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Article details Manuscript ID: ____430912________________________ First author: ____Andrea Amalfitano _______ Title: _____________Overcoming pre-existing Adenovirus immunity by genetic_________________ ________________________engineering of Adenovirus-based vectors____________________________ Journal title: Expert Opinion on Biological Therapy

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Journal: Expert Opinion on Biological Therapy (EOBT) Title: Overcoming pre-existing Adenovirus immunity by genetic engineering of Adenovirus-based vectors ID: 430912 Corresponding author: Andrea Amalfitano AUTHOR: Please complete the colour figure order form that accompanies your proof if you require figures to appear in colour in the print version of the journal; once you have completed your article and credit card details, email or fax the form back to the production editor. The following queries have arisen during the editing of your manuscript. Please answer the queries by making the necessary corrections on the CATS online corrections form. Once you have added all your corrections, please press the SUBMIT *button.

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Query AQ1: Please confirm that the declaration of interest is reflective of the submitted form and relevant to this article. AQ2: Ref [99] may be available in print by the time this review is published. AQ3: Please provide volume, page range for ref [94]. AQ4: Please check the page range is ok for ref [59].

Review 1

Overcoming pre-existing Adenovirus immunity by genetic engineering of Adenovirus-based vectors

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Introduction

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Ad capsid modifications

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Alternative Ad serotypes

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Genome modified Ad5 vectors

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Expert opinion and overall summary

Sergey S Seregin & Andrea Amalfitano† †

Michigan State University, Department of Microbiology and Molecular Genetics, 4194 Biomedical and Physical Sciences Bldg, East Lansing, MI 48823, USA

Adenovirus (Ad)-based vectors offer several benefits showing their potential for use in a variety of vaccine applications. Recombinant Ad-based vaccines possess potent immunogenic potential, capable of generating humoral and cellular immune responses to a variety of pathogen-specific antigens expressed by the vectors. Ad5 vectors can be readily produced, allowing for usage in thousands of clinical trial subjects. This is now coupled with a history of safe clinical use in the vaccine setting. However, traditional Ad5-based vaccines may not be generating optimal antigen-specific immune responses, and generate diminished antigen-specific immune responses when pre-existing Ad5 immunity is present. These limitations have driven initiation of several approaches to improve the efficacy of Ad-based vaccines, and/or allow modified vaccines to overcome pre-existing Ad immunity. These include: generation of chemically modified Ad5 capsids; generation of chimeric Ads; complete replacement of Ad5-based vaccine platforms with alternative (human and non-human origin) Ad serotypes, and Ad5 genome modification approaches that attempt to retain the native Ad5 capsid, while simultaneously improving the efficacy of the platform as well as minimizing the effect of pre-existing Ad immunity. Here we discuss recent advances in- and limitations of each of these approaches, relative to their abilities to overcome pre-existing Ad immunity.

35 Keywords: adaptive immunity, cellular responses, humoral responses, innate immunity, neutralizing antibodies, recombinant Adenovirus Expert Opin. Biol. Ther. (2009) 9(12):1-11

