Capsid-incorporation strategy to display antigens for an alternative

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Review

Capsid-incorporation strategy to display antigens for an alternative adenoviral vector vaccine approach Claudia AJ van Winkel, Alberto Moreno, and David T. Curiel Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00591 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Molecular Pharmaceutics

Capsid-incorporation strategy to display antigens for an alternative adenoviral vector vaccine approach Claudia AJ van Winkel1,2,*, Alberto Moreno3,4, David T Curiel1

1

Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri, United States 2

Department of Chemical and Pharmaceutical Biology, University of Groningen, Groningen, The Netherlands 3

Emory Vaccine Center, Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, United States

4

Division of Infectious Diseases, Department of Medicine, Emory University, Atlanta, Georgia, United States Keywords Vaccine; Antigen, Capsid-Incorporation; Adenovirus; Virus; Capsid; Epitope Display * Corresponding Author: David T Curiel 660 S Euclid Ave St. Louis, MO 63110 Email: [email protected] Telephone number: +1 314 747-5443 https://orcid.org/0000-0003-3802-6014 Auteurs: Claudia AJ van Winkel 660 S Euclid Ave St. Louis, MO 63110 Email: [email protected] https://orcid.org/0000-0001-7299-1390 Alberto Moreno 954 Gatewood Road Atlanta, GA 30329 Email: [email protected] Telephone number: +1 404 727-8611

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The adenovirus (Ad) is widely used as a vaccine because of its ability to induce a cellular and humoral immune response. In addition, human clinical trials have validated the safety and efficacy of Ad as a vaccine vector. The traditional approach for employing the adenovirus as vaccine is to configure the antigen genes into the expression cassette of the Ad genome. An alternative method for inducing an immune response is the “capsid incorporation” strategy. This strategy is based upon the incorporation of proteins or peptides into the capsid proteins. This review will focus on the established uses of this approach as well as highlighting the new developments regarding the capsid incorporation strategy.

Graphical Abstract


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Introduction Adenovirus (Ad) is a double-strand DNA virus with a naked icosahedral protein capsid. Of note, Ad has been employed for a wide range of gene transfer applications including gene therapy and vaccines.1-3 In this latter regard, a number of practical advantages have facilitated the use of Ad vectors as a vaccine platform for a diverse array of disease targets. This expanding employ of Ad for vaccine applications derives from key technical advantages. These include ease of genome manipulation, ability to package large quantities of DNA, and genetic stability allowing facile derivation of new vectors and vaccine constructs. In addition, Ad can be grown at high titers, allowing practical implementation of large-scale vaccine production. These considerations have led to the application of Ad vectors in expanding number of human clinical trials. Importantly, these trials have validated successful vaccine outcomes in selected instances.4-8 The conventional approach to exploit Ad vectors for vaccine development has been based upon the configuration of transgenes into the expression cassette of the Ad genome (Figure 1). This is generally endeavored in context of Ad which has been rendered replication-defective by deletion of its early E1A/B genes. The incorporation of a transgene encoding the antigen of interest into the Ad genome results in the production of the transgene product without integration. The immune system can thereby recognize the expressed antigen, eliciting adaptive immune responses.9, 10 Relevantly, the Ad capsid has the ability to trigger innate immune responses during cell entry enhancing the immunogenicity when used as a vector for vaccine delivery. An alternative method to deliver antigens using Ad vectors is the “capsid incorporation” strategy (Figure 1). This novel approach is based upon the incorporation of immunogens of interest into Ad5 capsid proteins. By configuring the antigen into the capsid, it will be processed via the exogenous pathway.11 This is the pathway whereby proteins are normally endocytosed and degraded by acid-dependent proteases in the endosomes. This processing will lead to quantitatively and qualitatively distinct active immunization in comparison to the conventional approach. Based on these potential differences, the immune response elicited by capsid-modified Ad vectors may complement the immunity induced by Ad vectors delivering a transgene.12 The capsid proteins of the Ad possess robust intrinsic plasticity, potentially allowing the incorporation of antigens (Ag) into the capsid. The capsid incorporation strategy can thus be accomplished via several distinct capsid proteins including the hexon (polypeptide II), penton base (polypeptide III), fiber (polypeptide IV), polypeptide VII (pVII) and polypeptide IX (pIX) (Figure 2).13, 14 The utility of any specific capsid site is limited to the incorporation feasibility of the antigen and its manner to be displayed on the capsid surface. We will review here the current strategies used for Ad capsid incorporation and discuss their relevance for the development of novel vaccines.



