A POETIC Phase II study of continuous oral everolimus in recurrent, radiographically progressive pediatric low-grade glioma.

BACKGROUND: To evaluate efficacy, pharmacokinetics (PK) and pharmacodynamics of single-agent everolimus in pediatric patients with radiographically progressive low-grade glioma (LGG). METHODS: Everolimus was administered at 5 mg/m2 once daily as a tablet or liquid for a planned 48-week duration or until unacceptable toxicity or disease progression. Patients with neurofibromatosis type 1 were excluded. PK and pharmacodynamic endpoints were assessed in consenting patients. RESULTS: Twenty-three eligible patients (median age 9.2 years) were enrolled. All patients received prior chemotherapy (median number of prior regimens two) and/or radiotherapy (two patients). By week 48, two patients had a partial response, 10 stable disease, and 11 clinical or radiographic progression; two discontinued study prior to 1 year (toxicity: 1, physician determination: 1). With a median follow up of 1.8 years (range 0.2-6.7 years), the 2-, 3-, and 5-year progression-free survivals (PFS) were 39 ± 11%, 26 ± 11%, and 26 ± 11%, respectively; two patients died of disease. The 2-, 3-, and 5-year overall survival (OS) were all 93 ± 6%. Grade 1 and 2 toxicities predominated; two definitively related grade 3 toxicities (mucositis and neutropenia) occurred. Grade 4 elevation of liver enzymes was possibly related in one patient. Predose blood levels showed substantial variability between patients with 45.5% below and 18.2% above the target range of 5-15 ng/mL. Pharmacodynamic analysis demonstrated significant inhibition in phospho-S6, 4E-BP1, and modulation of c-Myc expression. CONCLUSION: Daily oral everolimus provides a well-tolerated, alternative treatment for multiple recurrent, radiographically progressive pediatric LGG. Based on these results, everolimus is being investigated further for this patient population.

Targeting Refractory Sarcomas and Malignant Peripheral Nerve Sheath Tumors in a Phase I/II Study of Sirolimus in Combination with Ganetespib (SARC023).

PURPOSE: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas. Combining Hsp90 inhibitors to enhance endoplasmic reticulum stress with mTOR inhibition results in dramatic MPNST shrinkage in a genetically engineered MPNST mouse model. Ganetespib is an injectable potent small molecule inhibitor of Hsp90. Sirolimus is an oral mTOR inhibitor. We sought to determine the safety, tolerability, and recommended dose of ganetespib and sirolimus in patients with refractory sarcomas and assess clinical benefits in patients with unresectable/refractory MPNSTs. Patients and Methods. In this multi-institutional, open-label, phase 1/2 study of ganetespib and sirolimus, patients ≥16 years with histologically confirmed refractory sarcoma (phase 1) or MPNST (phase 2) were eligible. A conventional 3 + 3 dose escalation design was used for phase 1. Pharmacokinetic and pharmacodynamic measures were evaluated. Primary objectives of phase 2 were to determine the clinical benefit rate (CBR) of this combination in MPNSTs. Patient-reported outcomes assessed pain. RESULTS: Twenty patients were enrolled (10 per phase). Toxicities were manageable; most frequent non-DLTs were diarrhea, elevated liver transaminases, and fatigue. The recommended dose of ganetespib was 200 mg/m2 intravenously on days 1, 8, and 15 with sirolimus 4 mg orally once daily with day 1 loading dose of 12 mg. In phase 1, one patient with leiomyosarcoma achieved a sustained partial response. In phase 2, no responses were observed. The median number of cycles treated was 2 (1-4). Patients did not meet the criteria for clinical benefit as defined per protocol. Pain ratings decreased or were stable. CONCLUSION: Despite promising preclinical rationale and tolerability of the combination therapy, no responses were observed, and the study did not meet parameters for further evaluation in MPNSTs. This trial was registered with (NCT02008877).

Unusual Cause of Spontaneous Bilateral Pneumothorax Secondary to Osteosarcoma Metastasis.