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Introduction

Recombinant, Adenovirus (Ad)-based gene transfer vector platforms are heavily utilized in both gene therapy and vaccine applications. This utility is based on a number of positive attributes inherent to the use of recombinant Ads. Recombinant Ads are capable of transducing foreign genes into a wide range of cell types including antigen presenting cells; allow for efficient transgene expression, have large cloning capacities (8 – 36 kb); and can be easily produced to extremely high titers in a current good manufacturing practices (cGMP)-compliant fashion (up to 1 ! 1013 virus particles/ml). The ability to easily ‘scale-up’ traditional Ad vectors in a manner that is cGMP-compliant, has resulted in thousands of patients safely receiving recombinant, Ad5-based gene transfer vectors. In fact, it is the authors opinion that the proven ability to mass-produce Ad-based vectors makes them one of the most pragmatic of all gene transfer vectors currently proposed for direct in vivo human use. For example, the lack of simple, efficient, and large-scale cGMP production capabilities has limited the clinical utilization of a number of other 10.1517/14712590903307388 © 2009 Informa UK Ltd ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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proposed gene transfer platforms (i.e., Adeno-associated virus (AAV) or lentivirus based systems) to a handful of human clinical trials (see: http://www.wiley.co.uk/wileychi/genmed/ clinical/). There are, however, several limitations to the use of recombinant Ad vectors as a vaccine platform, the primary one of which is the decreased efficacy of these platforms, especially in Ad-immune individuals, concerns that are the focus of this review. Recently, another concern has been raised, regarding the use of Ad-based vectors, namely the possibility that administration of HIV-specific Ad vaccines into individuals with pre-existing anti-Ad immunity might ‘enhance’ or predispose the vaccinees to infection by HIV. We will discuss and address this issue first, and use the observations to validate the efforts being undertaken by several groups to overcome problems associated with the diminished efficacy of Ad-based vaccines, especially in the context of pre-existing Ad immunity. The STEP trial Most recently, the Merck sponsored STEP trial revealed that a E1 deleted ([E1-]) Ad5-based HIV specific vaccine (consisting of a cocktail of three [E1-] Ad5 vectors respectively expressing the HIV-1 derived gag, pol and nef antigens) failed in preventing HIV infection in vaccinees. This disappointing outcome positively correlated with poor elicitation of antiGag-specific T cell responses in the Ad5 vaccinees, responses that were especially blunted in Ad5-immune individuals [1,2]. Simultaneously, it was widely reported that Ad5-immune vaccinees appeared to have higher rates of HIV infection than the Ad5-immune placebo group [1]. The STEP trial was designed so as to result in 50 trial participants getting infected by HIV per subgroup (Ad immune or Ad nonimmune groups, based on baseline Ad5 specific antibody titer analysis). In placebo-treated Ad5 immune participants, only 13 out of 528 (~ 2.3%) of these individuals in the Ad immune group of patients treated with a placebo subsequently went on to develop HIV-AIDS, while 29 out of 532 (~ 5.5%) of the Ad5 immune patients vaccinated with the Ad5 vaccine went on to develop HIV-AIDS. While in isolation, this result suggests ‘enhanced’ acquisition of HIV in Ad5-immune Ad5-HIV vaccine recipients, it must be noted that in Ad5 non-immune individuals the rates of HIV-AIDS in placebo and Ad5 vaccinated individuals were also ~ 5.1% and ~ 5.2% respectively [3-5]. Thus the Ad-immune Ad5-HIV vaccinees had a rate of HIV-AIDS infection identical to the rate of HIV-AIDS infection that occurred in both of the Ad5-naı¨ve treatment groups. As has been pointed out by others, if one is to conclude that being Ad5 immune ‘enhances’ the rate of HIV-AIDS infection in Ad5 vaccinees, one would be equally justified in concluding that being Ad5 immune and being treated with a placebo may prevent HIV-AIDS [5] Thus one caveat of the STEP trial is that for some reason the placebo-treated group of Ad5-immune clinical trial participants had a lower than expected HIV infection ‘event rate’ than all other trial subgroups, confounding the 1.1

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data. Reasons for this are several, and have recently focused on circumcision rates being more closely correlated with HIV infection that Ad5 immune status [1]. Furthermore, the Ad5 immune status criteria used in the STEP trial may itself be at fault [6-8]. For example, the potential induction of T cell memory responses in Ad5 immune individuals by pre-existing Ad5 antibodies has been forwarded as a possible mechanism for enhanced HIV infection rates in Ad5 immune vaccinees [6-8]. However, STEP trial derived patient samples, supposedly Ad5 ‘antibody negative’ STEP trial participants (that did not get infected with HIV) did, in fact have preexisting T cell responses to Ad5 [6-8]. Although these latter studies have not assessed all potential immune responses present in the trial participants (i.e., pre-existing mucosal immune responses), the studies strongly suggest that preexisting Ad5 immunity did not correlate with acquisition of HIV in STEP trial vaccinees. Due to these and other considerations, we feel that the use of Ad5-based vaccines should not be limited due to this sole concern. However, many in the lay press and, in fact, the scientific theatre wrongly hold the view that Ad-based clinical trials are no longer being conducted or pursued due to this and/or other issues. Thus, it is very important to note the fact that Ad5-based vectors are currently, the most widely used gene transfer vector in human clinical trials (http://www.wiley. co.uk/wileychi/genmed/clinical/). More recently, a major advantage to the use of Ad-based vaccines has also become apparent, with the confirmation that integrating viral vectors (i.e., retrovirus, lentivirus, possibly AAVs) can cause insertional mutagenesis and cancer in animal models and in human clinical trial subjects, therefore vaccines based on such vectors may also be subject to similar risks [9-13]. The natural biology of the recombinant Ad genome, which does not integrate into the host chromosome, largely mitigates insertional mutagenesis risks [14]. Efficient administration of Ads has been reported utilizing: systemic intravenous delivery [15,16], intranasal administration [17] and intramuscular [18-20], footpad [21], and/or subcutaneous injections [22]. More relevant to the point of this review, Ad5-based vectors have been repeatedly proven to be a potent vaccine platform, capable of delivering and expressing significant amounts of Ad vaccine expressed target antigens to antigen-presenting cells, inducing rapid and robust antigen specific cellular as well as humoral immune responses [23-30]. Ad-based vaccines have been proven to generate not only systemic immunity, but can also induce robust mucosal immunity when enterically or intramuscularly administered [18,31]. This capability has now been verified in humans, as STEP trial subjects intramuscularly vaccinated with Ad5-based vaccines were found to have increased populations of HIV-antigen-specific cellular (T cell) immune responses, a feat not accomplished by previous use of DNA or poxvirus-based HIV vaccines expressing the same antigens [1,32-36]. These immune responses, were severely blunted in Ad5-immune individuals, and

