Rotavirus vaccination and infection induce VP6-specific IgA responses.

Rotavirus (RV) is the leading cause of severe gastroenteritis (GE) in young children, but RVGE has drastically been reduced with the introduction of live oral RV vaccines into childhood immunization program in many countries. Serum IgA antibody is a marker of clinical protection against severe RVGE after RV infection and vaccination. This study investigated VP6-specificity of anti-RV IgA antibody levels in Finnish children aged 6-23 months before and after introduction of RotaTeq® into national immunization program. Although RV inner capsid protein VP6 is considered as antigenic target in clinical and seroepidemiological studies, at present VP6 protein is not commonly employed as a primary ELISA-antigen. Thus, sera from 20 unvaccinated and 19 vaccinated children were examined in ELISA with recombinant VP6 (rVP6) protein, and the VP6-specific responses were compared to responses observed with human RV Wa and bovine RV WC3 cell culture antigens. Moreover, fecal antibodies were tested with rVP6 and Wa cell culture antigen. Equal levels of serum anti-RV IgA antibodies were detected by the three antigens. Fecal IgA titers against rVP6 and Wa antigen showed a correlation with the corresponding serum levels. The results suggest that the IgA response measured by virus-capture ELISA is mainly directed to VP6 protein, supporting the usage of rVP6 in detection of anti-RV IgA antibodies. Natural RV infections and vaccinations induced similar levels of serum VP6-specific IgA antibodies. Serum IgA antibodies after RotaTeq® vaccination showed sustained levels up to two years of age in line with long term protection. J. Med. Virol. 89:239-245, 2017. © 2016 Wiley Periodicals, Inc.

Rotavirus Recombinant VP6 Nanotubes Act as an Immunomodulator and Delivery Vehicle for Norovirus Virus-Like Particles.

We have recently shown that tubular form of rotavirus (RV) recombinant VP6 protein has an in vivo adjuvant effect on the immunogenicity of norovirus (NoV) virus-like particle (VLP) vaccine candidate. In here, we investigated in vitro effect of VP6 on antigen presenting cell (APC) activation and maturation and whether VP6 facilitates NoV VLP uptake by these APCs. Mouse macrophage cell line RAW 264.7 and dendritic cell line JAWSII were used as model APCs. Internalization of VP6, cell surface expression of CD40, CD80, CD86, and major histocompatibility class II molecules, and cytokine and chemokine production were analyzed. VP6 nanotubes were efficiently internalized by APCs. VP6 upregulated the expression of cell surface activation and maturation molecules and induced secretion of several proinflammatory cytokines and chemokines. The mechanism of VP6 action was shown to be partially dependent on lipid raft-mediated endocytic pathway as shown by methyl-ß-cyclodextrin inhibition on tumor necrosis factor α secretion. These findings add to the understanding of mechanism by which VP6 exerts its immunostimulatory and immunomodulatory actions and further support its use as a part of nonlive RV-NoV combination vaccine.

Simple and efficient ultrafiltration method for purification of rotavirus VP6 oligomeric proteins.

Bacterial endotoxins, DNA, live viruses, and viral proteins derived from bacterial and baculovirus (BV) expression vectors employed in recombinant protein production contaminate the final product. Density gradient centrifugation is commonly used to partially purify oligomeric proteins, but impurities from the expression system still remain. We describe a simple and rapid ultrafiltration method for final purification of rotavirus VP6 oligomeric nanotubes and nanospheres. Contamination originating from the BV vector used in VP6 production was undetectable. The method is highly efficient, fast and inexpensive and can be used for a small-scale laboratory purification of VP6 protein to replace technically demanding multi-step chromatographic procedures.

Rotavirus capsid VP6 protein acts as an adjuvant in vivo for norovirus virus-like particles in a combination vaccine.

