Rotavirus-specific Serum Research Articles

Rotavirus-Specific Maternal Serum Antibodies and Vaccine Responses to RV3-BB Rotavirus Vaccine Administered in a Neonatal or Infant Schedule in Malawi.

High titres of rotavirus-specific maternal antibodies may contribute to lower rotavirus vaccine efficacy in low- and middle-income countries (LMICs). RV3-BB vaccine (G3P[6]) is based on a neonatal rotavirus strain that replicates well in the newborn gut in the presence of breast milk. This study investigated the association between maternal serum antibodies and vaccine response in infants administered the RV3-BB vaccine. Serum was collected antenatally from mothers of 561 infants enrolled in the RV3-BB Phase II study conducted in Blantyre, Malawi, and analysed for rotavirus-specific serum IgA and IgG antibodies using enzyme-linked immunosorbent assay. Infant vaccine take was defined as cumulative IgA seroconversion (≥3 fold increase) and/or stool vaccine shedding. Maternal IgA or IgG antibody titres did not have a negative impact on vaccine-like stool shedding at any timepoint. Maternal IgG (but not IgA) titres were associated with reduced take post dose 1 (p < 0.005) and 3 (p < 0.05) in the neonatal vaccine schedule group but not at study completion (week 18). In LMICs where high maternal antibodies are associated with low rotavirus vaccine efficacy, RV3-BB in a neonatal or infant vaccine schedule has the potential to provide protection against severe rotavirus disease.

Safety and immunogenicity of the Rotavac and Rotasiil rotavirus vaccines administered in an interchangeable dosing schedule among healthy Indian infants: a multicentre, open-label, randomised, controlled, phase 4, non-inferiority trial

BackgroundRotavirus is the leading cause of severe dehydrating gastroenteritis among children younger than 5 years in low-income and middle-income countries. Two vaccines—Rotavac and Rotasiil—are used in routine immunisation in India. The safety and immunogenicity of these vaccines administered in a mixed regimen is not documented. We therefore aimed to compare the safety and seroresponse of recipients of a mixed regimen versus a single regimen.MethodsWe did a multicentre, open-label, randomised, controlled, phase 4, non-inferiority trial at two sites in India. We recruited healthy infants aged 6–8 weeks. Infants with systemic disorders, weight-for-height Z scores of less than minus three SDs, or a history of persistent diarrhoea were excluded. Eligible infants were randomly allocated to six groups in equal numbers to receive either the single vaccine regimen (ie, Rotavac–Rotavac–Rotavac [group 1] or Rotasiil–Rotasiil–Rotasiil [group 2]) or the mixed vaccine regimen (ie, Rotavac–Rotasiil–Rotavac [group 3], Rotasiil–Rotavac–Rotasiil [group 4], Rotavac–Rotasiil–Rotasiil [group 5], or Rotasiil–Rotavac–Rotavac [group 6]). Randomisation was done using an online software by site in blocks of at least 12. The primary outcome was seroresponse to rotavirus vaccine, measured using rotavirus-specific serum IgA antibodies 4 weeks after the third dose. The seroresponse rates were compared between recipients of the four mixed vaccine regimens (consisting of various combinations of Rotavac and Rotasiil) with recipients of the single vaccine regimens (consisting of Rotavac or Rotasiil only for all three doses). The non-inferiority margin was set at 10%. Safety follow-ups were done for the duration of study participation. This trial was registered with the Clinical Trials Registry India, number CTRI/2018/08/015317.FindingsBetween March 25, 2019, and Jan 15, 2020, a total of 1979 eligible infants were randomly assigned to receive a single vaccine regimen (n=659; 329 in group 1 and 330 in group 2) or a mixed vaccine regimen (n=1320; 329 each in groups 3 and 4, and 331 each in groups 5 and 6). All eligible participants received the first dose, 1925 (97·3%) of 1979 received the second dose, and 1894 (95·7%) received all three doses of vaccine. 1852 (93·6%) of 1979 participants completed the follow-up. The immunogenicity analysis consisted of 1839 infants (1238 [67·3%] in the mixed vaccine regimen and 601 [32·7%] in the single vaccine regimen; 13 samples were insufficient in quantity) who completed vaccination and provided post-vaccination sera. The seroresponse rate in the mixed vaccine regimen group (33·5% [95% CI 30·9–36·2]) was non-inferior compared with the single vaccine regimen group (29·6% [26·1–33·4]); the seroresponse rate difference was 3·9% (95% CI −0·7 to 8·3). The proportion of participants with any type of solicited adverse events was 90·9% (95% CI 88·4–93·0) in the single vaccine regimen group and 91·1% (89·5–92·6) in the mixed vaccine regimen group. No vaccine-related serious adverse events or intussusception were reported during the study.InterpretationRotavac and Rotasiil can be safely used in an interchangeable manner for routine immunisation since the seroresponse was non-inferior in the mixed vaccine regimen compared with the single vaccine regimen. These results allow for flexibility in administering the vaccines, helping to overcome vaccine shortages and supply chain issues, and targeting migrant populations easily.FundingMinistry of Health and Family Welfare, Government of India.TranslationFor the Hindi translation of the abstract see Supplementary Materials section.