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were also not potent enough to prevent HIV acquisition even in Ad-naı¨ve subjects. Pre-existing, Ad5 immunity Unfortunately, a majority of the human population has been infected by wild type Ads in childhood, and often these individuals develop pre-existing immunity to a number of common Ad serotypes (i.e., serotypes 2, 4, 5 and 7). The percentage of the human population with medium to high Ad-specific antibody titers largely depends upon geographic location, with 30 – 50% of the United States population, and up to 90% of the sub-Saharan African populations having significant Ad5 antibodies with an overall trend to having higher titers in developing countries [32,37,38]. Pre-existing immunity to Ad can include both neutralizing antibodies (NAb), as well as Ad-protein-specific T cell responses. Additionally, recent studies highlight a critical role for CD8+ T cells in pre-existing Ad immunity [39,40]. This latter point is very important, as these pre-existing responses can in fact still be harnessed when a host is exposed to a novel (previously unencountered) Ad serotype [41-44]. Therefore, in this review we discuss strategies that have been proposed to improve the efficacy or utility of Ad based vaccines. Sometimes these strategies can overcome specific aspects of the pre-existing Ad immune response, as well potentially allowing for improved vaccination of Ad-immune individuals with these same recombinant Ad-based vaccines [25,41,45]. 1.2

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Ad capsid modifications

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Chemical modifications of the Ad capsid

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Several groups have chemically modified the Ad5 capsid (i.e., in a non-covalent fashion) in an attempt to shield or hide highly antigenic epitopes on the viral capsid (mainly hexon protein) from the immune system of the Ad-immune host. This strategy primarily involves the addition of synthetic polymers onto the Ad capsid. These polymers can include PEG [46], polyactic glycolic acid (PLGA) [47] or other lipids [48]. PEGylation appears to be the most promising approach for non-covalent modification of Ad capsids. Ad5 PEGylation may allow for re-administration of such modified Ads in mice. For example, animals were given intravenous injections of an Ad5 vector expressing an irrelevant antigen, and then second injections of conventional or PEGylated Ad5 vectors each expressing the bacterial b-galactosidase gene (b-Gal) [16]. High levels of b-Gal expression were found only in Ad5-immune mice that received the the PEGylated Ad5 vector [16]. Moreover, it was shown that PEGylated Ad5 vectors also reduce the induction of Ad5-triggered innate toxicities, such as thrombocytopenia, plasma alanine aminotransferae (ALT) elevations, and release of pro-inflammatory cytokines such as IL-6 [16,49]. PEGylation with a smaller PEG (5 kDa) moiety increases transduction efficiency of the Ad5 capsid in Ad-immune mice [50]. Use of a 5 kDa PEGylation of Ad5