Rotavirus (RV) and norovirus (NoV) are the 2 leading causes of acute viral gastroenteritis worldwide. We have developed a non-live NoV and RV vaccine candidate consisting of NoV virus-like particles (VLPs) and recombinant polymeric RV VP6 protein produced in baculovirus-insect cell expression system. Both components have been shown to induce strong potentially protective immune responses. As VP6 nanotubes are highly immunogenic, we investigated here a possible adjuvant effect of these structures on NoV-specific immune responses in vivo. BALB/c mice were immunized intramuscularly with a suboptimal dose (0.3 µg) of GII.4 or GI.3 VLPs either alone or in a combination with 10 µg dose of VP6 and induction of NoV-specific antibodies in sera of experimental animals were measured. Blocking assay using human saliva or synthetic histo-blood group antigens was employed to test NoV blocking antibodies. Suboptimal doses of the VLPs alone did not induce substantial anti-NoV antibodies. When co-administered with the VP6, considerable titers of not only type-specific but also cross-reactive IgG antibodies against NoV VLP genotypes not included in the vaccine composition were induced. Most importantly, NoV-specific blocking antibodies, a surrogate for neutralizing antibodies, were generated. Our results show that RV VP6 protein has an in vivo adjuvant effect on NoV-specific antibody responses and support the use of VP6 protein as a part of the NoV-RV combination vaccine, especially when addition of external adjuvants is not desirable.

Protection against live rotavirus challenge in mice induced by parenteral and mucosal delivery of VP6 subunit rotavirus vaccine.

Live oral rotavirus (RV) vaccines are part of routine childhood immunization but are associated with adverse effects, particularly intussusception. We have developed a non-live combined RV - norovirus (NoV) vaccine candidate consisting of human RV inner-capsid rVP6 protein and NoV virus-like particles. To determine the effect of delivery route on induction of VP6-specific protective immunity, BALB/c mice were administered a vaccine containing RV rVP6 intramuscularly, intranasally or a combination of both, and challenged with murine RV. At least 65 % protection against RV shedding was observed regardless of delivery route. The levels of post-challenge serum VP6-specific IgA titers correlated with protection.

Genotype considerations for virus-like particle-based bivalent norovirus vaccine composition.

Norovirus (NoV) genogroup I (GI) and GII are responsible for most human infections with NoV. Because of the high genetic variability of NoV, natural infection does not induce sufficient protective immunity to different genotypes or to variants of the same genotype and there is little or no cross-protection against different genogroups. NoV-derived virus-like particles (VLPs) are promising vaccine candidates that induce high levels of NoV-specific humoral and cellular immune responses. It is believed that a bivalent NoV vaccine consisting of a representative VLP from GI and GII is a minimum requirement for an effective vaccine. Here, we compared the abilities of monovalent immunizations with NoV GI.1-2001, GI.3-2002, GII.4-1999, and GII.4-2010 New Orleans VLPs to induce NoV type-specific and cross-reactive immune responses and protective blocking antibody responses in BALB/c mice. All of the VLPs induced comparable levels of type-specific serum IgG antibodies, as well as blocking antibodies to the VLPs used for immunization. However, the abilities of different VLP genotypes to induce cross-reactive IgG and cross-blocking antibodies varied remarkably. Our results confirm previous findings of a lack of cross-protective immune responses between GI and GII NoVs. These data support the rationale for including NoV GI.3 and GII.4-1999 VLPs in the bivalent vaccine formulation, which could be sufficient to induce protective immune responses across NoV genotypes in the two common genogroups in humans.

Commonly circulating human coronaviruses do not have a significant role in the etiology of gastrointestinal infections in hospitalized children.

BACKGROUND: Human coronaviruses (HCoVs) OC43, 229E, NL63 and HKU1 are common causes of respiratory infections. Over the years, it has been proposed that HCoVs play a possible role in gastrointestinal infections. OBJECTIVES: To assess the role of HCoVs in acute gastroenteritis (AGE) in children. STUDY DESIGN: Study was conducted at Tampere University Hospital over 2 years. Both stool and nasal swab samples were collected from 172 children with AGE, 545 with acute respiratory tract infection (ARTI) and 238 with symptoms of both. The samples were tested for HCoVs by RT-PCR. RESULTS: HCoVs were detected in 52 (5.4%) children: in 6.4% of those with AGE, 4.4% with ARTI and 7.1% with symptoms of both. HCoVs OC43, HKU1, 229E and NL63 were encountered in 13, 11, 13 and 15 cases, respectively. HCoVs were detected simultaneously in stool and nasal swab samples in 17 children, in nasal swabs alone in 33 children, and in the stools alone in two children. HCoVs were present in the stools of eight (4.7%) of the 172 children with AGE; in six of these cases, the nasal swab sample was also positive for the respective HCoV. Additionally, in six of the eight cases, the stool sample contained either rotavirus or calicivirus. CONCLUSIONS: HCoVs can be detected in the stools of children with AGE, but usually together with well-known gastroenteritis viruses, and concomitantly in the respiratory tract. It appears that commonly circulating HCoVs do not have a significant role in the AGE of children admitted to hospital.

Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo.

Rotavirus (RV) is a common cause of severe gastroenteritis (GE) in children worldwide. Live oral RV vaccines protect against severe RVGE, but the immune correlates of protection are not yet clearly defined. Inner capsid VP6 protein is a highly conserved, abundant, and immunogenic RV protein, and VP6-specific mucosal antibodies, especially IgA, have been implicated to protect against viral challenge in mice. In the present study systemic and mucosal IgG and IgA responses were induced by immunizing BALB/c mice intranasally with a combination of recombinant RV VP6 protein (subgroup II [SGII]) and norovirus (NoV) virus-like particles (VLPs) used in a candidate vaccine. Following immunization mice were challenged orally with murine RV strain EDIMwt (SG non-I-non-II, G3P10[16]). In order to determine neutralizing activity of fecal samples, sera, and vaginal washes (VW) against human Wa RV (SGII, G1P1A[8]) and rhesus RV (SGI, G3P5B[3]), the RV antigen production was measured with an ELISA-based antigen reduction neutralization assay. Only VWs of immunized mice inhibited replication of both RVs, indicating heterotypic protection of induced antibodies. IgA antibody depletion and blocking experiments using recombinant VP6 confirmed that neutralization was mediated by anti-VP6 IgA antibodies. Most importantly, after the RV challenge significant reduction in viral shedding was observed in feces of immunized mice. These results suggest a significant role for mucosal RV VP6-specific IgA for the inhibition of RV replication in vitro and in vivo. In addition, these results underline the importance of non-serotype-specific immunity induced by the conserved subgroup-specific RV antigen VP6 in clearance of RV infection.

Human bocaviruses are commonly found in stools of hospitalized children without causal association to acute gastroenteritis.

UNLABELLED: Human bocaviruses (HBoVs) may be grouped into respiratory (HBoV1) and enteric (HBoV2-4) types. We examined this association of HBoV types and clinical symptoms in 955 children who had acute gastroenteritis (AGE, n = 172), acute respiratory tract infection (ARTI, n = 545) or symptoms of both (n = 238). Both nasal swab and stool specimens were studied for such patients. HBoV1 DNA was detected in 6.2 % of patients with ARTI and 9.2 % of patients with symptoms of both ARTI and AGE, but in only 1.7 % of patients with AGE alone. In about one half of the cases, HBoV1 was detected concomitantly in nasal swab and stool samples. HBoV2 was found in stool samples of patients with AGE (5.8 %), ARTI (5.1 %) and symptoms of both (5.5 %) but only rarely in nasal swabs. HBoV3 was found in the stools, but not in nasal swabs, in 0.6, 1.1 and 0.8 % of patients with, respectively, AGE, ARTI and both. HBoV4 was not found. All but one HBoV-positive stool sample of AGE patients contained a known gastroenteritis virus (rotavirus, norovirus, sapovirus, astrovirus or enteric adenovirus) that was probably responsible for the symptoms of the respective case. Sera of 30 HBoV-positive patients were available, and IgM antibodies for HBoVs were found in ten cases and HBoV DNA in eight of these. CONCLUSIONS: HBoV2 and HBoV3 were more commonly found in stool than in nasal swab samples, but the findings could not be causally linked with AGE. HBoV1 was commonly found in stool samples during ARTI, with or without gastrointestinal symptoms.

Trivalent combination vaccine induces broad heterologous immune responses to norovirus and rotavirus in mice.