Rotavirus specific maternal antibodies and immune response to RV3-BB rotavirus vaccine in central java and yogyakarta, Indonesia

BackgroundPlacental or breast milk maternal antibodies can potentially reduce oral rotavirus vaccine efficacy in developing countries. We aimed to examine the relationship between the level of rotavirus specific immunoglobulin A (IgA) and neutralising antibodies (NA) in colostrum and breast milk and cord IgG, with cumulative vaccine take following one and three doses of oral RV3-BB rotavirus vaccine within a Phase IIb trial in Indonesia. Methods196 infants received three doses of RV3-BB in a randomized, double-blinded trial, using a neonatal schedule (first dose at 0–5 days of age, n = 61), an infant schedule (first dose at ~ 8 weeks of age, n = 67) or placebo (n = 68). Rotavirus specific IgA and NA in colostrum and breast milk, rotavirus specific cord IgG, Serum IgA and stool excretion were measured. ResultsThere was little evidence of an association between IgA in colostrum or breast milk and cumulative vaccine take after three doses in the neonatal or infant groups. In the neonatal group, there was a negative association between IgG titre in cord blood and cumulative vaccine take (odds ratio [OR] 0.96; 95% confidence interval [CI] 0.92–1.00; p = 0.03) and serum IgA response (OR 0.94; 95%CI 0.89–0.99; p = 0.02) after one dose of vaccine, which were not evident after three doses in the neonatal or infant groups. ConclusionsAmongst Indonesian infants we did not find an association between IgA in colostrum or breast milk and vaccine take after 3 doses of RV3-BB vaccine. Maternal rotavirus antibodies in breast milk appear to have minimal impact on RV3-BB vaccine take when administered with a short delay in breast-feeding in settings with a high rotavirus disease burden.

Inactivated rotavirus vaccine by parenteral administration induces mucosal immunity in mice

To improve the safety and efficacy of oral rotavirus vaccines, we developed an inactivated rotavirus vaccine (IRV) for parenteral administration. Since it remains unknown whether parenteral vaccination can induce mucosal immunity, we performed a comprehensive assessment of immune responses to IRV in mice with an adjuvant-free dissolving polymer MN patch or by alum-adjuvanted IM injection. We demonstrated that IRV induced the expression of the gut homing receptor LPAM-1 on T and B cells in spleen and mLN of vaccinated mice. MN patch IRV vaccination induced a slight Th1 phenotype while IM vaccination induced a balanced Th1/Th2 phenotype. In addition, a dose-sparing effect was seen for rotavirus-specific serum IgG and neutralizing activity for both vaccination routes. Our study is the first to show that parenterally administered IRV can induce mucosal immunity in the gut, in addition to strong serum antibody response, and is a promising candidate vaccine in achieving global immunization against rotavirus.

