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. An N-terminal region of L2 protein identified by neutralizing monoclonal antibodies comprises a protective epitope conserved among HPV types, but it is weakly immunogenic compared to L1 VLP. The major antigenic capsid protein of adenovirus type 5 (Ad5) is hexon which contains 9 hypervariable regions (HVRs) that form the immunodominant neutralizing epitopes. Insertion of weakly antigenic foreign B cell epitopes into these HVRs has shown promise in eliciting robust neutralizing antibody responses. Thus here we sought to generate a broadly protective prophylactic HPV vaccine candidate by inserting a conserved protective L2 epitope into the Ad5 hexon protein for VLP-like display. METHODS: Four recombinant adenoviruses were generated without significant compromise of viral replication by introduction of HPV16 amino acids L2 12-41 into Ad5 hexon, either by insertion into, or substitution of, either hexon HVR1 or HVR5. RESULTS: Vaccination of mice three times with each of these L2-recombinant adenoviruses induced similarly robust adenovirus-specific serum antibody but weak titers against L2. These L2-specific responses were enhanced by vaccination in the presence of alum and monophoryl lipid A adjuvant. Sera obtained after the third immunization exhibited low neutralizing antibody titers against HPV16 and HPV73. L2-recombinant adenovirus vaccination without adjuvant provided partial protection of mice against HPV16 challenge to either the vagina or skin. In contrast, vaccination with each L2-recombinant adenovirus formulated in adjuvant provided robust protection against vaginal challenge with HPV16, but not against HPV56. CONCLUSION: We conclude that introduction of HPV16 L2 12-41 epitope into Ad5 hexon HVR1 or HVR5 is a feasible method of generating a protective HPV vaccine, but further optimization is required to strengthen the L2-specific response and broaden protection to the more diverse hrHPV.

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. falciparum sporozoite-neutralizing antibodies in mice. Human adenoviruses do not replicate in mice. Therefore, to examine immunogenicity in a system in which, as in humans, the recombinant replicates, we constructed a similar recombinant in an adenovirus mutant that replicates in monkey cells and immunized four Aotus nancymaae monkeys. The recombinant replicated in the monkeys after intratracheal instillation, the first demonstration of replication of human adenoviruses in New World monkeys. Immunization elicited antibodies both to the Plasmodium epitope and the Ad5 vector. Antibodies from all four monkeys recognized CSP on intact parasites, and plasma from one monkey neutralized sporozoites in vitro and conferred partial protection against P. falciparum sporozoite infection after passive transfer to mice. Prior enteric inoculation of two animals with antigenically wild-type adenovirus primed a response to the subsequent intratracheal inoculation, suggesting a route to optimizing performance. A vaccine is not yet available against P. falciparum, which induces the deadliest form of malaria and kills approximately one million children each year. The live capsid display recombinant described here may constitute an early step in a critically needed novel approach to malaria immunization.

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. falciparum CSP mAb into mice. VIP vector-transduced mice exhibited long-lived mAb expression at up to 1,200 µg/mL in serum, and up to 70% were protected from both i.v. and mosquito bite challenge with transgenic Plasmodium berghei rodent sporozoites that incorporate the P. falciparum target of the mAb in their CSP. Serum antibody levels and protection from mosquito bite challenge were dependent on the dose of the VIP vector. All individual mice expressing CSP-specific mAb 2A10 at 1 mg/mL or more were completely protected, suggesting that in this model system, exceeding that threshold results in consistent sterile protection. Our results demonstrate the potential of VIP as a path toward the elusive goal of immunization against malaria.

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. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines.

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. Alternatively, antigenic epitopes can be displayed on the rAd capsid itself, allowing presentation of the epitope to the immune system both prior to and during replication of the virus. Such capsid-display rAds offer a novel vaccine approach that could be used either independently of or in combination with transgene expression strategies to provide a new tool in the search for protection from infectious disease.

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. Significant variations in substrate specificities also exist among serotypes, as some degrade certain substrates very efficiently whereas others induce more modest or little degradation. As E1B55K is believed to function as the substrate acquisition component of the ligase, we undertook studies to compare the ability of representative E1B55K proteins to bind substrates with the efficacy of degradation by their respective E4orf6-based ligases. Interestingly, although efficient degradation in some cases corresponded to the ability of E1B55K to bind to or relocalize substrates, there were several examples of substrates that bound efficiently to E1B55K but were not degraded and others in which substrates were degraded even though binding to E1B55K was low or undetectable. These results suggest that transient interactions with E1B55K may be sufficient for efficient substrate degradation and that binding alone is not sufficient, implying that the orientation of the substrate in the ligase complex is probably crucial. Nevertheless, we found that the substrate specificity of certain E4orf6-based ligases could be altered through the formation of hybrid complexes containing E1B55K from another serotype, thus confirming identification of E1B55K as the substrate acquisition component of the complex.

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. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

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. We have identified a new set of proteins relocalized by 11 k: at least five protein components of cytoplasmic mRNA processing bodies (p-bodies) are found in 11 k-induced cytoplasmic aggresomes, sites where proteins are inactivated or destroyed. One of these p-body proteins, RNA helicase Ddx6, binds 11 k, suggesting a mechanism for relocalization. Because p-bodies are sites for mRNA degradation, their modification by 11 k may provide an explanation for the role of 11 in viral late mRNA accumulation.

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. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world.