Spontaneous pneumothorax is an emergency situation and early diagnosis and management is very important. Pneumothorax is rarely the first manifestation of lung metastasis. Spontaneous pneumothoraxes have been reported in some osteosarcoma patients. We hereby report a case of a bilateral spontaneous pneumothorax, revealing metastasis of an osteosarcoma.

Role of natriuretic peptides in cGMP production in fetal cardiac bypass.

BACKGROUND: We previously showed cyclic guanosine 3',5'-monophosphate (cGMP) levels increase with fetal cardiac bypass despite derangements in the placental nitric oxide pathway. The natriuretic peptides, atrial (ANP), brain (BNP), and c-type (CNP), are common indicators of cardiac distress, and an alternative pathway for cGMP generation. We hypothesized that these natriuretic peptides may account for the paradoxic rise in cGMP seen with fetal bypass. METHODS: Six ovine fetuses, 106 to 118 days' gestation, underwent cardiac bypass for 30 minutes and were followed for 120 minutes after bypass. Fetal plasma samples were collected before bypass, during bypass, and 30 and 120 minutes after bypass for natriuretic peptide analysis. Results were compared with 6 sham bypass fetuses and cGMP values from another 14 bypass fetuses (to avoid confounding effects of excess blood sampling). Fetal hemodynamics and metabolics were correlated to ANP, BNP, and CNP values. Statistical analysis was by analysis of variance, Student's t test, and best-fit correlations, with significance set at p = 0.05 or less. RESULTS: The ANP, BNP, and CNP increased with fetal bypass (674 +/- 133 pg/mL, 151 +/- 52 pg/mL, and 295 +/- 45 pg/mL, respectively), remaining elevated after bypass, whereas sham concentrations remained stable at pre-bypass levels. Changes in ANP, BNP, and CNP positively correlated with rising cGMP. There was positive correlation between ANP and CNP and rising fetal lactate levels, but not to other physiologic parameters associated with placental dysfunction. CONCLUSIONS: There is a substantial rise in natriuretic peptides seen with fetal bypass, likely in part a reflection of myocardial dysfunction. Further, the natriuretic peptide pathway may account for the paradoxic rise in cGMP seen with fetal bypass.

Rabeto plus: a valuable drug for managing functional dyspepsia.

The aim of the study was to evaluate and document the efficacy and tolerability of rabeto plus (FDC of rabeprazole and itopride) in management of functional dyspepsia. It was an open, prospective, non-comparative, multidose study. The patients with functional dyspepsia (NERD or non-erosive reflux disease) attending OPD of a leading, tertiary care, teaching hospital in West Bengal (BS Medical College, Bankura) were inducted in the study. A total of 46 adult patients of either sex with functional dyspepsia and a clinical diagnosis of NERD were given 1 capsule of rabeto plus before breakfast, for up to 4 weeks. Primary efficacy variables were relief from symptoms of heartburn, nausea, vomiting, waterbrash and fullness. Secondary efficacy variables were global assessment of efficacy and toleration by patients and treating physicians. The tolerability was assessed on the basis of record of spontaneously reported adverse events with their nature, intensity and outcome. Out of 55 patients enrolled in the study, 46 completed the study as planned, while 9 patients were lost to follow-up (dropped). Most patients reported near total symptom relief by the end of study. Total symptom score showed remarkable and significant improvement from baseline to end of the study. Importantly, none of the patients reported any side-effect. All participants tolerated the drug well. Moreover, response to study drug was rated as excellent or good by over 93% patients and their treating physicians. This means that 9 out 10 patients receiving rabeto plus reported desired symptom relief from dyspepsia. Thus it was concluded that rabeto plus is a valuable drug for treatment of functional dyspepsia or NERD.

IFN-gamma is the only anti-rotavirus cytokine found after in vitro stimulation of memory CD4+ T cells from mice immunized with a chimeric VP6 protein.

