Marli S.P. Azevedo

Pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup II.4-HS66 strain of human norovirus.

ABSTRACT We previously characterized the pathogenesis of two host-specific bovine enteric caliciviruses (BEC), the GIII.2 norovirus (NoV) strain CV186-OH and the phylogenetically unassigned NB strain, in gnotobiotic (Gn) calves. In this study we evaluated the Gn calf as an alternative animal model to study the pathogenesis and host immune responses to the human norovirus (HuNoV) strain GII.4-HS66. The HuNoV HS66 strain caused diarrhea (five/five calves) and intestinal lesions (one/two calves tested) in the proximal small intestine (duodenum and jejunum) of Gn calves, with lesions similar to, but less severe than, those described for the Newbury agent 2 (NA-2) and NB BEC. Viral capsid antigen was also detected in the jejunum of the proximal small intestine of one of two calves tested by immunohistochemistry. All inoculated calves shed virus in feces (five/five calves), and one/five had viremia. Antibodies and cytokine (proinflammatory, tumor necrosis factor alpha [TNF-α]; Th1, interleukin-12 [IL-12] and gamma interferon [IFN-γ]; Th2, IL-4; Th2/T-regulatory, IL-10) profiles were determined in serum, feces, and intestinal contents (IC) of the HuNoV-HS66-inoculated calves (n = 5) and controls (n = 4) by enzyme-linked immunosorbent assay in the acute (postinoculation day 3 [PID 3]) and convalescent (PID 28) stages of infection. The HuNoV-HS66-specific antibody and cytokine-secreting cells (CSCs) were quantitated by ELISPOT in mononuclear cells of local and systemic tissues at PID 28. Sixty-seven percent of the HuNoV-HS66-inoculated calves seroconverted, and 100% coproconverted with immunoglobulin A (IgA) and/or IgG antibodies to HuNoV-HS66, at low titers. The highest numbers of antibody-secreting cells (ASC), both IgA and IgG, were detected locally in intestine, but systemic IgA and IgG ASC responses also occurred in the HuNoV-HS66-inoculated calves. In serum, HuNoV-HS66 induced higher peaks of TNF-α and IFN-γ at PIDs 2, 7, and 10; of IL-4 and IL-10 at PID 4; and of IL-12 at PIDs 7 and 10, compared to controls. In feces, cytokines increased earlier (PID 1) than in serum and TNF-α and IL-10 were elevated acutely in the IC of the HS66-inoculated calves. Compared to controls, at PID 28 higher numbers of IFN-γ and TNF-α CSCs were detected in mesenteric lymph nodes (MLN) or spleen and Th2 (IL-4) CSCs were elevated in intestine; IL-10 CSCs were highest in spleen. Our study provides new data confirming HuNoV-HS66 replication and enteropathogenicity in Gn calves and reveals important and comprehensive aspects of the hosts local (intestine and MLN) and systemic (spleen and blood) immune responses to HuNoV-HS66.

Cytokine Responses in Gnotobiotic Pigs after Infection with Virulent or Attenuated Human Rotavirus

ABSTRACT To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma interferon (IFN-γ) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor β (Th3) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-γ, 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-α 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-γ responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-γ responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-γ-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-γ 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-γ (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.

Probiotic Lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs

We evaluated virus-specific B and T cell responses induced by the attenuated Wa (P1A[8]G1) human rotavirus (AttHRV) oral 2-dose vaccine with or without Lactobacillus acidophilus (LA) colonization in neonatal gnotobiotic (Gn) pigs. The AttHRV vaccinated and LA-fed pigs had a significantly higher magnitude of HRV-specific IFN-gamma producing CD8+ T cell responses in ileum and spleen, IgA and IgG antibody-secreting cell responses in ileum, and serum IgM, IgA and IgG antibody and virus neutralizing antibody titers compared to the AttHRV vaccinated pigs without LA colonization. These findings suggest that L. acidophilus has significant immunopotentiating effects and may be used as a safe oral adjuvant for rotavirus vaccines in neonates.

Cytokine and Antibody Responses in Gnotobiotic Pigs after Infection with Human Norovirus Genogroup II.4 (HS66 Strain)

