Anthony H.-C. Choi

CD4 T Cells Are the Only Lymphocytes Needed To Protect Mice against Rotavirus Shedding after Intranasal Immunization with a Chimeric VP6 Protein and the Adjuvant LT(R192G)

ABSTRACT Intranasal immunization of mice with a chimeric VP6 protein and the mucosal adjuvant Escherichia coli heat labile toxin LT(R192G) induces nearly complete protection against murine rotavirus (strain EDIM [epizootic diarrhea of infant mice virus]) shedding for at least 1 year. The aim of this study was to identify the protective lymphocytes elicited by this new vaccine candidate. Immunization of mouse strains lacking one or more lymphocyte populations revealed that protection was dependent on αβ T cells but mice lacking γδ T cells and B cells remained fully protected. Furthermore, depletion of CD8 T cells in immunized B-cell-deficient mice before challenge resulted in no loss of protection, while depletion of CD4 T cells caused complete loss of protection. Therefore, αβ CD4 T cells appeared to be the only lymphocytes required for protection. As confirmation, purified splenic T cells from immunized mice were intraperitoneally injected into Rag-2 mice chronically infected with EDIM. Transfer of 2 × 106 CD8 T cells had no effect on shedding, while transfer of 2 × 105 CD4 T cells fully resolved shedding in 7 days. Interestingly, transfer of naive splenic CD4 T cells also resolved shedding but more time and cells were required. Together, these results establish CD4 T cells as effectors of protection against rotavirus after intranasal immunization of mice with VP6 and LT(R192G).

Development of a Rotavirus-Shedding Model in Rhesus Macaques, Using a Homologous Wild-Type Rotavirus of a New P Genotype

ABSTRACT Although there are several reports on rotavirus inoculation of nonhuman primates, no reliable model exists. Therefore, this study was designed to develop a rhesus macaque model for rotavirus studies. The goals were to obtain a wild-type macaque rotavirus and evaluate it as a challenge virus for model studies. Once rotavirus was shown to be endemic within the macaque colony at the Tulane National Primate Research Center, stool specimens were collected from juvenile animals (2.6 to 5.9 months of age) without evidence of previous rotavirus infection and examined for rotavirus antigen. Six of 10 animals shed rotavirus during the 10-week collection period, and the electropherotypes of all isolates were identical to each other but distinct from those of prototype simian rotaviruses. These viruses were characterized as serotype G3 and subgroup 1, properties typical of many animal rotaviruses, including simian strains. Nucleotide sequence analysis of the VP4 gene was performed with a culture-grown isolate from the stool of one animal, designated the TUCH strain. Based on both genotypic and phylogenetic comparisons between TUCH VP4 and cognate proteins of representatives of the reported 22 P genotypes, the TUCH virus belongs to a new genotype, P[23]. A pool of wild-type TUCH was prepared and intragastrically administered to eight cesarean section-derived, specific-pathogen-free macaques 14 to 42 days of age. All animals were kept in a biocontainment level 2 facility. Although no diarrhea was observed and the animals remained clinically normal, all animals shed large quantities of rotavirus antigen in their feces after inoculation, which resolved by the end of the 14-day observation period. Therefore, TUCH infection of macaques provides a useful nonhuman primate model for studies on rotavirus protection.

Functional Mapping of Protective Domains and Epitopes in the Rotavirus VP6 Protein

ABSTRACT The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed inEscherichia coli was examined. Mice (BALB/c) immunized with two 9-μg doses of each of the chimeras and 10 μg of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively; P≤ 0.01) following challenge. Because CD produced almost complete protection, we prepared four E. coli-expressed, MBP-fused chimeras containing overlapping fragments of the CD region (i.e., CD1, CD2, CD3, and CD4) whose lengths ranged from 61 to 67 amino acid residues. Following i.n. immunization, CD1, CD2, and CD4 induced significant (P ≤ 0.004) protection (88, 84, and 92% reduction, respectively). In addition, 11 peptides (18 to 30 residues) of the CD region with between 0 and 13 overlapping amino acids were synthesized. Two 50-μg doses of each peptide with LT(R192G) were administered i.n. to BALB/c mice. Five peptides were found to elicit significant (P ≤ 0.02) protection. Moreover, a 14-amino-acid region within peptide 6 containing a putative CD4+ T-cell epitope was found to confer nearly complete protection, suggesting a protective role for CD4+ T cells. Mice that were protected by fragments BC and CD1 and four of the five protective synthetic peptides did not develop measurable rotavirus antibodies in serum or stool, implying that protection induced by these domains was not dependent on antibody. Together, these observations suggest that multiple regions of VP6 can stimulate protection, a region of VP6 as small as 14 amino acids containing a CD4+ T-cell epitope can stimulate nearly complete protection, and protection mediated by a subset of epitopes in the VP6 protein does not require antibodies in BALB/c mice.

Rotavirus subunit vaccine

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.

Intranasal or oral immunization of inbred and outbred mice with murine or human rotavirus VP6 proteins protects against viral shedding after challenge with murine rotaviruses.