Enhancement of VP6 immunogenicity and protective efficacy against rotavirus by VP2 in a genetic immunization

VP2/VP6 virus like particles (VLPs) are very effective in inducing protection against the rotavirus infection in animal models. Individually, VP6 can also induce protection. However, there is no information about the immunogenicity of VP2. The aim of this work was to evaluate the efficacy of DNA vaccines codifying for VP2 or VP6, alone or combined, to induce protection against the rotavirus infection. Murine rotavirus VP2 and VP6 genes were cloned into the pcDNA3 vector. Adult BALB/c mice were inoculated three times by intramuscular (i.m.) injections with 100 or 200µg of pcDNA3-VP2 or pcDNA3-VP6 alone or co-administered with 100µg of pcDNA3-VP2/100µg of pcDNA3-VP6. Two weeks after the last inoculation, mice were challenged with the wild type murine rotavirus strain epizootic diarrhea of infant mice (EDIMwt). We found that both plasmids, pcDNA3-VP2 and pcDNA3-VP6, were able to induce rotavirus-specific serum antibodies, but not intestinal rotavirus-specific IgA; only 200µg of pcDNA3-VP6 induced 35% protection against the infection. A similar level of protection was found when mice were co-administered with 100µg of pcDNA3-VP2/100µg of pcDNA3-VP6 (1:1 ratio). However, the best protection (up to 58%) occurred when mice were inoculated with 10µg of pcDNA3-VP2/100µg of pcDNA3-VP6 (1:10 ratio). These results indicate that the DNA plasmid expressing VP6 is a better vaccine candidate that the one expressing VP2. However, when co-expressed, VP2 potentiates the immunogenicity and protective efficacy of VP6.

Rotavirus specific maternal antibodies and immune response to RV3-BB neonatal rotavirus vaccine in New Zealand

ABSTRACTBackground: Maternal antibodies, acquired passively via placenta and/or breast milk, may contribute to the reduced efficacy of oral rotavirus vaccines observed in children in developing countries. This study aimed to investigate the effect of rotavirus specific maternal antibodies on the serum IgA response or stool excretion of vaccine virus after any dose of an oral rotavirus vaccine, RV3-BB, in parallel to a Phase IIa clinical trial conducted at Dunedin Hospital, New Zealand. At the time of the study rotavirus vaccines had not been introduced in New Zealand and the burden of rotavirus disease was evident.Methods: Rotavirus specific IgG and serum neutralizing antibody (SNA) levels in cord blood and IgA levels in colostrum and breast milk samples collected ∼4 weeks, ∼20 weeks and ∼28 weeks after birth were measured. Infants were randomized to receive the first dose of vaccine at 0–5 d (neonatal schedule) or 8 weeks (infant schedule). Breast feeding was with-held for 30 minutes before and after vaccine administration. The relationship between rotavirus specific IgG and SNA levels in cord blood and IgA in colostrum and breast milk at the time of first active dose of RV3-BB vaccine and level of IgA response and stool excretion after 3 doses of vaccine was assessed using linear and logistic regression.Results: Forty infants received 3 doses of RV3-BB rotavirus vaccine and were included in the analysis of the neonatal and infant groups. Rotavirus specific IgA in colostrum (neonatal schedule group) and breast milk at 4 weeks (infant schedule group) was identified in 14/21 (67%) and 14/17 (82%) of infants respectively. There was little evidence of an association between IgA in colostrum or breast milk IgA at 4 weeks, or between cord IgG or SNA level, and IgA response or stool excretion after 3 doses of RV3-BB, or after one dose (neonatal schedule) (all p>0.05).Conclusions: The level of IgA in colostrum or breast milk and level of placental IgG and SNA did not impact on the serum IgA response or stool excretion following 3 doses of RV3-BB Rotavirus Vaccine administered using either a neonatal or infant schedule in New Zealand infants.

Association of elevated rotavirus-specific antibody titers with HBGA secretor status in Swedish individuals: The FUT2 gene as a putative susceptibility determinant for infection