Rotavirus (RV) and norovirus (NoV) are the two major causes of viral gastroenteritis (GE) in children worldwide. We have developed an injectable vaccine design to prevent infection or GE induced with these enteric viruses. The trivalent combination vaccine consists of NoV capsid (VP1) derived virus-like particles (VLPs) of GI-3 and GII-4 representing the two major NoV genogroups and tubular RV recombinant VP6 (rVP6), the most conserved and abundant RV protein. Each component was produced in insect cells by a recombinant baculovirus expression system and combined in vitro. The vaccine components were administered intramuscularly to BALB/c mice either separately or in the trivalent combination. High levels of NoV and RV type specific serum IgGs with high avidity (>50%) as well as intestinal IgGs were detected in the immunized mice. Cross-reactive IgG antibodies were also elicited against heterologous NoV VLPs not used for immunization (GII-4 NO, GII-12 and GI-1 VLPs) and to different RVs from cell cultures. NoV-specific serum antibodies blocked binding of homologous and heterologous VLPs to the putative receptors, histo-blood group antigens, suggesting broad NoV neutralizing activity of the sera. Mucosal antibodies of mice immunized with the trivalent combination vaccine inhibited RV infection in vitro. In addition, cross-reactive T cell immune responses to NoV and RV-specific antigens were detected. All the responses were sustained for up to six months. No mutual inhibition of the components in the trivalent vaccine combination was observed. In conclusion, the NoV GI and GII VLPs combination induced broader cross-reactive and potentially neutralizing immune responses than either of the VLPs alone. Therefore, trivalent vaccine might induce protective immune responses to the vast majority of circulating NoV and RV genotypes.

Comparative immunogenicity in mice of rotavirus VP6 tubular structures and virus-like particles.

Rotavirus (RV) is the most important cause of severe gastroenteritis in children worldwide. Current live RV vaccines are efficacious but show lower efficacy in developing countries, as well as a low risk of intussusception. This has led to the development of parenteral non-live candidate vaccines against RV. RV capsid VP6 protein is highly conserved and the most abundant RV protein forming highly immunogenic oligomeric structures with multivalent antigen expression. Both recombinant VP6 (rVP6) or double-layered (dl) 2/6-virus-like particles (VLPs), might be considered as the simplest RV subunit vaccine candidates. Human rVP6 protein and dl2/6-VLPs were produced in Sf9 insect cells by baculovirus expression system. Formation of rVP6 tubules and VLPs were confirmed by electron microscopy. BALB/c mice were immunized intramuscularly, and immune responses were analyzed. Both rVP6 and dl2/6-VLPs induced a balanced Th1-type and Th2-type response and high levels of serum IgG antibodies with cross-reactivity against different RV strains (Wa, SC2, BrB, 69M, L26, WC3, and RRV). In addition, mucosal VP6-specific IgG and IgA antibodies were detected in feces and vaginal washes (VW) of immunized animals. Importantly, VWs of immunized mice inhibited RV Wa and RRV infection in vitro. Immunization with either protein preparation induced a similar level of VP6-specific, interferon-γ secreting CD4(+) T cells in response to different RVs or the 18-mer peptide (AA 242-259), a VP6-specific CD4(+) T cell epitope. RV rVP6 and dl2/6-VLPs induced equally strong humoral and cellular responses against RV in mice and therefore, may be considered as non-live vaccine candidates against RV.

Human bocavirus types 1, 2 and 3 in acute gastroenteritis of childhood.

AIM: Recently identified human bocavirus (HBoV) types 2 and 3 have been associated with acute gastroenteritis in children. We studied 878 stool specimens from children with acute gastroenteritis and 112 controls (43 children with unspecified fever, 33 with respiratory tract infection and 36 healthy children) for known HBoVs. The same specimens were previously studied for rotaviruses, noroviruses, sapoviruses, adenoviruses, coronaviruses and aichivirus. METHODS: HBoVs were detected by PCR and positive amplicons were sequenced to identify HBoV1, HBoV2, HBoV3 and HBoV4. RESULTS: HBoV of any type was found in 85 (9.7%) cases of acute gastroenteritis and in 6 (5.4%) controls. HBoV1 was detected in 49 (5.6%) cases and 2 (1.8%) controls, HBoV2 in 29 (3.3%) cases and 2 (1.8%) controls and HBoV3 in 8 (0.9%) cases and 2 (1.8%) controls. No HBoV4 was found. HBoV as a single infection was found in 16 (1.8%) cases and in 6 (5.4%) controls; in the remaining cases, a known gastroenteritis virus was also found. Among the single HBoV infections, HBoV2 was the most common type with 8 (50%) cases. CONCLUSION: HBoVs are rarely found alone in children with acute gastroenteritis. Further studies are warranted to confirm a possible specific association of HBoV2 with gastroenteritis.