Intranasal (i.n.) administration of an Escherichia coli-expressed chimeric VP6 protein from the EDIM strain of murine rotavirus to adult BALB/c (H-2(d)) mice along with LT(R192G), an attenuated mutant of the mucosal adjuvant E. coli heat-labile toxin, has been found to consistently stimulate ca. 99% reductions in rotavirus shedding after subsequent EDIM challenge. This study was designed to determine the robustness of this protection, i.e. can VP6 immunization consistently protect against shedding in this model, thus, providing an indication of its potential as a vaccine. Intranasal immunization with two 8.8 microg doses of EDIM VP6 and 10 microg of LT(R192G) was found to stimulate 99% reductions in EDIM shedding in four additional strains of inbred mice belonging to three haplotypes, i.e. DBA/2 (H-2(d)), C57BL/6 (H-2(b)), 129 (H-2(b)) and C3H (H-2(k)). Protection stimulated against EDIM antigen shedding following i.n. immunization with VP6 from the human CJN strain was less (P=0.02) than induced by EDIM VP6 (86% versus 99%), but no further loss of protection was observed when the dose of CJN VP6 was reduced 100-fold. Protection against EDIM shedding was also maintained after i.n. immunization of three strains of outbred mice (CF-1, CD-1 and Swiss Webster) with either EDIM or CJN VP6, i.e. EDIM VP6 immunization reduced EDIM shedding by 99% while CJN VP6 immunization produced reductions of 86-96%. Protection stimulated by oral immunization of BALB/c mice with two 8.8 microg doses of either VP6 chimera plus LT(R192G) was not significantly different from that induced by i.n. immunization. Finally, protection found after either oral or i.n. immunization with EDIM or CJN VP6 was no different when the mice were challenged with McN, another strain of murine rotavirus. These results support further evaluation of VP6 as a vaccine.

Association of Gamma Interferon and Interleukin-17 Production in Intestinal CD4+ T Cells with Protection against Rotavirus Shedding in Mice Intranasally Immunized with VP6 and the Adjuvant LT(R192G)

ABSTRACT Mucosal immunization of mice with chimeric, Escherichia coli-expressed VP6, the protein that comprises the intermediate capsid layer of the rotavirus particle, together with attenuated E. coli heat-labile toxin LT(R192G) as an adjuvant, reduces fecal shedding of rotavirus antigen by >95% after murine rotavirus challenge, and the only lymphocytes required for protection are CD4+ T cells. Because these cells produce cytokines with antiviral properties, the cytokines whose expression is upregulated in intestinal memory CD4+ T cells immediately after rotavirus challenge of VP6/LT(R192G)-immunized mice may be directly or indirectly responsible for the rapid suppression of rotavirus shedding. This study was designed to identify which cytokines are significantly upregulated in intestinal effector sites and secondary lymphoid tissues of intranasally immunized BALB/c mice after challenge with murine rotavirus strain EDIM. Initially, this was done by using microarray analysis to quantify mRNAs for 96 murine common cytokines. With this procedure, the synthesis of mRNAs for gamma interferon (IFN-γ) and interleukin-17 (IL-17) was found to be temporarily upregulated in intestinal lymphoid cells of VP6/LT(R192G)-immunized mice at 12 h after rotavirus challenge. These cytokines were also produced in CD4+ T cells obtained from intestinal sites specific to VP6/LT(R192G)-immunized mice after in vitro exposure to VP6 as determined by intracellular cytokine staining and secretion of cytokines. Although genetically modified mice that lack receptors for either IFN-γ or IL-17 remained protected after immunization, these results provide suggestive evidence that these cytokines may play direct or indirect roles in protection against rotavirus after mucosal immunization of mice with VP6/LT(R192G).

The Role of Interferons in Rotavirus Infections and Protection

Type I and type II interferons (IFNs) play a critical role in control of a number of viral infections. To study whether altered and reduced functional capacities of type I and type II IFNs would affect rotavirus-induced diarrhea and viral replication, we obtained signal transducers and activators of transcription 1 (Stat1) knock-out mice (Stat1(-/-)) that lack many IFN-induced responses. We found that suckling Stat1(-/-) and immunocompetent mice orally infected with rotavirus experienced diarrhea and shed rotavirus with similar intensity. However, adult Stat1(-/-) mice shed up to 100-fold more homologous murine rotavirus and heterologous rhesus rotavirus antigen in their stools than did immunocompetent mice 2-6 days after infection. Clearance of rotavirus in stools from adult Stat1(-/-) mice occurred at the same time as in wild-type (WT) control mice. Clearance in Stat1(-/-) mice correlated with a potent antibody response and a mixed Th1 and Th2 response, whereas in WT control mice, clearance correlated with a weaker antibody response and a polarized Th1 response. Stat1(-/-) mice were fully protected against subsequent challenge. Moreover, vaccination of adult Stat1(-/-) mice with a rotavirus VP6 protein and the mucosal adjuvant Escherichia coli heat-labile toxin LT (R192G) elicited 94% protection, as measured by the total reduction in viral shedding for the group in comparison to unimmunized controls. Thus, modulating IFN function through the loss of Stat1 caused a defective innate immune response in adult mice but had no effect on rotavirus-induced diarrhea and replication in suckling mice. Furthermore, adult Stat1(-/-), IFN-gamma, and IFN-alpha/beta receptor(-/-) (IFNAR-2(-/-)) mice infected with rotavirus or vaccinated with VP6 vaccine and adjuvant were fully protected against rotavirus shedding following a subsequent challenge with rotavirus.