Hexon capsid incorporation The hexon protein is the largest and most abundant protein of the Ad capsid proteins, with 720 monomers per virion (Figure 3). The hexon protein structures embody homo-trimers via the bundling of three monomers and their loops.15 Each hexon monomer contains seven flexible, serotype-specific loops, termed hypervariable regions (HVRs).16 These seven HVRs are categorized into nine HVRs. The HVRs are located on the surface of the hexon homo-trimer. This location enables the HVRs to interact with antibodies, receptors, cells, and proteins. In this regard, capsids with an Ag incorporated into their HVRs may be best positioned to allow effective antigen exposure.17 Consequently, the antigen incorporation strategy within different HVRs of the hexon protein has been explored. Incorporation of foreign peptides in the HVRs however, can affect the virion’s stability or function. To address this issue, Wu et al. studied different sites in the Ad hexon for feasibility to heterologous peptide incorporation. They found that a His6 epitope could be genetically incorporated into HVR2, HVR3, HVR5, HVR6, and HVR7, without interfering in virus formation and virion stability.18 Besides these HVRs, Shiratsuchi and colleagues reported that HVR1 also allowed capsid incorporation of heterologous peptides. This latter study incorporated a B cell epitope that is the target of neutralizing antibodies in a malaria model within the HVR1.19 Recently, Hansra et al. studied the insertion of a neutralizing epitope B corresponding to the porcine reproductive and respiratory syndrome (PRRS) virus into HVR7, HVR8 and HVR9. Their study showed that the insertion of the peptide into HVRs 8 and 9 was not tolerated. This is in contrast to the incorporation into HVR7 that resulted in the exposure of the epitope on the virion surface.20 In conclusion, HVR 1-3 and 5-7 tolerate Ag insertion. To investigate the maximum capacity of incorporation of peptides into the HVRs, Matthews et al. compared the insertion of identical epitopes of increasing size into HVR2 and HVR5. Their study showed that HVR2 allows insertion of 33 amino acids (aa) plus a 12 aa linker. The HVR5 was more permissive and allowed incorporation of 53 aa plus a 12 aa linker.12 Two other studies demonstrated higher permissibility; up to 57 aa in HVR2 and up to 77 aa in HVR5. These studies also showed that HVR1 accommodates insertions up to 90 aa.14, 21, 22 The noted study of the incorporation of a neutralizing B cell epitope from PRRS into HVR7 was the most extensive insertion in HVR7 performed. The epitope size was 9 amino acids (aa) with 3 linker aa on both sides, which makes the total incorporation 15 aa.20 In conclusion, HVR2 allows incorporation up to 57 aa, HVR5 up to 77 aa, HVR1 up to 90 aa and HVR7 up to 15 aa. With this knowledge, studies have evaluated the incorporation of different epitopes, of an expanded size, into the distinct HVRs. For example, Wu et al. employed the capsid incorporation strategy as a novel approach to delivering a human papillomavirus (HPV) vaccine. Their design was based on an Ad5 with the L2 protein incorporated in the hexon proteins. Current HPV prophylactic vaccines contain L1 virus-like particles. The disadvantage of using vaccine based on L1 is that the L1 protein is not crossreactive among HPV types. In contrast to L1, the L2 protein is highly conserved among HPV types. In this manner, Wu et al. constructed four viruses that have a part of HPV16 L2 protein inserted or substituted in HVR1 or HVR5. In all four recombinant hexon-modified vectors, the HPV L2 protein was successfully incorporated, without compromising virion replication. In addition, the vectors elicited sufficient


