Capsid display of a conserved human papillomavirus L2 peptide in the adenovirus 5 hexon protein: a candidate prophylactic hpv vaccine approach.

Background: Infection by any one of 15 high risk human papillomavirus (hrHPV) types causes most invasive cervical cancers. Their oncogenic genome is encapsidated by L1 (major) and L2 (minor) coat proteins. Current HPV prophylactic vaccines are composed of L1 virus-like particles (VLP) that elicit type restricted immunity.

A replicating adenovirus capsid display recombinant elicits antibodies against Plasmodium falciparum sporozoites in Aotus nancymaae monkeys.

Decades of success with live adenovirus vaccines suggest that replication-competent recombinant adenoviruses (rAds) could serve as effective vectors for immunization against other pathogens. To explore the potential of a live rAd vaccine against malaria, we prepared a viable adenovirus 5 (Ad5) recombinant that displays a B-cell epitope from the circumsporozoite protein (CSP) of Plasmodium falciparum on the virion surface. The recombinant induced P.

Vectored antibody gene delivery protects against Plasmodium falciparum sporozoite challenge in mice.

Malaria caused by Plasmodium falciparum kills nearly one million children each year and imposes crippling economic burdens on families and nations worldwide. No licensed vaccine exists, but infection can be prevented by antibodies against the circumsporozoite protein (CSP), the major surface protein of sporozoites, the form of the parasite injected by mosquitoes. We have used vectored immunoprophylaxis (VIP), an adeno-associated virus-based technology, to introduce preformed antibody genes encoding anti-P.

Immune responses in macaques to a prototype recombinant adenovirus live oral human papillomavirus 16 vaccine.

Immunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings.

Prospects for oral replicating adenovirus-vectored vaccines.

Orally delivered replicating adenovirus (Ad) vaccines have been used for decades to prevent adenovirus serotype 4 and 7 respiratory illness in military recruits, demonstrating exemplary safety and high efficacy. That experience suggests that oral administration of live recombinant Ads (rAds) holds promise for immunization against other infectious diseases, including those that have been refractory to traditional vaccination methods. Live rAds can express intact antigens from free-standing transgenes during replication in infected cells.

Role of E1B55K in E4orf6/E1B55K E3 ligase complexes formed by different human adenovirus serotypes.

The E4orf6 protein of serotypes representing all human adenovirus species forms Cullin-based E3 ubiquitin ligase complexes that facilitate virus infection by inducing degradation of cellular proteins that impede efficient viral replication. This complex also includes the viral E1B55K product believed to bind and introduce substrates for ubiquitination. Heterogeneity in the composition of these ligases exists, as some serotypes form Cul5-based complexes whereas others utilize Cul2.

The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection.

In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood.

The adenovirus E4 11 k protein binds and relocalizes the cytoplasmic P-body component Ddx6 to aggresomes.

The adenovirus E4 11 k protein, product of E4 ORF3, is required in infection for processes including normal accumulation of viral late mRNAs. 11 k restructures both the nucleus and cytoplasm of infected cells by relocalizing specific host cell target proteins, most strikingly components of nuclear PML oncogenic domains. It is likely that in many cases relocalization inactivates target proteins to produce 11 k's effects, although the mechanism and targets for stimulation of late mRNA accumulation is unknown.

Adenovirus particles that display the Plasmodium falciparum circumsporozoite protein NANP repeat induce sporozoite-neutralizing antibodies in mice.

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice.

The E4orf6/E1B55K E3 ubiquitin ligase complexes of human adenoviruses exhibit heterogeneity in composition and substrate specificity.

Although human adenovirus type 5 (Ad5) has been widely studied, relatively little work has been done with other human adenovirus serotypes. The Ad5 E4orf6 and E1B55K proteins form Cul5-based E3 ubiquitin ligase complexes to degrade p53, Mre11, DNA ligase IV, integrin α3, and almost certainly other targets, presumably to optimize the cellular environment for viral replication and perhaps to facilitate persistence or latency. As this complex is essential for the efficient replication of Ad5, we undertook a systematic analysis of the structure and function of corresponding E4orf6/E1B55K complexes from other serotypes to determine the importance of this E3 ligase throughout adenovirus evolution.

Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity.

A vaccine comprising human papillomavirus type 16 (HPV16) L2, E6 and E7 in a single tandem fusion protein (termed TA-CIN) has the potential advantages of both broad cross-protection against HPV transmission through induction of L2 antibodies able to cross neutralize different HPV types and of therapy by stimulating T cell responses targeting HPV16 early proteins. However, patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. Here we test TA-CIN formulated along with the adjuvant GPI-0100, a semi-synthetic quillaja saponin analog that was developed to promote both humoral and cellular immune responses.

E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection.

The ligase IV/XRCC4 complex plays a central role in DNA double-strand break repair by non-homologous end joining (NHEJ). During adenovirus infection, NHEJ is inhibited by viral proteins E4 34k and E1B 55k, which redirect the Cul5/Rbx1/Elongin BC ubiquitin E3 ligase to polyubiquitinate and promote degradation of ligase IV. In cells infected with E1B 55k-deficient adenovirus, ligase IV could not be found in XRCC4-containing complexes and was observed in a novel ligase IV/E4 34k/Cul5/Elongin BC complex.