The histo-blood group antigens (HBGAs) have recently been suggested to serve as attachment factors for rotavirus VP8* (P-genotype) in vitro and associated with susceptibility in vivo. We thus investigated whether rotavirus antibody titers and genotype specific neutralization titers correlate with HBGA status in Swedish individuals. We investigated the effect of inactivating mutations in the secretor FUT2 (rs601338) and Lewis FUT3 genes (rs28362459, rs3894326, rs812936 and rs778986) on serum IgG antibody titers and neutralizing antibody titers to rotavirus strains of the P[8] and P[6] genotypes in Swedish healthy blood donors and patients with IgA deficiency using genotyping, enzyme linked immunosorbent assay and a neutralization assay. Rotavirus-specific serum IgG and neutralizing antibody titers to the Wa strain (G1P[8]), but not to the ST3 (G4P[6]) strain, were significantly higher in secretors (with at least one functional FUT2 gene) than in non-secretors (P<0.001) (with homozygous nonsense mutation in the FUT2 gene). Thus, our results represent that secretors show elevated rotavirus specific serum antibodies, suggesting a higher susceptibility to rotavirus infections, as compared to non-secretors in Sweden.

Lactobacillus rhamnosus GG Dosage Affects the Adjuvanticity and Protection Against Rotavirus Diarrhea in Gnotobiotic Pigs.

The use of immunostimulatory strains of probiotics as adjuvants has been increasingly recognized as a promising approach in enhancing vaccine immunogenicity; however, dose effects of probiotic adjuvants are not well defined. In the present study, we examined dose effects of a commonly used probiotic strain, Lactobacillus rhamnosus GG (LGG), on immunomodulation with 2 different dosages. Neonatal gnotobiotic pigs were inoculated with 2 oral doses of attenuated human rotavirus (AttHRV) vaccines and fed with 5 doses (LGG5X; total 2.1 × 10(6) colony-forming units) or 9 doses (LGG9X; total 3.2 × 10(6) colony-forming units) of LGG, starting at 3 days of age. Both LGG feeding regimens enhanced the protection rate of AttHRV vaccine against diarrhea on virulent human rotavirus challenge. LGG5X, but not LGG9X, significantly enhanced rotavirus-specific intestinal memory B-cell responses to AttHRV; LGG5X also significantly enhanced virus-specific intestinal immunoglobulin A (IgA) antibody-secreting cell responses. Both regimens significantly enhanced rotavirus-specific serum IgA antibody responses to AttHRV. They also enhanced rotavirus-specific interferon-γ-producing effector/memory T-cell responses to AttHRV vaccine, with LGG9X being more effective than LGG5X, and both regimens downregulated CD4+CD25-FoxP3+ regulatory T (Treg) cell responses in most lymphoid tissues examined prechallenge and postchallenge and maintained the CD4+CD25+FoxP3+ Treg population in the ileum and intraepithelial lymphocyte postchallenge. LGG9X, however, did not significantly reduce total CD4+CD25-FoxP3+ Treg frequencies in the intestine and transforming growth factor-β-producing and interleukin (IL)-10-producing Treg frequencies in the blood. These results indicate that LGG at both dosages functioned as effective probiotic adjuvant for AttHRV vaccine, but different dosages differentially modulated immune responses to favor either the mucosal IgA response (LGG5X) or the T-cell response (LGG9X).

Human and bovine rotavirus strain antigens for evaluation of immunogenicity in a randomized, double-blind, placebo-controlled trial of a single dose live attenuated tetravalent, bovine-human-reassortant, oral rotavirus vaccine in Indian adults

A single dose of live attenuated tetravalent (G1–G4) bovine human reassortant rotavirus vaccine (BRV-TV) was administered to healthy Indian adult volunteers, who were assessed for safety and immunogenicity of the vaccine with 3:1 randomization to vaccine or placebo. All 20 adult male volunteers in the study had rotavirus specific serum IgA at baseline. There were no side effects or adverse events reported. Administration of BRV-TV was not associated with fever, diarrhea, or altered liver transaminases. Rotavirus IgA seroconversion post single dose administration was 27%. This study shows that BRV-TV is non-reactogenic, safe and immunogenic in adults. The IgA units estimated for the same sample using human G1P[8] rotavirus strain as the antigen were consistently higher than with the bovine G6P[5] WC3 strain and the human G2P[4] DS-1 strain antigen. The use of different human and bovine rotavirus strains as antigens in a quantitative rotavirus specific serum IgA assay resulted in different estimations of IgA antibody in the same sample.

Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses

B cell-dependent immunity to rotavirus, an important intestinal pathogen, plays a significant role in viral clearance and protects against reinfection. Human in vitro and murine in vivo models of rotavirus infection were used to delineate the role of primary plasmacytoid DCs (pDCs) in initiating B cell responses. Human pDCs were necessary and sufficient for B cell activation induced by rotavirus. Type I IFN recognition by B cells was essential for rotavirus-mediated B cell activation in vitro and murine pDCs and IFN-α/β-mediated B cell activation after in vivo intestinal rotavirus infection. Furthermore, rotavirus-specific serum and mucosal antibody responses were defective in mice lacking functional pDCs at the time of infection. These data demonstrate that optimal B cell activation and virus-specific antibody secretion following mucosal infection were a direct result of pDC-derived type I IFN. Importantly, viral shedding significantly increased in pDC-deficient mice, suggesting that pDC-dependent antibody production influences viral clearance. Thus, mucosal pDCs critically influence the course of rotavirus infection through rotavirus recognition and subsequent IFN production and display powerful adjuvant properties to initiate and enhance humoral immunity.

Rotavirus acceleration of murine type 1 diabetes is associated with a T helper 1-dependent specific serum antibody response and virus effects in regional lymph nodes

Rotavirus infection in at-risk children correlates with production of serum autoantibodies indicative of type 1 diabetes progression. Oral infection with rhesus monkey rotavirus (RRV) accelerates diabetes onset in mice. This relates to their rotavirus-specific serum antibody titre and local pro-inflammatory cytokine induction without pancreatic infection. Our aim was to further investigate the roles of serum antibodies and viral extra-intestinal spread in diabetes acceleration by rotavirus. Rotavirus-specific serum antibody production was detected by ELISA in diabetes-prone mice given either inactivated or low-dose RRV, in relation to their diabetes development. Serum anti-rotavirus antibody titres and infectious virus in lymph nodes were measured in mice given RRV or porcine rotavirus CRW-8. In lymph node cells, rotavirus antigen presence and immune activation were determined by flow cytometry, in conjunction with cytokine mRNA levels. Acceleration of diabetes by RRV required virus replication, which correlated with antibody presence. CRW-8 induced similar specific total immunoglobulin and IgA titres to those induced by RRV, but did not accelerate diabetes. RRV alone elicited specific serum IgG antibodies with a T helper (Th)1 bias, spread to regional lymph nodes and activated antigen-presenting cells at these sites. RRV increased Th1-specific cytokine expression in pancreatic lymph nodes. Diabetes onset was more rapid in the RRV-infected mice with the greater Th1 bias. Acceleration of murine diabetes by rotavirus is virus strain-specific and associated with virus spread to regional lymph nodes, activation of antigen-presenting cells at these sites and induction of a Th1-dominated antibody and cytokine response.

Lymphotoxin alpha-deficient mice clear persistent rotavirus infection after local generation of mucosal IgA.

Rotavirus is a major cause of pediatric diarrheal illness worldwide. To explore the role of organized intestinal lymphoid tissues in infection by and immunity to rotavirus, lymphotoxin alpha-deficient (LTα(-/-)) mice that lack Peyer's patches and mesenteric lymph nodes were orally infected with murine rotavirus. Systemic rotavirus was cleared within 10 days in both LTα(-/-) and wild-type mice, and both strains developed early and sustained serum antirotavirus antibody responses. However, unlike wild-type mice, which resolved the intestinal infection within 10 days, LTα(-/-) mice shed fecal virus for approximately 50 days after inoculation. The resolution of fecal virus shedding occurred concurrently with induction of intestinal rotavirus-specific IgA in both mouse strains. Induction of intestinal rotavirus-specific IgA in LTα(-/-) mice correlated with the (late) appearance of IgA-producing plasma cells in the small intestine. This, together with the absence of rotavirus-specific serum IgA, implies that secretory rotavirus-specific IgA was produced locally. These findings indicate that serum IgG responses are insufficient and imply that local intestinal IgA responses are important for the clearance of rotavirus from intestinal tissues. Furthermore, they show that while LTα-dependent lymphoid tissues are important for the generation of IgA-producing B cells in the intestine, they are not absolutely required in the setting of rotavirus infection. Moreover, the induction of local IgA-producing B cell responses can occur late after infection and in an LTα-independent manner.