CD4+ T cells are the only lymphocytes required for protection of mice against rotavirus shedding after mucosal immunization with chimeric VP6 (MBP::VP6) and the adjuvant LT(R192G). One possible effector of protection is CD4+ T-cell cytokines. To determine if memory CD4+ T cells of immunized mice produce cytokines with direct anti-rotavirus activity, an in vitro infection model was developed using mouse CMT-93 cells and rhesus rotavirus (RRV). Spleen and lamina propria (LP) cells, as well as purified splenic CD4T cells obtained after intranasal immunization of BALB/c mice with MBP::VP6/LT(R192G) released large quantities of two cytokines (IL-17 and IFN-gamma) into cell supernatants when stimulated with MBP::VP6. Production of these same cytokines is rapidly upregulated in intestinal lymphocytes after rotavirus inoculation of immunized mice. IL-17 pretreatment of CMT-93 cells had no effect on subsequent RRV replication, but IFN-gamma was the most potent inhibitor within a panel of nine cytokines tested. Supernatants obtained after in vitro stimulation of splenic CD4+ T cells of immunized mice had high levels of anti-RRV activity and their pretreatment with mAb against IFN-gamma caused essentially complete loss of activity. Thus, IFN-gamma was the only cytokine identified in stimulated CD4+ T cells from immunized mice that directly inhibited rotavirus replication.

Intrarectal immunization of mice with VP6 and either LT(R192G) or CTA1-DD as adjuvant protects against fecal rotavirus shedding after EDIM challenge.

Intranasal or oral delivery of the chimeric rotavirus VP6 protein MBP::VP6 to mice elicited >90% reductions in fecal rotavirus shedding after murine rotavirus challenge. Protection depended on co-administration of adjuvants, the most effective being bacterial toxins. Because of safety and efficacy concerns following intranasal or oral toxin delivery, protective efficacy of MBP::VP6 after intrarectal delivery with toxin adjuvants was determined and compared to that induced after intranasal and oral immunization. Adult BALB/c mice were orally challenged with the murine rotavirus strain EDIM 4 weeks after their second immunization with MBP::VP6 and either LT(R192G), an attenuated Escherichia coli heat-labile toxin, or CTA1-DD, a cholera toxin derivative. Reductions in fecal rotavirus shedding were then determined relative to mock-immunized mice. Immunization with MBP::VP6 and either adjuvant by any route (except oral immunization with CTA1-DD) significantly (P<0.0001) reduced rotavirus shedding. As was previously found after oral and intranasal immunization, intrarectal immunization with MBP::VP6 and adjuvant was associated with T cell responses (IFNgamma and IL-17) but not B cell (antibody) responses.

Identification of an immunodominant CD4+ T cell epitope in the VP6 protein of rotavirus following intranasal immunization of BALB/c mice.

The only lymphocytes required for protection against fecal rotavirus shedding after intranasal immunization of BALB/c (H-2(d)) mice with a chimeric rotavirus VP6 protein (MBPColon, two colonsVP6) and the mucosal adjuvant LT(R192G) are CD4(+) T cells. The purpose of this study was to identify CD4(+) T cell epitopes within VP6 that might be responsible for this protection. To make this determination, spleen cells obtained from BALB/c mice following intranasal immunization with MBPColon, two colonsVP6/LT(R192G) were stimulated in vitro with either MBPColon, two colonsVP6 or overlapping VP6 peptides containing

Protection of mice against rotavirus challenge following intradermal DNA immunization by Biojector needle-free injection.

Mucosal administration (intranasal or oral) of a VP6 rotavirus vaccine to mice consistently elicits high levels of protection after rotavirus challenge (93->99% reductions in fecal rotavirus shedding) but only when co-administered with an effective adjuvant such as LT(R192G). Here, we showed that Biojector needle-free injection of VP6-encoded plasmids also induced protection (85-93%) when they were co-administrated with LT(R192G)-encoded plasmids. A reduction in the amount of VP6 plasmid from 50 to 10 microg reduced protection from 93 to 70%, but the immunized mice remained significantly (P<0.05) protected. Intramuscular needle injection of VP6/LT(R192G)-plasmids also induced significant protection (66%).