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antibody titers to confer protection against HPV16. However, the L2-recombinant viruses were not able to induce the expected cross-immunity against other HPV types, like HPV56.23 A similar study for a distinct disease target has been endeavored by Farrow et al. In this study, the Ag capsid-incorporation strategy was used to design a vaccine for Chagas’ disease based on the glycoprotein 83 (gp83) of Trypanosoma cruzi the causative agent. This antigen is a ligand that is used by T. cruzi to attach the host cell for infection. The Ag together with a His-tag were incorporated into HVR1 to generate the vector Ad5-HVR1-gp83-18. The recombinant vector was tested for immunogenicity in mice and protective efficacy elicited by immunization assessed after experimental challenge with a lethal dose of T. cruzi. Ad5-HVR1-gp83-18 was able to induce a robust immune responses with a significant reduction in parasitemia levels, induction of neutralizing antibodies, and increased survival rate. Relevantly, gp83 amino acid sequences from isolates from various geographical regions exhibit high identity suggesting that Ad5-HVR1-gp83-18 could have cross-reactivity against other T. cruzi strains.24 Comparable studies with an human immunodeficiency virus (HIV) domain within HVR1 in combination with a His-tag was reported by Gu and colleagues. . The design of this study was based on targeting domains that are involved in HIV binding and entry, specifically the well-defined V3 domain. Vaccine candidates based on the V3 peptide have proven to be safe and able to induce humoral and cellmediated immune responses in two different phase I clinical trials.25 On this basis, Gu et al. designed four different Ad vectors containing two different lengths of the V3 domain and with or without His6 tags incorporated into the HVR1: Ad-HVR1-lgs-His6-V3, Ad-HVR1-V3, Ad-HVR1-long-V3, and Ad-HVR1-lgs-V3His6-lgs. The Ad-HVR1-V3 and Ad-HVR1-lgs-V3-His6-lgs both failed to induce anti-V3 immune responses suggesting that a more extended peptide, and a proper spacer-linked peptide, may help V3 exposure and antigenicity. The two other vectors (Ad-HVR1-lgs-His6-V3 and Ad-HVR1-long-V3) showed promising results in neutralizing assays. However, only Ad-HVR1-lgs-His6-V3 significantly triggered V3-specific binding antibodies.21 Incorporation of His-tag Ag into HVR5 has been also reported by Gu et al. The cloning of the shuttle plasmid with a His-tag in HVR5 along with a shuttle plasmid containing the HIV epitope ELDKWAS within HVR1 was used by these authors to generate a recombinant Ad5 with ELDKWAS in HVR1 and His6 in HVR5. Both, the HIV epitope and the His-tag, were exposed on the capsid surface. The dual hexonmodified vector induced a robust humoral immunity specific to the HIV epitope and His6 in mice. This study, therefore, demonstrated the feasibility of capsid incorporation of multiple epitopes into different HVR in the same vector.22 Dual capsid incorporation has been also reported by Xue and colleagues using Ad3 and two epitopes incorporated into different HVRs. This multivalent Ad3 displays two neutralizing epitopes from enterovirus type 71 in HVR1 and HVR2. The goal of this Ad vector is to seek if antibodies were raised against one or both epitopes. Immunological assays showed that the recombinant vector elicited antibodies against both exogenous epitopes. In addition, this assay showed no significant difference between antibody titers. Furthermore, immunization with the multivalent Ad resulted in higher IgG titers and higher neutralization titers in comparison to an Ad vector with a single epitope within HVR1.26