Immune Mediators of Rotavirus Antigenemia Clearance in Mice

The immunological mediators that clear rotavirus antigenemia or viremia remain undefined. Immunodeficient mice and antibody transfer were used to test whether lymphocytes or rotavirus-specific serum antibodies are essential for resolving antigenemia. Clearance of antigenemia required lymphocytes, but neither T nor B lymphocytes were absolutely required. Transfer of convalescent-phase or nonneutralizing rotavirus-specific serum antibodies to the systemic compartment of severe-combined-immunodeficient (SCID) mice temporarily suppressed the onset or level of chronic rotavirus antigenemia. Our findings provide the first report demonstrating that clearance of rotavirus antigenemia and possibly viremia are mediated by multiple effector lymphocyte subsets and serum antibodies.

Parenteral administration of RF 8-2/6/7 rotavirus-like particles in a one-dose regimen induce protective immunity in mice

Rotavirus virus-like particles (RV-VLPs) represent a novel strategy for development of a rotavirus subunit vaccine. In this study, RF 8-2/6/7-VLPs with rotavirus VP8 protein (amino acid 1–241 of VP4) fused to the amino terminal end of a truncated VP2, were evaluated for their immunogenic and protective properties. A single intramuscular dose of, either 2 or 20 μg, RF 8-2/6/7-VLPs alone or combined with a potent adjuvant poly[di(carboxylatophenoxy)]phosphazene] (PCPP) induced rotavirus-specific serum IgG and IgA, fecal IgG titers that were enhanced 5–90-fold by adjuvant. Passive protective immunity was achieved in offspring to dams vaccinated with 2 and 20 μg RV-VLPs in presence of adjuvant and 20 μg RV-VLP without adjuvant.

Rotavirus Antigenemia in Children Is Associated with Viremia

BackgroundAntigenemia is commonly detected in rotavirus-infected children. Although rotavirus RNA has been detected in serum, definitive proof of rotavirus viremia has not been shown. We aimed to analyze a defined patient population to determine if infectious virus could be detected in sera from children with rotavirus antigenemia.Methods and FindingsSerum samples obtained upon hospitalization from children with gastroenteritis (57 stool rotavirus-positive and 41 rotavirus-negative), children with diagnosed bronchiolitis of known (n = 58) or unknown (n = 17) viral etiology, children with noninfectious, nonchronic conditions (n = 17), and healthy adults (n = 28) were tested for rotavirus antigen by enzyme immunoassay (EIA). Results of serum antigen testing were assessed for association with clinical and immunological attributes of the children. Rotavirus antigenemia was detected in 90% (51/57) of children with rotavirus-positive stools, in 89% (8/9) of children without diarrhea but with rotavirus-positive stools, in 12% (2/17) of children with bronchiolitis of unknown etiology without gastroenteritis, and in 12% (5/41) of children with gastroenteritis but with rotavirus-negative stools. Antigenemia was not detected in sera from children with noninfectious nonchronic conditions, children with bronchiolitis of known etiology and no gastroenteritis, or healthy adults. Neither age nor timing of serum collection within eight days after onset of gastroenteritis significantly affected levels of antigenemia, and there was no correlation between antigenemia and viral genotype. However, there was a negative correlation between serum rotavirus antigen and acute rotavirus-specific serum IgA (r = −0.44, p = 0.025) and IgG (r = −0.40, p = 0.01) titers. We examined 11 antigen-positive and nine antigen-negative sera for infectious virus after three blind serial passages in HT-29 cells using immunofluorescence staining for rotavirus structural and nonstructural proteins. Infectious virus was detected in 11/11 (100%) sera from serum antigen-positive children and in two out of nine (22%) sera samples from antigen-negative children (p = 0.002).ConclusionsMost children infected with rotavirus are viremic. The presence of viremia is directly related to the detection of antigenemia and is independent of the presence of diarrhea. Antigenemia load is inversely related to the titer of antirotavirus antibody in the serum. The finding of infectious rotavirus in the blood suggests extraintestinal involvement in rotavirus pathogenesis; however, the impact of rotavirus viremia on clinical manifestations of infection is unknown.