ABSTRACT A human norovirus genogroup II.4 strain HS66 (HuNoV-HS66) infects and causes mild diarrhea in gnotobiotic (Gn) pigs (S. Cheetham, M. Souza, T. Meulia, S. Grimes, M. G. Han, and L. J. Saif, J. Virol. 80:10372-10381, 2006). In this study we evaluated systemic and intestinal humoral and cellular immune responses to HuNoV-HS66 in orally inoculated pigs. Antibodies and type I interferon (IFN-I or IFN-α), proinflammatory interleukin-6 (IL-6), Th1 (IL-12 and IFN-γ), Th2 (IL-4), and Th2/regulatory T ([Treg] IL-10) cytokine profiles in serum and intestinal contents (IC) of the HuNoV-HS66-inoculated pigs and controls were assessed by enzyme-linked immunosorbent assay at selected postinoculation days (0 to 28). Using an enzyme-linked immunospot assay, we evaluated immunoglobulin M (IgM), IgA, and IgG antibody-secreting cells (ASC) and cytokine-secreting cells (CSC) in intestine, spleen, and blood. In the HuNoV-inoculated pigs, antibody titers in serum and IC were generally low, and 65% seroconverted. Pigs with higher diarrhea scores were more likely to seroconvert and developed higher intestinal IgA and IgG antibody titers. The numbers of IgA and IgG ASC were higher systemically than in the gut. In serum, HuNoV induced persistently higher Th1 (low transient IFN-γ and high IL-12) than the other cytokines, but also low Th2 (IL-4) and Th2/Treg (IL-10) levels; low, transient proinflammatory (IL-6) cytokines; and, notably, a delayed IFN-α response. In contrast, intestinal innate (IFN-α early and late) and Th1 (IL-12 late) cytokines were significantly elevated postinfection. HuNoV-HS66 also elicited higher numbers of Th1 (IL-12 and IFN-γ) CSC than Th2 (IL-4) and proinflammatory (IL-6) CSC, with the latter responses low in blood and intestine, reflecting low intestinal inflammation in the absence of gut lesions. These data provide insights into the kinetics of cytokine secretion in serum and IC of HuNoV-inoculated Gn pigs and new information on intestinal humoral and cellular immune responses to HuNoV that are difficult to assess in human volunteers.

Virus-specific intestinal IFN-γ producing T cell responses induced by human rotavirus infection and vaccines are correlated with protection against rotavirus diarrhea in gnotobiotic pigs

We examined rotavirus-specific IFN-gamma producing CD4+, CD8+ and CD4+CD8+ T cell responses in gnotobiotic pigs infected with a virulent human rotavirus (VirHRV) or vaccinated with an attenuated (Att) HRV vaccine (AttHRV3x or AttHRV2x) or an AttHRV oral priming and 2/6-virus-like particle (VLP) intranasal boosting (AttHRV-2/6VLP) regimen. In VirHRV infected pigs, HRV-specific IFN-gamma producing T cells reside primarily in ileum. AttHRV-2/6VLP induced similar frequencies of intestinal IFN-gamma producing T cells as the VirHRV, whereas AttHRV3x or 2x vaccines were less effective. Protection rates against rotavirus diarrhea upon VirHRV challenge significantly correlated (r=0.97-1.0, p<0.005) with frequencies of intestinal IFN-gamma producing T cells, suggesting their role in protective immunity.

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

ABSTRACT 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/VLP2×) 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 (VLP2×/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 (AttHRV1× and Mock2×/AttHRV, respectively), three oral doses of attenuated Wa HRV (AttHRV3×), three i.n. doses of 2/6-VLPs plus mLT (VLP3×), three i.n. doses of purified double-layered inactivated Wa HRV plus mLT (InactHRV3×), 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/VLP2× 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 VLP2×/AttHRV regimen was less efficacious than the AttHRV/VLP2× regimen in inducing IgA ASC responses and protection against diarrhea (25% protection rate) but was more efficacious than VLP3× or InactHRV3× (no protection). In conclusion, the AttHRV/VLP2× 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.

Lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs

Abstract Despite accumulating knowledge of porcine macrophages and dendritic cells (DCs) from in vitro studies, information regarding monocytes/macrophages and DCs in lymphoid tissues of enteric pathogen-infected neonatal animals in vivo is limited. In this study we evaluated the influence of commensal bacterial [two strains of lactic acid bacteria (LAB), Lactobacillus acidophilus and L. reuteri] colonization and rotavirus infection on distribution and frequencies of monocytes/macrophages and conventional DCs (cDCs) in ileum, spleen and blood. Gnotobiotic pigs were inoculated with LAB and virulent Wa strain human rotavirus (HRV) (LAB+HRV+), HRV only (LAB−HRV+), LAB only (LAB+HRV−) or mock (LAB−HRV−). The cDCs were characterized as SWC3+CD11R1+, whereas monocytes/macrophages were identified as SWC3+CD11R1− by flow cytometry in the gnotobiotic pigs at 10 days of age. Infection with HRV alone activated/recruited significantly more monocytes/macrophages to the intestine than LAB colonization and 56% versus 28% of these cells expressed CD14. Colonization with LAB alone also significantly increased the frequencies of monocytes/macrophages and cDCs and the CD14 expression on monocytes/macrophages in ileum and spleen compared to the controls. LAB colonization plus HRV infection significantly reduced macrophage and cDC frequencies in spleen compared to LAB colonization or HRV infection alone, suggesting that LAB colonization down-regulated HRV− infection-induced monocyte/macrophage activation/recruitment at the systemic lymphoid tissue. These results illustrated the distribution of porcine monocytes/macrophages and cDCs and the frequencies of CD14 expression on these cells in intestinal and systemic lymphoid tissues in the early stage of immune responses to intestinal colonization by LAB versus infection by an enteric pathogen HRV and will facilitate further in vivo studies on functional characterization of these immune cells in neonates.