An alternative approach has been explored by Fonseca and colleagues. They combined the capsid incorporation strategy with the conventional method in order to design a malaria vaccine candidate that targets both the liver and the blood stages of the infection. This malaria vaccine is an Ad5 with the Plasmodium yoelli T helper epitope (PyT53) in HVR2 and a Plasmodium yoelli chimeric multistage protein (PyCMP) driven by a CMV promoter in the E1 region. After immunization with this combinate virus, the antibody titers were not significantly different from the ones generated by the same virus with only transgene PyCMP inserted. However, the combined virus was able to induce a better humoral and cellular immune response. Specifically, a higher IgG2 to IgG1 ratio was observed, and the quality of the cellular immune responses was better resulting in the induction of multifunctional CD8+ T cells with the ability to produce INF-y and TNF-a simultaneously.27 Penton complex capsid incorporation In addition to the hexon HVR antigen incorporation, the capsid incorporation strategy has also been explored for the penton complex. This complex contains two of the major capsid proteins, the penton base, and the fiber. The penton base sits on the vertices of the icosahedral capsid and is involved in viral internalization. This internalization is mediated by binding to α√β3/5 integrins on the cell surface via the Arg-Gly-Asp (RGD) epitope in the penton base. The binding to the integrins receptor contributes to virus internalization.28, 29 The fiber extends from the penton base at each vertex and forms a knob at the end. The globular knob contains a receptor-binding domain, which can bind to coxsackievirus and adenovirus receptor (CAR). By binding of the knob domain to the CAR, the virus is attached to the cell surface, and the process of endocytosis can occur. 30 There have only been a few attempts to incorporate foreign peptides into the penton base. This fact may be due to structural constraints at the capsid base.31, 32 However, the capsid incorporation strategy is frequently performed in the fiber. The most common modification in the fiber is the addition of an RGD sequence in the HI loop of the knob. This insertion enhances infection of cells that express high levels of α√β3/5 integrins, in particular dendritic cells and cancer cells.33-37 Recently, Krause et al. combined the insertion of an RGD motif in the fiber with the display of the outer membrane protein F (OprF) of Pseudomonas aeroginosa on the capsid. They used a non-human primate-based AdC7 vector to develop the recombinant AdC7OprF.RGD vector. This vector resulted in higher and more persistent humoral immunity compared to Ad5-based vectors. Addition of the RGD motif did not interfere with the capacity of the vector to induce humoral immunity. Furthermore, the fiber-modified vector including the RGD motif induced high levels of mucosal antibodies, suggesting that the RGD enhanced the mucosal humoral response. However, the enhancement of Th1 responses by an Ad5 fiber with RGD motif was not seen with the AdC7OprF.RGD. Nevertheless, protective immunity was increased by the addition of RGD to the AdC7 fiber. The target cells responsible for this effect are nonetheless unknown.38 Besides the RGD motif incorporation into the Ad fiber, other foreign proteins can also be inserted. For example, Klein and colleagues incorporated the Epidermal Growth Factor Receptor Variant III (EGFRvIII), a deletion mutant expressed in a variety of tumoral cells, in the fiber of an oncolytic Ad. This recombinant virus was constructed to test if autophagy regulates the processing of adenoviral proteins


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for antigen presentation. They found that this fiber-modified Ad elicited the production of anti-EGFRvIII antibodies,that recognized infected cancer cells. Moreover, their study showed that autophagy of viral antigens was necessary for antigen presentation. Inhibition of autophagy therefore drastically decreased that capability of antibodies to recognize the infected cancer cells. This data suggested that combination of Ad with autophagy inducers may enhance the presentation of antigens in the capsid proteins.39 In an effort to define Ad interactions with its receptors and the influence of innate immune pathways equivalent studies have been performed by Anchim and colleagues. They designed an Ad with an ovalbumin-derived B-cell epitope in the fiber. Firstly, their results showed that the efficacy of the capsid incorporation strategy does not depend on Ad infection and interaction with its natural receptors. Secondly, the toll-like receptor (TLR)/MyD88 and the RIG-I/mitochondrial antiviral-signaling (MAVS) innate immunity pathways were not required to produce an epitope antibody response. Thirdly, their results also show that the TLR/MyD88 pathways, not the MAVS pathway, determines the nature of the antibodies against the presented epitope.40 pIX capsid incorporation pIX is one of the minor proteins of the Ad capsid. There are 240 copies of the pIX protein per virion, positioned between nine hexons in each facet of the capsid. Because of this, the pIX protein functions as “cement” for the Ad capsid and stabilizes the structure.41 In contrast to the major capsid proteins, the Cterminal of pIX tolerates fusion of relatively large and functional proteins up to 30-120 kDa.42, 43 Moreover, incorporation of distinct proteins does not impact the function of the pIX protein.44 In this regard, Gu et al. developed Ad vectors with HIV-1 epitopes incorporated within HVR1 or pIX to assess immune responses elicited in comparative experiments. The authors used the variable loop 2 (V2) of HIV-1 gp120 for these experiments. Three different sizes of the V2 loop were explored 20 aa, fulllength (43 aa) and a combination of V1/V2 (67aa). The HVR1 allowed incorporation of the 20 aa and fulllength V2 and pIX allowed insertion of V1V2. V2-specific antibodies from Ad-HVR1-V2 immunization were elicited in the following order of the Ad vectors tested, starting with the highest magnitude AdHVR1-V2 followed by Ad-pIX-V1V2 and Ad-HVR1-V2-20aa representing the less immunogenic. . This difference can be explained by the fact that the hexon has a three-fold more molecular copy per virion. However, Ad-HVR1-V2 and Ad-pIX-V1V2 both induced robust levels of anti-V2 antibodies to both linear and discontinuous V2 epitopes.45 Mattews et al. incorporated the amastigote protein (ASP-M) in pIX of Ad5 in an attempt to design an effective vaccine for the Chagas’ disease. This latter study was an extension of their earlier mentioned study of a capsid-modified vector where the gp83 neutralizing epitope was inserted into HVR1. Ad5-pIXASP-M elicited T. cruzi-specific effector CD8+ T-cell responses and neutralizing antibodies that reduced the parasitemia and extend the survival rates. Furthermore, their study demonstrates the efficacy of coimmunization with both adenoviruses, Ad5-pIX-ASP-M and Ad5-gp83. Co-immunization generated ASPM activated CD8+ T lymphocytes that reduce T. cruzi parasitism by secreting IFNγ and TNFα induced by Ad5-pIX-ASP-M and Ad5-gp83 reduced parasitemia by eliciting neutralizing antibodies. Moreover, the high significant identity, as described earlier, between epitope gp83 and ASP-M suggest that the vaccines can be applied for geographically diverse T. cruzi strain proteins.46



