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Many viral structural proteins and their truncated domains share a common feature of homotypic interaction forming dimers, trimers, and/or oligomers with various valences. We reported previously a simple strategy for construction of linear and network polymers through the dimerization feature of viral proteins for vaccine development. In this study, technologies were developed to produce more sophisticated polyvalent complexes through both the dimerization and oligomerization natures of viral antigens. As proof of concept, branched-linear and agglomerate polymers were made via fusions of the dimeric glutathione-s-transferase (GST) with either a tetrameric hepatitis E virus (HEV) protruding protein or a 24-meric norovirus (NoV) protruding protein. Furthermore, a monomeric antigen, either the M2e epitope of influenza A virus or the VP8* antigen of rotavirus, was inserted and displayed by the polymer platform. All resulting polymers were easily produced in Escherichia coli at high yields. Immunization of mice showed that the polymer vaccines induced significantly higher specific humoral and T cell responses than those induced by the dimeric antigens. Additional evidence in supporting use of polymer vaccines included the significantly higher neutralization activity and protective immunity of the polymer vaccines against the corresponding viruses than those of the dimer vaccines. Thus, our technology for production of polymers containing different viral antigens offers a strategy for vaccine development against infectious pathogens and their associated diseases., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The extracellular domain of the matrix protein 2 (M2e) of influenza viruses is highly conserved among all influenza A subtypes, making it a suitable target for a universal influenza vaccine. In this study, we demonstrated an enhanced immune response and protection of a chimeric M2e vaccine against influenza A viruses using our newly developed vaccine platform, the norovirus P particle, to present the M2e peptide. The 23-amino acid peptide was inserted into one of the surface loops of the P protein, resulting in 24 copies of M2e presented on each P particle. Significantly (P<0.001) increased antibody responses to M2e were observed in mice immunized with the P particle-M2e chimera compared with those immunized with the free M2e peptides. Mice immunized with the P particle-M2e vaccine were fully protected (100% survived) against lethal challenge of a mouse adapted human influenza virus PR8 (H1N1), while only low survival rates (<12.5%) were found in mice immunized with the free M2e peptides or wild type P particle. In addition, the mouse sera collected after immunization with the P particle-M2e vaccine were able to block the binding of norovirus virus-like particle and P particle to histo-blood group antigen receptors. These results suggest that the P particle-M2e chimera can be used as dual vaccine against both noroviruses and influenza viruses., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

I.m. immunization of mice with inactivated rotavirus particles protects against subsequent infection. To optimize protection, the effects of different adjuvants (QS-21, QS-7, QUIL A, PCPP and RAS) with potential for human use were compared. Twenty-eight days after i.m. immunization with 20 microg of purified, UV/psoralen-inactivated murine rotavirus (EDIM), either with or without adjuvant, BALB/c mice were orally challenged with live EDIM and virus shedding was measured. All five adjuvants stimulated large (P < 0.001) increases in rotavirus antibody, but significant differences were found between adjuvants. The order of rotavirus IgG responses, i.e. no adjuvant < RAS < QS-7 < Quil A < QS-21 < PCPP, was the same as the order of protection except that QS-21 and PCPP were reversed. These results establish the importance of adjuvants during i.m. immunization with rotavirus and identify those with the greatest potential.

Antibody responses and protection against shedding following oral challenge with murine rotavirus (EDIM) were determined in mice after sequential oral parenteral immunization. Oral immunization of 4-day-old BALB/c mice with live, heterologous rotavirus (RRV) stimulated serum rotavirus IgG but little serum or intestinal rotavirus IgA and small but significant (P < 0.001) reductions in EDIM shedding. Intraperitoneal immunization with inactivated EDIM at 29 days of age had similar effects. Sequential oral-parenteral immunization under these conditions stimulated small but significant (P < 0.001) increases in both rotavirus IgG and IgA titers and reduced shedding (P < 0.001) compared to individual immunizations. However, these responses were essentially additive, indicative of separate inductive/effector sites for mucosal and systemic immunity.