Simultaneous presence of human herpesvirus 6 and adenovirus infections in intestinal intussusception of young children.

AIM: This prospective study investigated the role of viral infections in the pathogenesis of intussusception, including human herpesvirus 6 (HHV-6), a known lymphotropic virus. METHODS: Fifty-three children with intussusception treated in hospital were enroled, and children of comparable age and gender served as controls. Blood, stool and throat swab specimens, as well as mesenteric lymph nodes and pieces of intestine from patients requiring surgery were tested for various viruses by PCR methods. RESULTS: Altogether, 85% of intussusception cases showed evidence of a recent or ongoing viral infection. Among the 53 intussusception cases, adenovirus was detected in 25 (47%), HHV-6 in 24 (45%), rhinovirus in 12 (23%), cytomegalovirus in 7 (13%), enterovirus in 4 (8%) and rotavirus in 3 (6%) patients. Of the 50 whole blood samples, 44% were positive for HHV-6 and of the 16 resected mesenteric lymph nodes, 50% were positive for HHV-6. Simultaneous presence of HHV-6 and adenovirus infection correlated significantly with intussusception (OR 12.1, 95% CI 2.2 to 66.5). CONCLUSIONS: A statistically significant association was observed between adenovirus and childhood intussusception. HHV-6 was a common finding and occurred concomitantly with other viruses. A simultaneous infection with HHV-6 and adenovirus carried the highest risk for intussusception.

A comparison of immunogenicity of norovirus GII-4 virus-like particles and P-particles.

Norovirus (NoV) -derived virus-like particles (VLPs) resemble empty shells of the virus and NoV P-particles contain only protruding domains of the NoV capsid. Both NoV-derived subviral particles show similar functionality and antigenicity in vitro and are considered to be potential vaccine candidates against NoV gastroenteritis. BALB/c mice were immunized with baculovirus-produced GII-4 VLPs or the corresponding Escherichia coli-produced P-particles by the intramuscular or intradermal route and the NoV-specific antibody and T-cell immune responses were compared. Elevated antibody levels were induced with a single VLP immunization, whereas P-particle immunization required a boost. High avidity antibodies were raised only by VLP immunization. VLP immunization resulted in a balanced T helper type 1/type 2 immune response whereas P-particles induced a T helper type 2-biased response. Only VLP immunization primed T cells for interferon-γ production. Most importantly, cross-reactive B and T cells were induced solely by VLP immunization. In addition, VLP antiserum blocked the binding of heterotypic VLPs to human histo-blood group antigen receptor and saliva. The findings in this study are relevant for the development of NoV vaccines.

Norovirus VLPs and rotavirus VP6 protein as combined vaccine for childhood gastroenteritis.

Noroviruses (NoVs) and rotaviruses (RVs) are the two most important viral causes of severe gastroenteritis in young children worldwide. Live oral RV vaccines are already part of routine childhood immunization in many countries, but may be associated with low risk of intussusception and other potential risks associated with live vaccines. NoV capsid-derived virus-like particles (VLPs) are in early phase clinical trials, but there is no vaccine available yet. We suggest that there is a need for non-live vaccines against both enteric pathogens. We have combined NoV GII-4 VLPs and human RV recombinant VP6 (rVP6) protein produced by recombinant baculovirus (BV) expression system in insect cells and used this combination vaccine to immunize BALB/c mice parenterally. Strong systemic cross-reactive and cross-blocking antibody responses towards NoV and RV were induced, and there was no interference of the immune response to either antigen given in combination. Rather, we observed an adjuvant effect of rVP6 on the NoV-specific homologous and heterologous immune responses to genotypes not included in a vaccine formulation.