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).

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-gamma) 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-gamma 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).

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.

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 >or= 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.

Protection against rotavirus shedding after intranasal immunization of mice with a chimeric VP6 protein does not require intestinal IgA.

Intranasal immunization of mice with chimeric VP6 and the adjuvant LT(R192G) consistently elicits >95% reductions in fecal rotavirus shedding following challenge. To determine the association between mucosal antibody and protection, we immunized BALB/c wt and J chain knockout (Jch-/-) mice with VP6 and either LT(R192G) or cholera toxin (CT). Both strains developed nearly equal levels of serum rotavirus IgG, but Jch-/- mice, which cannot transport dimeric IgA across epithelial cell surfaces, developed >4-fold higher levels of serum rotavirus IgA. Stool rotavirus IgA was present in wt but undetectable in Jch-/- mice. When challenged with rotavirus strain EDIM, reductions in rotavirus shedding were nearly identical in VP6-immunized wt and Jch-/- mice (i.e., 97% and 92%, respectively; P > 0.01). Th1 CD4 T cell responses were also detected in VP6-immunized animals based on high levels of IFN-gamma and IL-2 found after in vitro VP6 stimulation of spleen cells. Therefore, protection induced by intranasal immunization of mice with VP6 and adjuvant does not depend on intestinal rotavirus IgA antibody but appears to be associated with CD4 T cells.

Vanadium, a versatile biochemical effector in chemical rat mammary carcinogenesis.

Recent studies indicate the role of the micronutrient vanadium in chemoprevention in many animal models, human cancer cell lines, and also in xenografted human carcinomas of the lung, breast, and gastrointestinal tract. The present studies were undertaken to ascertain the antineoplastic potential of vanadium in a defined model of mammary carcinogenesis. Female Sprague-Dawley rats, at 50 days of age, were treated with 7,12-dimethylbenz(alpha) anthracene (DMBA) (0.5 mg/100 g body weight) by a tail vein injection in oil emulsion. Vanadium (ammonium metavanadate) at a concentration of 0.5 ppm (4.27 micromol/l) was supplemented in drinking water and given ad libitum to the experimental group after the carcinogen treatment, and it continued until the termination of the study. In vivo studies of DNA chain breaks demonstrated that vanadium offered significant (61%, P<0.005) protection against generation of single-strand breaks when compared with the DMBA control group. Supplementation of vanadium normalizes the level of zinc, iron, and copper as revealed by proton-induced X-ray emission analysis to a substantial extent (P<0.01). In vitro study of chromosomal aberrations (CAs) revealed that vanadium triggered a protective effect (62.9%) on induction of CAs, which was maximum on structural aberrations followed by numerical and physiological types. Histopathological and morphological analyses were done as end-point biomarkers. We conclude herein that vanadium has the potential to reduce genomic instability in mammary carcinoma in rats.

Induction of immune responses and partial protection in mice after skin immunization with rotavirus VP6 protein and the adjuvant LT(R192G).

Oral or intranasal administration of mice with rotavirus VP6/LT(R192G) vaccine induces between 95 and 99% protection against fecal shedding of rotavirus after challenge. However, mucosal administration of LT(R192G) is controversial. Subcutaneous, intradermal or Biojector injection induced high titers of serum VP6-specific IgG, eliciting only partial to no protection (73, 0 and 26%, respectively), while transcutaneous delivery using gauze pad induced both poor immune responses and no protection (13%). A mixture of VP6-derived synthetic peptides induced >97, 48 and 33% protection after intranasal, gauze pad or Biojector administration, respectively. For needle-free delivery methods to be viable, improvements to these methods must be made to enhance the efficacy of the VP6 vaccine.