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. E4orf6 and E1B55K coding sequences from serotypes representing all subgroups were cloned, and each pair was expressed and analyzed for their capacity to assemble the Cullin-based ligase complex and to degrade substrates following plasmid DNA transfection. The results indicated that all formed Cullin-based E3 ligase complexes but that heterogeneity in both structure and function existed. Whereas Cul5 was present in the complexes of some serotypes, others recruited primarily Cul2, and the Ad16 complex clearly bound both Cul2 and Cul5. There was also heterogeneity in substrate specificity. Whereas all serotypes tested appeared to degrade DNA ligase IV, complexes from some serotypes failed to degrade Mre11, p53, or integrin α3. Thus, a major evolutionary pressure for formation of the adenovirus ligase complex may lie in the degradation of DNA ligase IV; however, it seems possible that the degradation of as-yet-unidentified critical targets or, perhaps even more likely, appropriate combinations of substrates plays a central role for these adenoviruses.

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. Subcutaneous administration to mice of TA-CIN (20 microg) with 50microg GPI-0100, three times at biweekly intervals, elicited high titer HPV16 neutralizing serum antibody, robust neutralizing titers for other HPV16-related types, including HPV31 and HPV58, and neutralized to a lesser extent other genital mucosatropic papillomaviruses like HPV18, HPV45, HPV6 and HPV11. Notably, vaccination with TA-CIN in GPI-0100 protected mice from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP without adjuvant. Formulation of TA-CIN with GPI-0100 enhanced the production of E7-specific, interferon gamma producing CD8(+) T cell precursors by 20-fold. Vaccination with TA-CIN in GPI-0100 also completely prevented tumor growth after challenge with 5x10(4) HPV16-transformed TC-1 tumor cells, whereas vaccination with TA-CIN alone delayed tumor growth. Furthermore, three monthly vaccinations with 125 microg of TA-CIN and 1000 microg GPI-0100 were well tolerated by pigtail macaques and induced both HPV16 E6/E7-specific T cell responses and serum antibodies that neutralized all HPV types tested.

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. These observations suggest that dissociation of the ligase IV/XRCC4 complex occurs at an early stage in E4 34k-mediated degradation of ligase IV and indicate a role for E4 34k in dissociation of the ligase IV/XRCCC4 complex. Expression of E4 34k alone was not sufficient to dissociate the ligase IV/XRCC4 complex, which indicates a requirement for an additional, as yet unidentified, factor in E1B 55k-independent dissociation of the ligase IV/XRCC4 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. We show that in Ad5-infected cells, loss of ligase IV is accompanied by loss of DNA binding by XRCC4. Expression of E1B 55k and E4 34k was sufficient to cause loss of ligase IV and loss of XRCC4 DNA binding. Using ligase IV mutant human cell lines, we determined that the absence of ligase IV, and not expression of viral proteins, coincided with inhibition of DNA binding by XRCC4. In ligase IV mutant human cell lines, DNA binding by XLF was also inhibited. Expression of both wild-type and adenylation-mutant ligase IV in ligase IV-deficient cells restored DNA binding by XRCC4. These data suggest that the intrinsic DNA-binding activities of XRCC4 and XLF may be subject to regulation and are down regulated in human cells that lack ligase IV.

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. Here, we describe a small-scale method for production of WCE that can be used to study NER. We also describe a rapid, small-scale method for the preparation of WCE that can be used in the study of NHEJ. These methods require less time, 20- to 1000-fold fewer cells than large-scale extracts, facilitate examination of numerous samples and are ideal for such applications as the study of host-virus interactions and analysis of mutant cell lines.

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., E1A, E1B). We report here that E1A inhibits androgen receptor (AR) target gene induction and, correspondingly, activated AR inhibits adenoviral replication. This mutual inhibition appears to be an indirect effect, possibly through competition for shared transcriptional co-activators. The net effect is that the oncolytic effect of prostate-specific CRAds is attenuated by these interactions. Fusion of the E1A to AR ameliorates this inhibition, while enhancing specificity. These findings have significant implications in the development of prostate-specific CRAd therapies.

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. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.

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. Protocols are presented for manipulation of E4, incorporation of E4 mutations into the viral genome, and growth of E4 mutants on complementing cell lines. A compilation of the described E4-complementing cell lines is included.

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.

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. Furthermore, we have identified a lentiviral Vif-like BC-box motif in E4orf6 that is highly conserved among adenoviruses from multiple species. More importantly, we have shown that this Vif-like BC-box is essential for the recruitment of Cul5-ElonginB-ElonginC E3 ubiquitin ligase by E4orf6 and is also required for E4orf6-mediated p53 degradation. E4orf6 selectively recruited Cul5 despite the lack of either a Cul5-box, which is used by cellular substrate receptors to recruit Cul5, or a newly identified HCCH zinc-binding motif, which is used by primate lentiviral Vif to recruit Cul5. Therefore, adenovirus E4orf6 molecules represent a novel family of viral BC-box proteins the cellular ancestor of which is as yet unknown.

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. Purified HPV16 VLPs are recognized by HPV16 neutralizing antibodies and induce high neutralizing titers when injected intraperitoneally into mice. Canine oral papillomavirus VLPs derived from previously described recombinants also induce strong antibody responses in mice. These data support our suggestion that viable adenovirus recombinants will be able to induce protective immunity to papillomavirus infection during replication in human vaccinees.