Recent studies have demonstrated the importance of coagulation factor X (FX) in adenovirus (Ad) serotype 5-mediated liver transduction in vivo. FX binds to the adenovirus hexon hypervariable regions (HVRs). Here, we perform a systematic analysis of FX binding to Ad5 HVRs 5 and 7, identifying domains and amino acids critical for this interaction. We constructed a model of the Ad5-FX interaction using crystallographic and cryo-electron microscopic data to identify contact points. Exchanging Ad5 HVR5 or HVR7 from Ad5 to Ad26 (which does not bind FX) diminished FX binding as analyzed by surface plasmon resonance, gene delivery in vitro, and liver transduction in vivo. Exchanging Ad5-HVR5 for Ad26-HVR5 produced deficient virus maturation. Importantly, defined mutagenesis of just 2 amino acids in Ad5-HVR5 circumvented this and was sufficient to block liver gene transfer.In addition, mutation of 4 amino acids in Ad5-HVR7 or a single mutation at position 451 also blocked FX-mediated effects in vitro and in vivo. We therefore define the regions and amino acids on the Ad5 hexon that bind with high affinity to FX thereby better defining adenovirus infectivity pathways. These vectors may be useful for gene therapy applications where evasion of liver transduction is a prerequisite. (Blood. 2009;114:965-971) IntroductionAdenovirus (Ad)-based vectors are used frequently for preclinical gene delivery and therapy and have been used in more than 25% of gene therapy clinical trials conducted to date. Although adenovirus serotype 5 is the most commonly used serotype, the human and nonhuman adenovirus families are large, and many of these are being exploited in diverse clinical applications, such as cancer gene therapy and vaccination. [1][2][3][4] However, the use of Ad vectors as gene delivery tools has raised several safety concerns. The importance of such issues was highlighted in the recent STEP trial in which patients were vaccinated against human immunodeficiency virus using an Ad5 gene delivery vector. The trial was terminated because the vaccine did not function as expected, but actually increased infection rates in those patients with preexisting antibodies to Ad5. Together with other adverse events in humans transduced with Ad5, 5 this highlights the importance of understanding fundamental aspects of Ad biology.In vitro, the interaction of the Ad5 fiber and the coxsackie and adenovirus receptor (CAR) is the major pathway for Ad cell binding. 6,7 Similarly, engagement with integrins by the penton base protein mediates internalization after cell binding. 8 Although other candidate receptors for Ad5 have emerged since the interaction with CAR was identified, 9,10 the role of these receptors in gene transfer after intravascular gene delivery has not been substantiated.It is well established that Ad5 predominantly transduces rodent liver after intravascular injection, 11 however mutations of the Ad5 fiber and/or penton show limited effects on liver gene transfer mediated by Ad5 (reviewed in Nicklin et al 12 ). Thereafter, it w...

Replication-defective adenovirus (rAd) vectors are powerful inducers of cellular immune responses and have therefore come to serve as useful vectors for gene-based vaccines, particularly for lentiviruses and filoviruses, as well as other nonviral pathogens (14,34,39,40,43,44,46). Adenovirus-based vaccines have several advantages as human vaccines-they can be produced to high titers under good manufacturing practice (GMP) conditions and have proven to be safe and immunogenic in humans (2,6,12,16,18). While most of the initial vaccine work was conducted using rAd serotype 5 (rAd5) due to its significant potency in eliciting broad antibody and CD8 ϩ T-cell responses, preexisting immunity to rAd5 in humans may limit efficacy (5-7, 29). This property might restrict the use of rAd5 vectors in clinical applications for many vaccines that are currently in development, including those for Ebolavirus (EBOV) and Marburg virus (MARV).To circumvent the issue of preexisting immunity to rAd5, several alternative vectors are currently under investigation. These include adenoviral vectors derived from rare human serotypes and vectors derived from other animals, such as chimpanzees (1,39,49). Research on the use of animal-derived adenoviral vectors is relatively nascent, while human adenoviruses possess the advantages of having well-characterized biology and tropism on human cells, as well as documented manufacturability (48). Immunogenicity of these vectors and their potential as vaccines has been demonstrated with animal models, primarily as prime-boost combinations with heterologous vectors (1, 41).Adenovirus seroprevalence frequencies are cohort dependent (28), but among the large group of 51 human adenoviruses tested, Ad35 and Ad11 were the most rarely neutralized by sera from six geographic locations (49). rAd35 vector vaccines have been shown to be immunogenic in mice, nonhuman primates (NHPs), and humans and are able to circumvent Ad5 immunity (4,30,31,36,47). rAd35 vectors grow to high titers in cell lines suitable for production of clinical-grade vaccines (13) and have been formulated for injection as well as stable inhalable powder (15). These vectors show efficient transduction of human dendritic cells (8,26) and thus have the capability to mediate high-level antigen delivery and presentation.Prime-boost regimens based on vectors derived from closely related adenovirus serotypes, such as Ad11 and Ad35, both from subgroup B, are less immunogenic than combinations of more genetically and immunologically distinct adenoviral vec-* Corresponding author. Mailing address:

