21 on, with significantly fewer IgA ASC at PCD 7. IgG ASC in the duodenum and mesenteric lymph nodes of group 3 and 4 pigs were significantly fewer than in group 5 at PCD 7. These decreases in ASC emphasize the role of passive antibodies in impairing induction of ASC rather than in merely suppressing the function of differentiated B cells. To be successful, vaccines intended for populations with high titers of maternal antibodies (infants in developing countries) may require higher titers of virus, multiple doses, or improved delivery systems, such as the use of microencapsulation or immune stimulating complexes, to overcome the suppressive effects of maternal antibodies.     

Formation of rhinovirus-soluble ICAM-1 complexes and conformational changes in the virion.

Viral receptors serve both to target viruses to specific cell types and to actively promote the entry of bound virus into cells. Human rhinoviruses (HRVs) can form complexes in vitro with a truncated soluble form of the HRV cell surface receptor, ICAM-1. These complexes appear to be stoichiometric, with approximately 60 ICAM molecules bound per virion or 1 ICAM-1 molecule per icosahedral face of the capsid. The complex can have two fates, either dissociating to yield free virus and free ICAM-1 or uncoating to break down to an 80S empty capsid which has released VP4, viral RNA, and ICAM-1. This uncoating in vitro mimics the uncoating of virus during infection of cells. The stability of the virus-receptor complex is dependent on temperature and the rhinovirus serotype. HRV serotype 14 (HRV14)-ICAM-1 complexes rapidly uncoat, HRV16 forms a stable virus-ICAM complex which does not uncoat detectably at 34 degrees C, and HRV3 has an intermediate phenotype. Rhinovirus can also uncoat after exposure to mildly acidic pH. The sensitivities of individual rhinovirus serotypes to ICAM-1-mediated virus uncoating do not correlate with uncoating promoted by incubation at low pH, suggesting that these two means of virus destabilization occur by different mechanisms. Soluble ICAM-1 and low pH do not act synergistically to promote uncoating. The rate of uncoating does appear to be inversely related to virus affinity for its receptor.     

Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains.

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.     

Group A Rotavirus Infection and Age-Dependent Diarrheal Disease in Rats: a New Animal Model To Study the Pathophysiology of Rotavirus Infection

Group A rotaviruses are major pathogens causing acute gastroenteritis in children and animals. To determine if group A rotavirus replicates and induces disease in rats, antibody-negative Lewis neonatal or adult rats were inoculated orally with tissue culture-adapted human (Wa, WI61, and HAL1166), simian (rhesus rotavirus [RRV] and SA11), bovine (WC3), lapine (ALA), or porcine (OSU) rotavirus strains, wild-type murine (EC(wt)) rotavirus strain, or phosphate-buffered saline (PBS). Rotavirus infection in rats was evaluated by (i) clinical findings, (ii) virus antigen shedding or infectious virus titers in the feces or intestinal contents measured by enzyme-linked immunosorbent assay or fluorescent-focus assay, (iii) histopathological changes in the small intestine, (iv) distribution of rotavirus antigen in small-intestine sections by immunofluorescence, and (v) growth rate. Rotavirus infection of 5-day-old but not > or =21-day-old rats resulted in diarrhea that lasted from 1 to 10 days postinoculation. The severity of disease and spread of infection to naIve littermates differed depending on the virus strain used for inoculation. The duration of virus antigen shedding following infection was considerably prolonged (up to 10 days) in neonatal rats compared to that in 21-day-old rats (1 or 2 days). Based on lack of virus antigen shedding and disease induction, the murine EC(wt) rotavirus was the only strain tested that did not infect rats. Histopathological changes in the small-intestine mucosa of 5-day-old RRV-inoculated rats but not of PBS-inoculated rats was limited to extensive enterocyte vacuolation in the ileum. In RRV-inoculated neonatal rats, rotavirus antigen was detected in the epithelial cells on the upper half of the intestinal villi of the jejunum and ileum. In addition, infection of neonatal rats with RRV but not with PBS resulted in reduced weight gain. Rats infected with group A rotaviruses provide a new animal model with unique features amenable to investigate rotavirus pathogenesis and the molecular mechanisms of intestinal development, including physiological factors that may regulate age-dependent rotavirus-induced diarrhea.