Development of a rotavirus-shedding model in rhesus macaques, using a homologous wild-type rotavirus of a new P genotype.

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.

Intranasal administration of an Escherichia coli-expressed codon-optimized rotavirus VP6 protein induces protection in mice.

We are developing rotavirus vaccines based on the VP6 protein of the human G1P[8] [corrected] [J. Virol. 73 (1999) 7574] CJN strain of rotavirus. One prototype candidate consisting of MBP::VP6::His6, a chimeric protein of maltose-binding protein, VP6 and hexahistidine, was expressed mainly as truncated polypeptides in Escherichia coli BL21(DE3) cells. A possible reason for this extensive truncation is the high frequencies of rare bacterial codons within the rotavirus VP6 gene. Expression of truncated recombinant VP6 was found to be reduced, and expression of complete VP6 protein was simultaneously increased, when the protein was expressed in Rosetta(DE3)pLacI E. coli cells that contain increased amounts of transfer RNAs for a selection of rare codons. The same observation was made when a synthetic codon-optimized CJN-VP6 gene was expressed in E. coli BL21 or Rosetta cells. To increase protein recovery, recombinant E. coli cells were treated with 8M urea. Denatured, full-length MBP::VP6::His6 protein was then purified and used for intranasal vaccination of BALB/c mice (2 doses administered with E. coli heat-labile toxin LT(R192G) as adjuvant). Following oral challenge with the G3P[16] [corrected] [J. Virol. 76 (2002) 560] EDIM strain of murine rotavirus, protection levels against fecal rotavirus shedding were comparable (P>0.05) between groups of mice immunized with denatured codon-optimized or native (not codon-optimized) immunogen with values ranging from 87 to 99%. These protection levels were also comparable to those found after immunization with non-denatured CJN VP6. Thus, expression of complete rotavirus VP6 protein was greatly enhanced by codon optimization, and the protection elicited was not affected by denaturation of recombinant VP6.

Discovery of a new strain of murine rotavirus that is consistently shed in large quantities after oral inoculation of adult mice.

In 1990, we developed the adult mouse model for studies on active immunity against shedding of the EDIM strain of murine rotavirus. Low and inconsistent levels of EDIM shedding in some strains of adult mice, particularly those on C57BL/6 backgrounds, established the need for an alternative murine rotavirus strain for these studies. Fortuitously, such a rotavirus strain was obtained from mice housed within the conventional colony at Children's Hospital. This strain, named EMcN, was clearly distinguishable from EDIM based on electropherotype. Furthermore, sequence analyses of VP4 and VP7 genes of EMcN revealed non-identities in 5% of the amino acids of both proteins relative to EDIM but established EMcN as another G3P[16] strain of murine rotavirus. Subgroup analysis showed EMcN belonged to SG1 while EDIM was found to be non-SG1/SG2. Similarly, unlike EDIM, the EMcN strain was identified as serotype G3 based on neutralization by hyperimmune antisera developed against prototype human and simian G3 rotavirus strains. Although EDIM produced more days of diarrhea and was shed in greater quantities in neonatal BALB/c mice, EMcN was shed in much greater quantities in adult BALB/c mice. More importantly, in contrast to the EDIM strain, EMcN was shown to be consistently shed in large quantities in adult C57BL/6 mice and ko mice on this background. Therefore, it is recommended that the EMcN strain be used for future challenge studies with mice on this background.

Functional mapping of protective epitopes within the rotavirus VP6 protein in mice belonging to different haplotypes.