Instead of utilizing an Ad5 vector for pIX foreign protein incorporation, Salisch et al. explored the use of an Ad35 vector. They designed human Ad35 with two different lengths of the P. falciparum, circumsporozoite protein (CSP), and a CSP-short, and two different linkers named glycerine linker and 45Å-spacer. In contrast to the expectation, incorporation of full-size CSP (376 aa) into pIX failed. This fact indicates that other factors than size may influence the successful incorporation of foreign proteins into pIX. Furthermore, the glycine-linker may be able to make the CSP more flexible in its interaction with the B-cell receptor. The 45Å-spacer may be able to present the antigen at the hexon surface, which may enhance the full-length display of the antigen at an optimal distance from the Ad capsid. Overall, this study demonstrated an increased humoral immune response and a robust cellular response. Specifically, the Ad35 with CSP-short displayed in pIX significantly increased the potency of the antigen to induce CS-specific antibody titers in comparison to those induced by the CSP-transgene expressed Ad35.42 The three prior mentioned articles showed pIX incorporation in human Ads. To optimize the capsid incorporation strategy in pIX, Matteson and colleagues investigated the structural differences between the human adenovirus D26 (HAdV-D26) and the bovine adenovirus 3 (BAdV3) in pIX organization and antigen display. The C-terminal molecules of the HAdV-D26 are assembled into a tetrameric coiled-coil structure termed 4 helix bundle (4-HLXB), while the C-terminus of BAdV3 forms compact trimeric structures composed of triskelions and trimeric coiled-coil structures. Incorporation of foreign proteins in pIX in the HAdV-D26 could disrupt the formation of 4-HLXB due to sterical hindrance and therefore potentially affect the capsid stability. Nevertheless, the capsid incorporation strategy into pIX in BAdV3 is unlikely to influence the trimeric coiled-coil and triskelions structures. Also, the antigen is more efficiently displayed on the BAdV3 surface. These results suggested that a BAdV3 is more advantageous to present antigen in the C-terminal of pIX than HAdV-D26.47 pVII capsid incorporation pVII is the most abundant core protein of the Ad with approximately 800 copies per virion. It is associated with the Ad DNA by packing the genome in nucleosome-like structures.48 By this means, the pVII has several functions related to the Ad DNA. It promotes nuclear import of viral DNA and protects incoming viral DNA from the damage mechanism of the cellular DNA. Also, pVII can introduce changes in the viral DNA.49 A single article by Shiratsuchi et al has been published regarding pVII capsid incorporation. They designed a triple modified Ad that expressed the CSP of the human malaria parasite P. falciparum (PfCSP) as a transgene, a PfCSP B-cell epitope (NANP) in HVR1 and a PfCSP-specific CD4+ T-cell epitope in PVII. Incorporation of the PfCSP epitope in pVII seemed to strengthen the humoral response induced by the NANP epitope. Also, the level of CD4+ T-cell responses was higher with the combination of epitopes in the hexon and pVII. However, the insertion of the CD4+ T-cell epitope into pVII reduced the infectivity or transgene expression in vivo. Nevertheless, the triple modified Ad protected more mice than mice that received the conventional approach Ad or those who received the hexon-modified Ad.14