The field of adenovirology is undergoing rapid change in response to increasing appreciation of the potential advantages of adenoviruses as the basis for new vaccines and as vectors for gene and cancer therapy. Substantial knowledge and understanding of adenoviruses at a molecular level has made their manipulation for use as vaccines and therapeutics relatively straightforward in comparison with other viral vectors. In this review we summarize the structure and life cycle of the adenovirus and focus on the use of adenovirus-based vectors in vaccines against infectious diseases and cancers. Strategies to overcome the problem of preexisting antiadenovirus immunity, which can hamper the immunogenicity of adenovirus-based vaccines, are discussed. When armed with tumor-associated antigens, replication-deficient and oncolytic adenoviruses can efficiently activate an antitumor immune response. We present concepts on how to use adenoviruses as therapeutic cancer vaccines and consider some of the strategies used to further improve antitumor immune responses. Studies that explore the prospect of adenoviruses as vaccines against infectious diseases and cancer are underway, and here we give an overview of the latest developments.

The search for a universal filovirus vaccine that provides protection against multiple filovirus species has been prompted by sporadic but highly lethal outbreaks of Ebolavirus and Marburgvirus infections. A good prophylactic vaccine should be able to provide protection to all known filovirus species and as an upside potentially protect from newly emerging virus strains. We investigated the immunogenicity and protection elicited by multivalent vaccines expressing glycoproteins (GP) from Ebola virus (EBOV), Sudan virus (SUDV), Taï Forest virus (TAFV) and Marburg virus (MARV). Immune responses against filovirus GP have been associated with protection from disease. The GP antigens were expressed by adenovirus serotypes 26 and 35 (Ad26 and Ad35) and modified Vaccinia virus Ankara (MVA) vectors, all selected for their strong immunogenicity and good safety profile. Using fully lethal NHP intramuscular challenge models, we assessed different vaccination regimens for immunogenicity and protection from filovirus disease. Heterologous multivalent Ad26-Ad35 prime-boost vaccination regimens could give full protection against MARV (range 75%-100% protection) and EBOV (range 50% to 100%) challenge, and partial protection (75%) against SUDV challenge. Heterologous multivalent Ad26-MVA prime-boost immunization gave full protection against EBOV challenge in a small cohort study. The use of such multivalent vaccines did not show overt immune interference in comparison with monovalent vaccines. Multivalent vaccines induced GP-specific antibody responses and cellular IFNγ responses to each GP expressed by the vaccine, and cross-reactivity to TAFV GP was detected in a trivalent vaccine expressing GP from EBOV, SUDV and MARV. In the EBOV challenge studies, higher humoral EBOV GP-specific immune responses (p = 0.0004) were associated with survival from EBOV challenge and less so for cellular immune responses (p = 0.0320). These results demonstrate that it is feasible to generate a multivalent filovirus vaccine that can protect against lethal infection by multiple members of the filovirus family.

The filoviruses Marburgvirus (MARV) and Ebolavirus (EBOV) are endemic primarily to central Africa and cause a severe form of viral hemorrhagic fever. Of all the filovirus strains or species, the Angola strain of MARV is associated with the highest mortality rate (90%) in humans observed to date (26). An increase in natural filovirus outbreak frequency over the past decade and the potential for use to cause deliberate human mortality have focused attention on the need for therapeutics and vaccines against filoviruses. While regulatory pathways have been proposed to facilitate licensing of a preventive vaccine against potently lethal pathogens such as these, there is as yet no licensed vaccine for use in humans, and efforts remain targeted to the optimization of vaccine performance in nonhuman primates (NHP) since this animal model recapitulates many aspects of disease pathogenesis observed in humans.Genetic vaccines are a promising approach for immunization against pathogens that are rapidly changing due to natural evolution, cross-species transmission, or intentional modification. Gene-based vaccines are produced rapidly and can be delivered by a variety of vectors. DNA vectors are advantageous because they are inherently safe and stable and can be used repeatedly without inducing antivector immune responses. However, while filovirus DNA vaccines have demonstrated efficacy in small animal models, efforts to induce protective immunity by injection of plasmid DNA alone into NHP have yielded less encouraging results. EBOV DNA vectors generate immune protection in mice and guinea pigs, but this has not been demonstrated in NHP unless DNA immunization is boosted with a viral vector vaccine (23). MARV DNA fully protects mice and guinea pigs but provides only partial protection in NHP (17). The discordant results between rodent and primate species may be due to the use of slightly modified infectious challenge viruses in rodent models or may reflect underlying differences in vaccine performance and the mechanisms of immune protection between rodents and NHP.In the current study, we examined whether DNA plasmidbased vaccines could be improved to increase potency in NHP and compared immunogenicity of this vaccine modality with those of viral vector and prime-boost approaches. DNA-vectored vaccines were modified by codon optimizing gene target inserts for enhanced expression in primates. These vectors induced antigen-specific cellular and humoral immune responses similar to immunization using a recombinant adenoviral vector and provided protection after lethal challenge with MARV Angola. However, macaques vaccinated with DNA