can also reduce Ad5-specific neutralizing antibody (Nab) titers generated after intranasal injection into Ad-naive BALB/c mice [17]. However, no significant differences in generating antigen-specific immune responses were found when different size PEGylated Ads (3 – 35 kDa PEG) and/or different injection routes were analyzed [17]. Several promising trends were noted, mainly highlighting that PEGylated Ads may be better as a ‘boosting’ vaccine relative to conventional Ad vaccines [17]. How PEGylation may affect the efficiency or tropism of Ad5-mediated gene transduction in vivo has not been fully delineated. For example, a dramatic reduction of liver transduction was reported after Ad PEGylation [51], while other studies, reported equal and/or increased liver transduction by PEGylated Ads [16,49]. In summary, characterization of how PEGylation affects the ability of so modified Ad vaccines relative to tropism changes, ability to transduce lymphoid tissues, ability to evade pre-existing Ad5 NAb and/or T cell immunity, is still required to determine if these strategies afford improved utility of PEGylated Ad vaccines, especially in the Ad-immune host.

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Genetic modification of the Ad capsid Insertion of novel peptides into Ad capsid proteins 2.2

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The non-enveloped Ad virion is composed of a large capsid containing nine proteins. The hexon (protein II), penton base (protein III) and fiber (protein IV) proteins are the most abundant, and are so termed the major capsid proteins, while proteins IIIa, VI, VIII and IX are referred to as the minor capsid proteins [52-54]. The fiber, penton, protein IX and hexon proteins have been exploited for genetic insertion of foreign peptides into these specific capsid proteins, either as ‘in-frame’ insertions within the proteins, or as ‘in-frame’ C-terminal fusions. If the insertion does not cause a deleterious change in the biology of the so-modified protein, (i.e., causes misfolding, lack of stability, lack of trimerization or lack of incorporation into the tertiary structure of the Ad capsid) novel, recombinant Ad vectors now ‘capsid-displaying’ the respective foreign peptide can be isolated. The penton and hexon proteins tolerate relatively small peptide insertions: up to 18 amino acids (aa) for penton base [55,56], and up to 36 aa within the hypervariable region 5 (HVR5) of hexon [19,57], whereas the HI loop of the fiber knob and C-terminus of protein IX allow for incorporation of peptides of substantial size: up to 83 aa for fiber [58] and up to 1018 aa for pIX [59]. Issues regarding capability of generating these recombinant viruses consistently, to very high titers, or in a cGMP-compliant fashion have many times not been addressed in studies published to date, but will need to be if clinical deployment of these vectors is to be achieved. The insertion of foreign peptides into capsid proteins in attempts to change the natural tropism of the Ad vector have been well-described [60,61]. However, in this review, we will shift our focus to studies using Ad ‘capsid-display’ as a method to introduce pathogen-derived antigens into a host to generate


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immune responses to the respective antigens. For example, antigenic epitopes derived from the hemagglutinin (HA) protein of the influenza A virus were incorporated into and displayed from the Ad capsid as hexon, penton base, fiber knob or protein IX fusions [23]. The immunogenicity of these novel vaccines was then cross-compared. The fiberdisplaying Ads induced the highest levels of HA-specific immunity, as determined by measuring production of HA-specific IgM and IgG humoral responses, as well as HA-specific IL-4- or IFN-g-producing CD4+ T cellular immune responses [23]. In another example, the immunodominant portions of the Bacillus anthracis protective antigen (PA) were genetically inserted and displayed from the HVR5 site of the Ad5 hexon [19,57]. Intramuscular injections of the novel vector into BALB/c mice resulted in generation of PA-specific IgG1 and IgG2a antibodies, possibly indicating that TH1 and TH2 immunity to the antigen was generated, surpassing the efficacy of synthetic peptide based vaccination strategies [19]. The capabilities of these novel vaccine platforms in the context of pre-existing Ad5 immunity have not been extensively reported. However, in one study, the HVR5 site of hexon has also been exploited for display of Pseudomonas aeruginosa B cell epitope-encoding peptide. Footpad vaccinations with the novel vaccines induced antibody responses to P. aeruginosa 2 – 4 weeks post injection [21]. Both IFNg-positive CD4+ and CD8+ P. aeruginosa specific T cell responses were also generated. Most convincingly, mice vaccinated with the P. aeruginosa B cell epitope displaying Ad were protected against subsequent lethal pulmonary challenge with several P. aeruginosa strains. Subsequent studies with Ad vaccines simultaneously expressing (as a transgene encoded by the Ad vaccine genome) and displaying P. aeruginosa antigens suggested an improved ability to increase humoral responses to the bacterially derived antigens upon boosting injection relative to repeated vaccinations with a conventional Ad5 vaccine expressing the same antigen [21,62]. More extensive future studies are required to shed light on whether ‘capsid-display’ is a viable approach to overcome pre-existing Ad immunity. Ad vaccine chimeras – modifications of hexon and/or fiber