We recently reported that epidermal immunization using the PowderJet particle delivery device with plasmid vector pcDNA1/EDIM6 encoding rotavirus VP6 of murine strain EDIM induced high levels of serum rotavirus IgG but failed to protect mice against EDIM infection (Choi, A. H., Knowlton, D. R., McNeal, M. M., and Ward, R. L. (1997) Virology 232, 129-138.). This was extended to determine whether pcDNA1/EDIM4 or pcDNA1/EDIM7, which encode either rotavirus VP4 or VP7, the rotavirus neutralization proteins, could also induce rotavirus-specific antibody responses and if these responses resulted in protection. Titers of rotavirus serum IgG increased with the first dose in mice immunized with pcDNA1/EDIM7, but little or no serum rotavirus IgG was detected in mice immunized with pcDNA1/EDIM4. In vitro assays with these plasmids in rabbit reticulocyte lysates showed that VP4 was expressed but the amount was considerably lower than VP6 or VP7. To improve expression of VP4 and induction of rotavirus-specific humoral responses, the coding region of VP4 was cloned into the high-expression plasmid WRG7054 as a fusion protein containing the 22-amino-acid secretory signal peptide of tissue plasminogen activator (tPA) at its N terminus. In vitro expression of tPA::VP4 was significantly higher than unmodified VP4, and mice inoculated with WRG7054/EDIM4 generated high titers of rotavirus IgG. The coding sequence of VP7 without the first 162 nucleotides was also cloned into WRG7054, but no difference was observed between titers of serum rotavirus IgG in mice immunized with this plasmid (WRG7054/EDIM7Delta1-162) and pcDNA1/EDIM7. The rotavirus-specific IgG titers in all immune sera were predominantly IgG1 indicating induction of Th 2-type responses. None of the mice immunized with any of the VP4 or VP7 plasmids developed serum or fecal rotavirus IgA or neutralizing antibody to EDIM. When immunized mice were challenged with EDIM virus, there was no significant reduction in viral shedding relative to unimmunized controls. Therefore epidermal immunization with VP4 or VP7 alone elicited rotavirus IgG responses but did not protect against homologous rotavirus challenge., (Copyright 1998 Academic Press.)

The rotavirus inner capsid protein VP6 contains conserved epitopes that are potential targets for eliciting protective immunity against different serotypes within the same group of rotavirus. In order to determine whether VP6 alone can induce protective immunity, an expression vector pcDNA1/EDIM6 containing gene 6 of rotavirus EDIM strain was constructed and used as a vaccine in an adult mouse model. Cloned gene 6 was determined to be 1356 nucleotides long and contained a 5' noncoding region of 23 nucleotides, a 3' noncoding region of 139 nucleotides, and a coding frame of 1194 nucleotides for a polypeptide of 397 amino acid residues. Recombinant VP6 was expressed in rabbit reticulocyte lysate and the heat-denatured recombinant VP6 migrated in SDS-gels with an apparent molecular weight of approximately 43 kDa. Five additional polypeptide bands corresponding to oligomers of recombinant VP6 were observed when the expressed product was not heat denatured. To determine the immunogenicity of recombinant VP6, female BALB/c mice were injected intramuscularly or intradermally with pcDNA1/EDIM6, or were inoculated epidermally with plasmid-coated gold beads using the Geniva Accell particle delivery device. Only intradermal injection and particle delivery elicited measurable serum anti-rotavirus IgG responses, but responses developed following particle delivery were significantly (P < 0.001) greater. However, none of the delivery methods induced serum or stool anti-rotavirus IgA responses and, when challenged with EDIM no protection against infection was observed in the immunized mice. Therefore, parenteral immunization with VP6 alone elicited large anti-rotavirus IgG responses but did not elicit protection against murine rotavirus infection in this model.