Mice Develop Effective but Delayed Protective Immune Responses When Immunized as Neonates either Intranasally with Nonliving VP6/LT(R192G) or Orally with Live Rhesus Rotavirus Vaccine Candidates

ABSTRACT Rotavirus vaccines are delivered early in life, when the immune system is immature. To determine the effects of immaturity on responses to candidate vaccines, neonatal (7 days old) and adult mice were immunized with single doses of either Escherichia coli-expressed rotavirus VP6 protein and the adjuvant LT(R192G) or live rhesus rotavirus (RRV), and protection against fecal rotavirus shedding following challenge with the murine rotavirus strain EDIM was determined. Neonatal mice immunized intranasally with VP6/LT(R192G) were unprotected at 10 days postimmunization (dpi) and had no detectable rotavirus B-cell (antibody) or CD4+ CD8+ T-cell (rotavirus-inducible, Th1 [gamma interferon and interleukin-2 {IL-2}]-, Th2 [IL-5 and IL-4]-, or ThIL-17 [IL-17]-producing spleen cells) responses. However, by 28 and 42 dpi, these mice were significantly (P ≥ 0.003) protected and contained memory rotavirus-specific T cells but produced no rotavirus antibody. In contrast, adult mice were nearly fully protected by 10 dpi and contained both rotavirus immunoglobulin G and memory T cells. Neonates immunized orally with RRV were also less protected (P = 0.01) than adult mice by 10 dpi and produced correspondingly less rotavirus antibody. Both groups contained few rotavirus-specific memory T cells. Protection levels by 28 dpi for neonates or adults were equal, as were rotavirus antibody levels. This report introduces a neonatal mouse model for active protection studies with rotavirus vaccines. It indicates that, with time, neonatal mice develop full protection after intranasal immunization with VP6/LT(R192G) or oral immunization with a live heterologous rotavirus and supports reports that protection depends on CD4+ T cells or antibody, respectively.

Role for T cell-independent B cell activity in the resolution of primary rotavirus infection in mice

We examined the importance of T cell‐independent B cell activity in the resolution of primary murine (EDIM) rotavirus infection in adult mice. We showed that Rag 1 (C57BL / 6 background) and Rag 2 (BALB / c background) knockout mice, which lack both T and B cells, chronically shed high levels of rotavirus Ag in stool samples following oral inoculation. However, nude mice (BALB / c and C57BL / 6 backgrounds) and α β TCR knockout mice (C57BL / 6 background) chronically shed 100‐fold lower levels of virus in stool samples. Thus, B cells appeared to sharply reduce the level of chronic rotavirus shedding by a T cell‐independent mechanism. C57BL / 6 mice depleted of CD4+ cells or both CD4+ and CD8+ cells were also unable to resolve primary rotavirus infection but chronically shed equally low levels of rotavirus Ag in stool samples, whereas mice depleted of only CD8+ cells resolved infection. Similar results were obtained with a second rotavirus strain (ECw) in which virus was shed chronically in stool samples at low levels in α β TCR knockout mice and at high levels in Rag 1 knockout mice. Virus‐specific intestinal IgA was readily detected in mice lacking thymic T cells and α β T cells and in mice depleted of CD4+ cells but levels were 95 % reduced in comparison to immunocompetent controlmice. Together, these results show that B cells lacking CD4+ T cell help have the capacity to substantially reduce rotavirus shedding, possibly through the production of T cell‐independentIgA to rotavirus, but full resolution requires α β T cells.

Defining T-Cell-Mediated Immune Responses in Rotavirus-Infected Juvenile Rhesus Macaques

ABSTRACT The appearance of virus-specific CD4+ and/or CD8+ T lymphocytes in peripheral blood of captive juvenile rhesus macaques (Macaca mulatta) was observed following rotavirus infection. These cell-mediated immune responses were measured following experimental or natural infection after rotavirus was isolated from stool specimens of asymptomatic animals. The virus isolated was a new strain of simian rotavirus that we named TUCH (for Tulane University and Cincinnati Childrens Hospital). Restimulation of peripheral T lymphocytes by inactivated double- or triple-layered TUCH rotavirus particles containing either VP6 or VP4 and VP7 on their respective surfaces resulted in increased quantities of interleukin-6 (IL-6) and IL-12 in cell culture supernatants. Recall responses to rotavirus by CD4+ and CD8+ T lymphocytes were associated with accumulation of intracellular IL-6 and gamma interferon. Antigen presentation of TUCH rotavirus to lymphocytes was mediated via differentiated cultures of monocyte-derived dendritic (HLA-DR+) cells. This is the first report demonstrating cell-mediated immune responses to rotavirus in nonhuman primates. Further exploration of rhesus macaques in vaccine trials with human rotavirus vaccine candidates is the major objective of future studies.