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Pre-existing immunity against Ad Supplementary to the enhanced humoral and cellular immune response, the capsid incorporation strategy exhibit the ability to circumvent preexisting immunity (PEI) against Ad5. 50-90% of the adult population carry neutralizing antibodies (NAbs) against the Ad5.50 These NAbs generated against capsid proteins play a primary part in the contribution to Ad5 PEI. The effect of the use of Ad5-based vectors in individuals with PEI limits the expression of the antigen due to Ad5 clearance by the immune system.51 The capsid incorporation strategy disguises the Ad5 capsid proteins, which lead to a modification of Ad5 neutralizing epitopes. Accordingly, this modification leads to a decrease in recognition of the neutralizing epitopes by the NAbs.52-54 Gu et al. combined the capsid incorporation strategy with an Ad5-based chimera to enhance the reduction of NAbs against the Ad5. An Ad5-based chimera was generated by substitution of the hexon of Ad5 with the hexon from Ad3 (H3). This viral chimera was further modified by incorporation of an His6-tag into HVR2 of the H3. The ELISA assay showed lower binding to Ad5-positive sera by Ad5H3HVR2-His and an in vitro neutralizing assay demonstrated the ability to escape Ad5 sera neutralizing. Furthermore, a robust immune response was generated against the His-tag. These results suggest that Ad5H3-HVR2-His can elicit a robust immune response and circumvent in vivo Ad5 PEI.55 Rojas et al. developed a unique approach to enhance the circumvention of PEI by developing an Ad with albumin incorporated into HVR1. Similar to the incorporation of distinct foreign proteins into the capsid, albumin shields the Ad5 capsid proteins from recognition by the immune system. The Ad was designed to contain the albumin-binding domain 3 (ABD) from the protein G of Streptococcus into HVR1. This virus allowed same organ transduction and oncolysis in naïve mice and adenovirus pre-immune mice, while a non-modified virus was neutralized entirely in adenovirus pre-immune mice. These results suggest that the ABD incorporated Ad5 can shield the capsid proteins and evade the formation of NAbs. The new ‘capsid incorporation strategy’ for adenoviruses described in this review article could potentially be useful strategies for vaccination for a variety of pathogens.56 Conclusion remarks and future directions The technological opportunities offered by the capsid incorporation approach are fully commensurate with other strategies linked to capsid modification of Ad. In this regard, capsid modification can alter Ad tropism allowing enhanced and targeted delivery to key immunoregulatory dendritic cells for enhanced vaccine efficacy.57 It would thus be technically feasible to combine Ad vector targeting with the capsid incorporation approach. In addition, the recent use of non-human Ad serotypes has provided an effective means to circumvent pre-formed immunity to vaccine vectors based on human Ad serotype5 (hAd5).58 It should thus be likewise feasible to accomplish capsid incorporation of antigens into such non-human Ad, to gain the benefits of both engineering technologies. Overall, the very recent advent of this technology as well as the limited number of reports comparing the capsid-incorporation strategy to the conventional approach, must presently limit a full understanding of precise advantages. Nonetheless, the expanding number of studies, and the diversifying range of evaluations will define the precise utilities embodied in this vaccine technology. 59



Acknowledgments We would like to thank the Stripendia Fond of the Royal Dutch Pharmacists Association, Marco Polo fund, and Groningen University Fund of the University of Groningen for support of Claudia van Winkel expenses. We also would like to thank Dr. S Peter Goedegebuure for his thoughtful insights.


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Figure 1

Figure 1) Design strategies for employment of adenoviral vectors for vaccine applications. A) Conventional approach whereby the gene of interest is configured into E1A/B region of the Ad genome. B) Capsid incorporation strategy whereby the protein or peptide of interest (green) is incorporated into the hexon (orange).

Figure 2

Figure 2) Schematic representation of adenovirus capsid proteins that permit foreign protein or peptide incorporation. The hexon, penton base, fiber and pIX are capsid proteins and pVII is a core protein.



Figure 3

Figure 3) Crystal structure of adenovirus highlighting utility of hexon for capsid incorporation strategy. Hexon hypervariable regions (HVRs) that allow foreign protein capsid incorporation, only one region of every HVR marked. One the right is an example showing incorporation into HVR1 and HVR7.


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