Antibody responses to human rotavirus (HRV) in gnotobiotic pigs following a new prime/boost vaccine strategy using oral attenuated HRV priming and intranasal VP2/6 rotavirus-like particle (VLP) boosting with ISCOM.

Safer and more effective human rotavirus (HRV) vaccines are needed. We evaluated oral priming with attenuated WaHRV (AttHRV) followed by boosting with two intranasal (IN) doses of VP2/6 virus‐like particles (2/6 VLP) with immunostimulating complexes (ISCOM) to determine if this regimen induces protection against diarrhoea and viral shedding in the gnotobiotic pig model. IgM, IgA and IgG antibody titres in serum and intestinal contents were quantified by enzyme‐linked immunosorbent assay (ELISA) and serum neutralizing antibody titres were measured by a virus neutralization (VN) test. Seven groups of neonatal gnotobiotic pigs were vaccinated at post‐inoculation days (PID) 0, 10 and 21 and challenged with virulent WaHRV at PID 28. The vaccine groups included: (1, 2) oral priming with AttHRV and boosting with two IN immunizations with 2/6 VLP–ISCOM (Att + 2/6 VLP–ISCOM) at VLP concentrations of 250 µg or 25 µg; (3, 4) three IN immunizations with 2/6 VLP–ISCOM at VLP concentrations of 250 µg or 25 µg (2/6 VLP–ISCOM); (5) three oral immunizations with AttHRV (3×AttHRV); (6) one oral immunization with AttHRV (1×AttHRV); (7) controls (ISCOM matrix and/or diluent). The pigs that received 3×AttHRV or Att + 2/6 VLP250–ISCOM had the highest protection rates against diarrhoea upon challenge at PID 28 with virulent WaHRV. The IgA antibody titres to HRV in intestinal contents were significantly higher in the Att + 2/6 VLP250–ISCOM group than in all other groups prechallenge (PID 28). Serum VN antibody titres were statistically similar after the first inoculation among the groups given AttHRV, but at PID 28 VN antibody titres were significantly higher for the 3×AttHRV and Att + 2/6 VLP250–ISCOM groups than for the 1×AttHRV group suggesting that boosting with 2/6 VLP also boosted VN antibody responses. In humans, intestinal IgA antibodies have been correlated with protection against symptomatic reinfection. Thus the vaccine regimen of one oral dose of AttHRV and two IN immunizations with 2/6 VLP250–ISCOM may be an alternative to multiple‐dose live oral vaccines in humans.

Inactivated rotavirus vaccine induces protective immunity in gnotobiotic piglets

Live oral rotavirus vaccines that are effective in middle and high income countries have been much less immunogenic and effective among infants in resource-limited settings. Several hypotheses might explain this difference, including neutralization of the vaccine by high levels of maternal antibody in serum and breast milk, severe malnutrition, and interference by other flora and viruses in the gut. We have pursued development of an alternative parenteral rotavirus vaccine with the goal of inducing comparable levels of immunogenicity and efficacy in populations throughout the world regardless of their income levels. In the present study, we assessed the immunogenicity and protection of a candidate inactivated rotavirus vaccine (IRV), the human strain CDC-9 (G1P[8]) formulated with aluminum phosphate, against rotavirus infection in gnotobiotic piglets. Three doses of IRV induced high titers of rotavirus-specific IgG and neutralizing activity in the sera of gnotobiotic piglets and protection against shedding of rotavirus antigen following oral challenge with a homologous virulent human strain Wa (G1P[8]). Our findings demonstrate the proof of concept for an IRV in a large animal model and provide evidence and justification for further clinical development as an alternative candidate vaccine.

Systemic and intestinal antibody secreting cell responses and protection in gnotobiotic pigs immunized orally with attenuated Wa human rotavirus and Wa 2/6-rotavirus-like-particles associated with immunostimulating complexes

The undesirable side effects and variable efficacy of some oral live rotavirus vaccines in infants have necessitated alternative vaccine approaches. We evaluated a recombinant RFVP2/WaVP6 rotavirus-like-particle (2/6VLP) oral vaccine, using an immunostimulating complex (ISCOM) matrix as adjuvant, in a gnotobiotic (Gn) pig model of human rotavirus (HRV) disease. The 2/6VLPs adhered to the ISCOM-matrix (2/6VLP-ISCOM ) and were antigenic, but they failed to induce protection. However, when combined with attenuated (Att) HRV for oral priming, the 2/6VLP-ISCOM vaccine was effective as a booster and induced partial protection against virulent Wa HRV. The 250 microg 2/6VLP dose was more effective than 100 microg. The highest mean numbers of IgA antibody secreting cells evaluated by ELISPOT in intestinal lymphoid tissues were in pigs receiving AttHRV+2/6VLP-ISCOM or three doses of AttHRV and were associated with the highest protection rates.