We recently used "functional mapping" to locate protective epitopes in the carboxyl terminus (aa 197-397) of the VP6 protein (designated CD) of the EDIM strain of murine rotavirus [J. Virol. 74 (2000) 11574]. For this, H-2(d) BALB/c mice were given two intranasal (i.n.) immunizations (separated by 2 weeks) with VP6 or CD genetically-fused to maltose-binding protein, or with overlapping synthetic CD peptides, along with LT(R192G), a genetically-attenuated E. coli heat-labile toxin. The protective efficacies, i.e., percentage reductions in rotavirus shedding relative to control mice during 7 days following oral challenge with EDIM, were determined 4 weeks after the second immunization. Five of the 11 overlapping CD peptides stimulated significant protection (57-85%, P<0.05). Furthermore, chimeric VP6, the CD fragment, and a 14-amino-acid VP6 peptide within CD (RLSFQLMRPPNMTP), identified as a H-2(d)-restricted CD4 T cell epitope, were highly protective (93-98%, P<0.05). In this study, we continued to utilize functional mapping to show that the 14-mer peptide elicited significant protection (97.0%, P<0.05) in another H-2(d) mouse strain (DBA/2) but partial protection in H-2(b) 129 (39.2%) and C57Bl/6 (53.6%) as well as H-2(k) C3H (44.6%) mice. The first 13 amino acids of this 14-mer were necessary to induce maximal protection in H-2(d) mice. In addition, the H-2(b) 129 mice were immunized intranasally (i.n.) with 10 of the synthetic CD peptides and 5 were found to induce significant protection (90-97%, P<0.05). We also performed functional mapping to identify MHC class I epitopes in rotavirus proteins. A class I-binding epitope for H-2(b) C57Bl/6 mice had previously been mapped by ex vivo CTL assays within the VP6 protein and two additional class I epitopes were identified by computer-based prediction. When examined for their protective efficacies by functional mapping, two of the three were found to be partially but not significantly protective (44 and 46%, P>0.05). To better determine the usefulness of our in vivo methods to identify MHC class I-binding epitopes, four epitopes from the outer capsid VP7 rotavirus protein determined in ex vivo assays were evaluated for their protective efficacies and two were found to be partially protective. Together, these studies show that functional mapping is useful in locating epitopes that are relevant to the development of subunit rotavirus vaccines.

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.

The level of protection against rotavirus shedding in mice following immunization with a chimeric VP6 protein is dependent on the route and the coadministered adjuvant.

Intranasal (i.n.) immunization of BALB/c mice with chimeric murine rotavirus EDIM (epizootic diarrhea of infant mice) VP6 and attenuated E. coli heat-labile toxin (LT), LT(R192G), stimulated >99% protection against rotavirus shedding after EDIM challenge. Here, we evaluated other potential adjuvants with chimeric VP6 administered by two mucosal routes: i.n. and oral. Besides LT(R192G), the adjuvants examined included Adjumer, CpG oligodeoxynucleotides (CpG ODN), chimeric A1 subunit of cholera toxin (CTA1)-DD, and QS-21. All except QS-21 significantly (P<0.05) increased VP6-specific serum IgG responses after i.n. immunization, but none significantly increased these responses when administered orally. The i.n. delivery of chimeric VP6 alone induced both rotavirus IgG1 and IgG2a whose relative titers suggested a skewed Th2-like response. Inclusion of Adjumer greatly increased Th2-like responses, while CpG ODN shifted the response to a less Th2-like response. The adjuvants CTA1-DD, LT(R192G), QS-21 had no significant effect on ratios of IgG1/IgG2a titers. Following EDIM challenge of mice immunized i.n. with chimeric VP6 and either LT(R192G), CTA1-DD, Adjumer or CpG ODN, shedding was reduced >99, 95, 80, 74, respectively, relative to that found in unimmunized mice (P<0.05). QS-21 induced less protection (43%, not significant (N.S.)) while immunization with chimeric VP6 alone reduced shedding by only 16% (N.S.). Oral immunization with chimeric VP6 and all selected adjuvants except QS-21 was less effective than after i.n. immunization, with protection levels of 94 (P<0.05), 71 (P<0.05), 55, 35 and 28% for LT(R192G), QS-21, CpG ODN, CTA1-DD, and Adjumer, respectively, while immunization with chimeric VP6 alone gave no protection. Thus, different adjuvants induced different degrees of protection and oral immunization was generally less effective then the i.n. route.