Inhibitory Activities of Bovine Macromolecular Whey Proteins on Rotavirus Infections In Vitro and In Vivo

Rotavirus is a major cause of infantile viral gastroenteritis and can lead to severe and sometimes lethal dehydration. Previous studies have shown that breast-fed children are better protected against symptomatic infections, and that the milk fat globule protein lactadherin might be at least partly responsible for this effect. In vitro studies have shown that human lactadherin, in contrast to the bovine ortholog, could inhibit rotavirus infectivity, and that bovine MUC1 and a commercially available bovine macromolecular whey protein (MMWP) fraction proved to be effective. The present work describes the versatility of MMWP against the infection of 2 human intestinal cell lines (Caco-2 and FHs 74 Int) by 4 different rotavirus strains (Wa, RRV, YM, RF). Isolation of a protein fraction (CM3Q3) from MMWP that effectively inhibits rotavirus infectivity in vitro is documented. Purification was achieved by monitoring the rotaviral inhibitory activity in fractions obtained from 2 consecutive steps of ion-exchange chromatography. The major component of CM3Q3 was shown to be bovine IgG, and the attenuating capacity of this fraction is most properly linked to this component. The capacity of MMWP, MUC1, lactadherin, and the CM3Q3 fraction to inhibit the infectivity of the murine EMcN rotavirus strain was analyzed in adult BALB/c mice by using 2 different amounts of virus (10 and 100 times more than 50% the viral shedding doses). Only CM3Q3 was able to significantly affect the shedding of rotavirus in the stools of experimentally infected mice when the high viral dose was given. Detection of rotavirus-specific serum antibodies showed that the high dose infected all groups of mice. Experiments with the low dose of virus implied that all the tested milk proteins could affect the viral shedding in stools; in addition, use of MUC1, MMWP, and CM3Q3 prevented the appearance of serum viral antibodies. The advantages of using bovine immunoglobulins to induce passive immunity against rotavirus have been substantially investigated, although studies have mainly focused on the use of derivatives from immunized cows, especially colostrum. This report associates considerable activity against rotavirus infectivity with an ordinary whey product, suggesting that there might be alternatives to colostral-derived products.

Experimental infection of pigtailed macaques with a simian rotavirus, YK‐1

Experimental rotavirus infection was investigated in pigtailed macaques to study the infectivity, immunity, and pathogenesis of rotavirus. A challenge virus, YK-1, was administered intragastrically to four seronegative macaques (age: 11-16 months). Although none of the monkeys developed diarrhea, an active infection occurred with high titers of rotavirus antigen detected in stools 2-10 days after challenge. These animals developed rotavirus-specific antibody responses similar to those seen following primary exposure to rotavirus. YK-1 was then inoculated in four seropositive macaques (age: 14-16 months). All animals shed viral antigen in their stool, but the titers and duration were significantly less when compared to seronegative macaques. When rechallenged 28 days after initial YK-1 challenge, the macaques demonstrated significant protection against reinfection. All seropositive animals developed a rise in rotavirus-specific serum and fecal antibodies during YK-1 challenge and rechallenge. To independently assess the role of age and preexisting IgG titers to rotavirus, a 4-month-old seronegative and 6-month-old seropositive macaque were inoculated with YK-1. The seronegative macaque shed high titers of virus for 9 days, while the seropositive macaque shed only 3 days and in low titer. These data suggest that a primate model of rotavirus infection using the YK-1 strain may be useful in examining the immune response and protection from infection in pigtailed macaques and indicate that levels and duration of shedding may provide a good measure of protection from natural infection and from that induced by oral or parenteral vaccines.

Immune responses and protection obtained with rotavirus VP6 DNA vaccines given by intramuscular injection

Intramuscular (i.m.) injection of murine VP6 DNA vaccines raised high titers of rotavirus-specific serum IgG and IgA antibodies in BALB/c mice. A Th1-like antibody response was generated based on the ratio of serum IgG2a to IgG1 antibodies. Rotavirus-specific serum IgA but not fecal IgA was detected in mice prior to rotavirus challenge. Partial protection against rotavirus challenge was achieved as measured by reduction of rotavirus antigen shedding in feces. A similar level of protection was found with a bovine rotavirus VP6 DNA vaccine against a murine rotavirus challenge, suggesting that heterologous protection can be obtained by immunizing with VP6 DNA vaccines. We did not directly test for cytotoxic T lymphocyte (CTL) activity, but in vivo depletion of CD8+ T cells in mice immunized with a murine VP6 DNA vaccine did not significantly change the duration of virus shedding or the pattern of protection obtained. This finding suggested that CD8+ CTL activity was not essential for the partial protection we obtained by i.m. immunization of mice with VP6 DNA vaccines.