Loss of DNA ligase IV prevents recognition of DNA by double-strand break repair proteins XRCC4 and XLF.

The repair of DNA double-strand breaks by nonhomologous end-joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Central to the molecular mechanism of NHEJ is DNA ligase IV/XRCC4/XLF complex, which rejoins the DNA. During adenovirus (Ad5) infection, ligase IV is targeted for degradation in a process that requires expression of the viral E1B 55k and E4 34k proteins while XRCC4 and XLF protein levels remain unchanged.

Small-scale extracts for the study of nucleotide excision repair and non-homologous end joining.

The repair of DNA by nucleotide excision repair (NER) and non-homologous end joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Examination of NHEJ and NER in vitro using cell-free extracts has led to a deeper understanding of the biochemical mechanisms that underlie these processes. Current methods for production of whole-cell extracts (WCEs) to investigate NER and NHEJ start with one or more liters of culture containing 1-5 x 10(9) cells.

Androgen receptor attenuation of Ad5 replication: implications for the development of conditionally replication competent adenoviruses.

Conditionally replication competent adenoviruses (CRAds) represent one of the most intensely studied gene therapy strategies for a variety of malignancies, including prostate cancer. These viruses can be generated by placing a tissue or cancer-specific promoter upstream of one or more of the viral genes required for replication (e.g.

Adenovirus E4 34k and E1b 55k oncoproteins target host DNA ligase IV for proteasomal degradation.

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation.

Isolation, growth, and purification of defective adenovirus deletion mutants.

Defective adenovirus deletion mutants can be grown by complementation in the presence of helper viruses that supply essential functions missing in the deletion mutant. In general, the deletion mutant then must be separated physically from the helper for use in subsequent experiments. This chapter includes suggestions for selection of helper viruses, protocols for the production of stocks by complementation, and procedures for physical separation of deletion mutants from their helpers.

Manipulation of early region 4.

Adenovirus early region 4 (E4) regulates processes in infected cells that include viral late gene expression, nonhomologous end joining, responses to DNA damage, and apoptosis. E4 is essential for viral growth in most cell lines. In this chapter, the current knowledge of the functions of six E4 products is summarized briefly.

Adenovirus E4orf6 assembles with Cullin5-ElonginB-ElonginC E3 ubiquitin ligase through an HIV/SIV Vif-like BC-box to regulate p53.

The adenovirus protein E4orf6 targets p53 for polyubiquitination and proteasomal degradation and is known to form a complex with the Cul5-ElonginB-ElonginC E3 ubiquitin ligase. However, whether Cul5 is directly responsible for the E4orf6-mediated degradation of p53 remains unclear. By using a dominant-negative mutant of Cul5 and silencing Cul5 expression through RNA interference, we have now demonstrated that E4orf6-mediated p53 degradation requires Cul5.

HPV16 L1 capsid protein expressed from viable adenovirus recombinants elicits neutralizing antibody in mice.

Immunization against human papillomavirus (HPV) infection promises to reduce the worldwide burden of cervical cancer. To evaluate the potential of live recombinant adenoviruses for induction of HPV infection-blocking immunity, we prepared viable adenovirus recombinants that express the HPV16 L1 gene from the adenovirus major late transcriptional unit. Adenovirus-produced HPV16 L1 assembles into virus-like particles (VLPs) in infected cells in culture.

Viable adenovirus vaccine prototypes: high-level production of a papillomavirus capsid antigen from the major late transcriptional unit.

Safe, effective, orally delivered, live adenovirus vaccines have been in use for three decades. Recombinant derivatives of the live adenovirus vaccines may prove an economical alternative to current vaccines for a variety of diseases. To explore that possibility, we constructed a series of recombinants that express the major capsid protein (L1) of canine oral papillomavirus (COPV), a model for mucosal human papillomavirus (HPV) infection.

Both BC-box motifs of adenovirus protein E4orf6 are required to efficiently assemble an E3 ligase complex that degrades p53.

Small DNA tumor viruses typically encode proteins that either inactivate or degrade p53. Human adenoviruses encode products, including E4orf6 and E1B55K, that do both. Each independently binds to p53 and inhibits its ability to activate gene expression; however, in combination they induce p53 degradation by the ubiquitin pathway.

Expression of the adenovirus E4 34k oncoprotein inhibits repair of double strand breaks in the cellular genome of a 293-based inducible cell line.

The human adenovirus E4 ORF 6 34 kDa oncoprotein (E4 34k), in concert with the 55 kDa product of E1b, prevents concatenation of viral genomes in infected cells, inhibits the repair of double strand breaks (DSBs) in the viral genome, and inhibits V(D)J recombination in a plasmid transfection assay. These activities are consistent with a general inhibition by the E4 34k and E1b 55k proteins of DSB repair by non-homologous end joining (NHEJ) on extrachromosomal substrates. To determine whether inhibition of NHEJ extends to repair of DSBs in the cell chromosome, we have examined the effects of E4 34k on repair of chromosomal DSBs induced by ionizing radiation in a cell line in which E4 34k expression and biological activity is inducible and E1b 55k is produced constitutively.

Neonatal sex steroids affect responses to Seoul virus infection in male but not female Norway rats.

Previous studies illustrate that after inoculation with Seoul virus (i.e., the naturally occurring hantavirus found in Norway rats), adult male rats produce higher antibody responses, exhibit higher Th1 responses (i.