Characterization of polymorphism displayed by the coat protein mutants of tomato bushy stunt virus

AbstractExpression of full-length and N-terminal deletion mutants of the coat protein (CP) of tomato bushy stunt virus (TBSV) using the recombinant baculovirus system resulted in spontaneously assembled virus-like particles (VLPs). Deletion of the majority of the R-domain sequence of the CP, residues 1–52 (CP-NΔ52) and 1–62 (CP-NΔ62), produced capsids similar to wild-type VLPs. Interestingly, the CP-NΔ62 mutant that retains the last 3 residues of R-domain is capable of forming both the T = 1 and T = 3 particles. However, between the two types of VLPs, formation of the T = 1 capsids appears to be preferred. Another mutant, CP-NΔ72, in which R-domain (residues 1–65) was completely removed but contains most of the β-annulus and extended arm (βA) regions exclusively formed T = 1 particles. These results suggest that as few as 3 residues (63–65) of the R-domain, which includes 2 basic amino acids together with the arm (βA) and β-annulus regions, may be sufficient for the formation of T = 3 particles. However, anywhere between 4 to 13 residues of the R-domain may be required for proper positioning of βA and β-annulus structural elements of the C-type subunits to facilitate an error free assembly of T = 3 capsids

Pre-Clinical Development of a Recombinant, Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

BackgroundThere is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.MethodsThe recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.ResultsRobust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.ConclusionsThe Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials

Pre-Clinical Evaluation of a Replication-Competent Recombinant Adenovirus Serotype 4 Vaccine Expressing Influenza H5 Hemagglutinin

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis.The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus.Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine

Evaluation of a one-tube RT-PCR system for detection of enteroviruses

Background: A highly sensitive PCR assay for early and rapid detection of enteroviral (EV) RNA in CSF is necessary to investigate the role of EV in acute neurological illnesses. Objectives: To evaluate and compare two PCR protocols (Titan one-tube RT-PCR and random primed RT-PCR) for detection of enteroviral RNA in CSF. Study design: The PCR protocols were evaluated for lower limit of input detection using log dilutions of five stock EV strains and an isolate of enterovirus-71 in minimum essential medium and three EV stock strains in CSF. The tests were also applied on 77 CSF samples, 46 from patients with suspected acute EV neurological illness and 31 from 'disease controls'. Results: Even though in the initial virus titration assays there was no statistically significant difference in the limit of input detection by Titan system and the random primed two-step PCR, the latter had a higher positivity rate when used on CSF samples from patients (20/46 vs. 10/46, P<0.01). Conclusions: Random primed RT-PCR assay is superior to Titan one-tube RT-PCR for detection of EV RNA in CSF

A viral nanoparticle with dual function as an anthrax antitoxin and vaccine.

The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax

Pre-clinical development of a recombinant, replication-competent adenovirus serotype 4 vector vaccine expressing HIV-1 envelope 1086 clade C.

There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials

Vaccine-induced H5HA-specific humoral response in the presence and absence of pre-existing Ad4-specific immunity.

<p>Mice were immunized i.n. with 1×10⁹ vp of Ad4wt virus per mouse to establish pre-existing immunity to the vector. Four weeks following the immunization, ten individual mice were bled and Ad4-specific neutralizing antibody titers were determined (A). Mice immunized with Ad4wt virus and naïve mice were subsequently immunized i.n. with a dose titration of the Ad4-H5-Vtn vaccine; 1×10⁹, 1×10⁸, 1×10⁷ and 1×10⁶ vp per mouse and bled 6 weeks after vaccine immunization and again 5 days later following H5N1 reassortant virus challenge to determine HAI antibody titers (B). The immune responses are represented by post-Ad4-H5-Vtn vaccine immunization (open bar) and post-H5N1 reassortant challenge (cumulative of open and black fill bar). Three mice from the group were bled and sera pooled to determine HAI antibody titers.</p

Mice immunized with Ad4-H5-Vtn vaccine presented with a reduction of H5N1 reassortant virus in the lungs.

<p>Groups of mice were immunized with Ad4wt virus to establish pre-existing immunity as previously described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031177#s2" target="_blank">Materials and Methods</a> section. Mice were subsequently immunized intranasally with a dose titration of the Ad4-H5-Vtn vaccine. Six weeks following Ad4-H5-Vtn vaccine immunization, the mice were challenged with a lethal dose of H5N1 reassortant virus. Lungs were recovered from a subset of mice 5 days post-challenge to determine influenza-specific viral titers.</p

Ad4-H5-Vtn vector design.

<p>The H5HA native coding sequence, with the polybasic domain removed (B), was derived from A/Vietnam/1194/2004 influenza virus and inserted into the Ad4 virus E3 gene region. The Ad4 virus E3 24.8K, E3 6.3K and E3 29.7K genes were deleted to accommodate the HA transgene and the splice acceptor site of E3 24.8K was retained to drive expression of the HA transgene. The E3A polyadenylation signal sequence, derived from Ad5, was placed downstream of the HA coding sequence. The use of a shuttle plasmid encoding the H5HA sequence and the Ad4 plasmid to obtain the final vaccine product is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031177#s2" target="_blank">Materials and Methods</a>.</p