Protective Efficacy of Rotavirus 2/6-Virus-Like Particles Combined with CT-E29H, a Detoxified Cholera Toxin Adjuvant

Identifying a safe and efficacious mucosal adjuvant is crucial for the development of subunit vaccines against rotavirus and other mucosal pathogens. Moreover, recognition of determinants of protective immunity to rotavirus infection is essential to the design of the means to prevent or control this viral gastrointestinal disease. We have studied the kinetics of systemic and mucosal antibody responses elicited upon mucosal immunization of mice with rotavirus recombinant virus-like particles (rVLPs) alone or combined with a detoxified version of cholera toxin, CT-E29H. CT-E29H has been shown to maintain the adjuvant effect of parental cholera holotoxin. Both inbred BALB/c and outbred CD-1 mice were immunized with rotavirus VP2/6-rVLPs (2/6-VLPs) combined with CT-E29H, orally or intranasally (i.n.), and the comparative efficacy of different formulations was then determined. Rotavirus-specific serum and fecal IgA, IgM, and IgG antibodies were determined by enzyme-linked immunoadsorbent assay (ELISA) weekly (or every other week) following vaccination. Animals then were challenged with a murine rotavirus strain, EDIM. The degree to which vaccinated animals were protected from the wild-type rotavirus challenge was reflected in the levels of viral antigen shed in stools (percent reduction in antigen shedding, PRAS). BALB/c mice immunized by either route produced rotavirus-specific serum IgA, IgM and IgG, as well as fecal IgA and IgG, but not IgM; however, the intranasal immunization induced stronger systemic IgG and IgM responses than did oral immunization. Similar levels of prechallenge rotavirus-specific fecal and serum IgA were detected in both the orally and the i.n. immunized groups. Two immunizations with 2-6VLPs and CT-E29H were sufficient to protect BALB/c mice, regardless of the route of administration. PRAS was 99.6, 98.8, and 98.8% for oral, i.n. and the oral + i.n. groups, respectively; in contrast vaccination with 2/6-VLPs alone was not protective (PRAS = 39%), indicating the critical role of CT-E29H in inducing protective levels of immune responses. Two of four outbred CD-1 mice that were immunized orally with 2/6-VLPs-CT-E29H showed no humoral responses (PRAS, 65%), but four of four i.n. immunized CD-1 mice displayed humoral responses (PRAS, 97.9%). Serum anti-VP6 and VP2 antibodies were detected in all immunoresponsive mice. The combined results in two strains of mice indicate that CTE29H is an effective mucosal adjuvant capable of inducing protective immune responses and suggest that intranasal administration is the preferred route of immunization.

Immune Responses and Protection Obtained by Oral Immunization with Rotavirus VP4 and VP7 DNA Vaccines Encapsulated in Microparticles

Protective immune responses in mice were obtained after oral immunization with rotavirus DNA vaccines encapsulated in poly(lactide-co-glycolide) (PLG) microparticles. The DNA vaccines used encoded outer capsid proteins VP4 and VP7; proteins that are the basis for rotavirus serotyping and the generation of virus neutralizing antibodies. One dose of vaccine was given to BALB/c mice by oral gavage (75 μg DNA/mouse). Rotavirus-specific serum antibodies and intestinal IgA antibodies were detectable by 6 weeks postimmunization. After challenge with homologous murine rotavirus at 12 weeks postimmunization, fecal rotavirus antigen was reduced significantly in immunized mice compared with controls. Protective immunity also was generated by oral delivery of unencapsulated VP 7 DNA vaccine but to a lesser degree. These results demonstrate that the oral route is effective for generating protective immune responses with rotavirus DNA vaccines targeting neutralization antigens.