Noroviruses in children seen in a hospital for acute gastroenteritis in Finland.

UNLABELLED: Noroviruses (NoVs) are second only to rotaviruses (RVs) as causative agents of acute gastroenteritis (AGE) in children. The proportional role of NoVs is likely to increase after control of RV by vaccination. We investigated NoVs in children seen in Tampere University Hospital either treated as outpatients or hospitalized because of AGE before universal RV vaccination was implemented in Finland. This prospective study was conducted from September 2006 to August 2008. A total of 1,128 children <15 years of age with symptoms of AGE were enrolled either in the hospital clinic or in a ward, and stool samples for NoV studies were obtained from 759 children. NoVs were found in 196 (26%) cases. In the first year, NoVs were found in 116 (34%) out of 341, and in the second year, in 80 (19%) out of 418 cases. RVs were found respectively in 128 (38%) and 260 (62%) cases in these two seasons. Both RV and NoV were present in 24 cases. NoV genotype GII.4 predominated with a 96% share of the NoV cases in the first season and an 80% share in the second season. Other NoV genotypes seen infrequently were GII.7, GIIb, GI.6, GII.1, GII.2, and GIIc. The median clinical severity of NoV AGE was 14 compared to 16 for RV AGE on a 20-point scale. CONCLUSION: NoVs were nearly as common as RVs as causative agents of severe AGE in children seen in hospital. After implementing universal RV vaccination, the importance of NoVs will still increase further.

Rotavirus gastroenteritis in Finnish children in 2006-2008, at the introduction of rotavirus vaccination.

Rotaviruses (RV) are major causative agents of acute gastroenteritis (AGE) requiring hospitalization in children; RV hospitalizations may be largely eliminated by universal mass vaccination with RV vaccine. We conducted a hospital-based prospective survey of AGE in children over 2 RV epidemic seasons, from 2006 to 2008, when the coverage of RV vaccination in Finland increased to 35% of the birth cohort. RVs were detected by reverse transcription polymerase chain reaction (RT-PCR). In the first season, only 38% of AGE cases were RV-positive, and the onset of the RV season was delayed. Type G1P[8], RVs accounted for 40%, G2P[4] for 19%, G3P[8] for 2%, G4P[8] for 2% and G9P[8] for 38%. In the second season, 63% of AGE cases were RV-positive: G1P[8] accounted for 73%, G2P[4] for only 3%, G3P[8] for 4%, G4P[8] for 13%, and G9P[8] had almost disappeared. G2P[4] RV did not become predominant at the coverage level of 29% of G1P[8] human RV vaccine. RV-associated hospitalizations were seen in children up to the age of 9 y. This study forms the epidemiological background for the follow-up of the impact of universal RV vaccination in Finland introduced in 2009.

Detection of human coronaviruses in children with acute gastroenteritis.

BACKGROUND: Human coronaviruses (HCoVs) are known respiratory pathogens. Moreover, coronavirus-like particles have been seen by electron microscope in stools, and SARS-HCoV has been isolated from intestinal tissue and detected in stool samples. OBJECTIVES: To find out if HCoVs can be found in stools of children with acute gastroenteritis and to assess the significance of HCoVs in the etiology of acute gastroenteritis in children. STUDY DESIGN: 878 stool specimens from children with acute gastroenteritis and 112 from control children were tested by RT-PCR to detect HCoV groups 1B, 2A and SARS. HCoVs were typed by sequencing all PCR positive samples. RESULTS: Twenty-two (2.5%) of the 878 stool specimens of children with acute gastroenteritis were positive for HCoVs. The following HCoV types were detected: OC43 (10 cases, 45.5%), HKU1 (6 cases, 27.3%), 229E (2 cases, 9.1%) and NL63 (4 cases, 18.2%). In 4 of the cases a HCoV was the only detected virus; in the remaining cases rotavirus or norovirus was found in the same sample. In control groups there were two HCoV positive samples of 112 tested. CONCLUSIONS: This study shows that all known non-SARS HCoVs can be found in stools of children with acute gastroenteritis. On the basis of this study, the significance of coronaviruses as gastrointestinal pathogens in children appears minor, since most of the coronavirus findings were co-infections with known gastroenteritis viruses.