It has been found that mice infected with murine rotavirus can be protected against subsequent murine rotavirus infection for up to 2 months. It was also reported that protection against rotavirus infection in adult mice correlated with serum and stool rotavirus IgA titers. The present study was conducted to determine the duration of rotavirus antibody production and protection against rotavirus infection in this mouse model and its possible correlation with rotavirus antibody titers. It was found that protection of mice against subsequent infection following a single oral immunization with the murine rotavirus strain EDIM was 100% effective for at least 14 months, most of the lifetime of a mouse. During this period, serum and stool rotavirus antibody titers which included serum IgA, IgG, and neutralizing antibody to EDIM, as well as stool IgA, remained elevated. Of particular note, stool rotavirus IgA titers gradually decreased to levels that were approximately 10% of their peak at 1 month after infection but did not decrease further, while serum rotavirus IgG titers continuously increased during the 14 months of the study. Serum rotavirus IgA titers varied from month to month but overall remained relatively constant throughout the 14-month period. Thus, both serum and stool rotavirus antibody was retained at substantial levels long after a single rotavirus immunization in the absence of reexposure, and mice remained protected against reinfection.

Using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge with the murine rotavirus strain EDIM. To determine if a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of 18 reassortants between the fully protective EDIM strain and a partially protective heterologous rotavirus strain (RRV-G). Reassortants that contained genes for EDIM proteins responsible for protection were anticipated to provide complete protection; however, no EDIM proteins were found to be both necessary and sufficient for full protection. Instead, protection was found to be highly correlated with viral shedding (P = 0.005) and with serum rotavirus IgA titers stimulated by the different reassortants (P < 0.001). This indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of EDIM proteins. This conclusion was supported by the finding that the titers of serum rotavirus IgA, but not IgG, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against EDIM. If these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization.

Active immunity to rotavirus has been demonstrated following oral inoculation with live virus but little is known about the effects of parenteral immunization. In this study, adult mice were immunized by intraperitoneal (ip) inoculation with live rotaviruses and later orally challenged with murine rotavirus (EDIM) to measure active immunity against infection. Three doses of EDIM (8 micrograms/dose) given intraperitoneally (ip) provided full protection against EDIM infection, whether administered with or without Freund's adjuvant. Only partial protection was found when the quantity of immunogen was reduced to < 2 micrograms/dose. Reduction of the number of doses from three to one (8 micrograms/dose), however, still resulted in protection of all mice. Significant protection was also observed after inoculation with one or three doses (2 micrograms/dose) of heterologous rotaviruses. Protection provided by the heterologous strains did not correlate with neutralizing antibody to EDIM, which indicated that neutralizing antibody to the challenge virus was not required for protection. uv-Inactivated EDIM also provided significant protection against EDIM, thus demonstrating that viral replication was not required for protection. These results suggest that parenteral immunization may be an effective method to vaccinate against rotavirus disease.

An evaluation of the safety and immunogenicity of WC3 rotavirus vaccine was evaluated in adult volunteers. Pre- and post-vaccination titers of neutralizing antibody to WC3 and to the four human rotavirus serotypes as well as serum and stool rotavirus IgA levels were measured. Vaccination was safe and did not induce elevation of liver enzymes. None of the 12 volunteers receiving WC3 vaccine shed detectable amounts of virus although antibody rises were detected in 11 of 12 vaccines. Nine developed and increase in WC3 neutralizing antibody, one additional subject had a rise in Wa (human serotype 1) neutralizing antibody while another subject only developed a rise in stool rotavirus IgA. All of the vaccine recipients with a rise in WC3 neutralizing antibody also developed a rise in neutralizing antibody against at least one of the four most common human rotavirus serotypes. A stool IgA rotavirus antibody response was detected in 6 of 9 WC3 recipients with measurable stool antibody. None of the control subjects developed significant rises in any of the antibody titers measured. WC3 rotavirus vaccine appears to be safe and induces systemic and local immune responses in adults suggesting that further evaluation of WC3 should be considered in infants.