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Classification of Ad serotypes have been historically based upon biological, biochemical and structural properties, including the lack of cross-neutralization between serotypes from different species. In fact, neutralization and hemagglutination tests are the most common clinical tests utilized to distinguish infections by different Ad serotypes. Subsequently such parameters as oncogenicity, length of fiber protein and genomic sequence similarity were taken into consideration to further sub-classify Ads [63,64]. The capsid proteins hexon and fiber are the major targets of Ad specific antibodies, many of which become neutralizing [40,65-69]. Some investigators have therefore begun to ‘swap’ portions of the Ad5 capsid with 4

homologous proteins from alternative serotype Ads, generating ‘chimeric’ vectors that have new properties as a result of the modifications. The Ad fiber and hexon proteins have been the two main targets for generation of chimeric Ads. Several fiber-chimeric Ads [70,71], including Ad5 chimeric Ads with fibers from non-human Ads [72] have been described. These novel vectors were primarily generated to alter the tissue tropism of the resultant vectors, since the fiber protein primarily interacts with specific cellular receptors, initiating virus transduction and thus directing vector tropism [72]. Subsequent studies revealed that Ad5 vectors lacking the fiber knob domain have reduced susceptibility to pre-existing Ad5 antibodies, supporting the notion that the fiber protein is one of the major targets of Ad antibodies in humans [69]. Incidentally, systemically administered Ad5/7 and Ad5/41 fiber chimeras had dramatically reduced activation of Ad triggered innate toxicities as compared to unmodified Ad5, but identical levels of transgene specific adaptive responses [15]. Although theoretically possible, the ability of fiberchimeric Ads to evade pre-existing Ad immunity has not been extensively reported. Initial studies suggested increased ability of a chimp AdC23/C24 fiber chimera to deliver and express a transgene in AdC23 pre-immune mice as compared with a AdC23 unmodified vector [73]. Subcutaneously injected Ad5/3 fiber knob chimeric vectors had an increased ability to evade pre-existing Ad5 immunity in immune-competent mice pre-immunized with unmodified Ad5 [22]. Hexon is the most abundant protein of the Ad capsid, and thus it stands to reason that hexon is also a major target for host neutralizing antibodies against Ads [40,65-68]. Therefore several reports have attempted to replace the Ad5 hexon, with hexon moieties derived from alternative Ad serotypes to evade pre-existing Ad5-neutralizing antibodies [74]. For example, chimpanzee derived AdC68-hexon-specific monoclonal antibodies predominantly recognize a small hexon loop region of the AdC68 hexon. Introduction of a mutation into this region allowed the modified virus to not only escape neutralization by the same monoclonal antibodies, but also reduced its neutralization by polyclonal sera [75]. While this study clearly provided evidence that the hexon R1 loop region is a major site for neutralizing antibodies, it did not directly address in vivo efficiency of the modified-hexon-containing vector, both in vector-naı¨ve and vector-immune hosts [75]. It was also recently shown that after intramuscular administration into Ad5 pre-immunized BALB/c mice, a modified Ad5 vaccine deleted for the HVR5 region of the Ad5 hexon was able to increase HIV env-specific humoral and cellular immune responses as compared with a conventional Ad5 vaccine expressing the same antigen [20]. More extensive modification of the hexon protein has also been described. In one study, hexon proteins, derived from 18 different serotypes were tested for their ability to replace the Ad5 hexon protein. However, only four serotypes yielded hexons capable of allowing for generation of viable chimeras [76]. Several groups now report the ability to produce


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Ad5 vectors with the Ad5 hexon replaced with hexons derived from Ad1 [76], Ad2 [66], Ad3 [68], Ad6 [76], and Ad12 [67]. Each of these chimeric vectors has shown an ability to evade pre-existing Ad5 immunity. While these studies provide proof of concept that hexonor fiber- swapped chimeric Ads can be viable, Ad5/48 or Ad5/26 hexon chimeric vaccines were also found to outperform Ad5-based conventional vaccine when administered into Ad5-immune animals. Most dramatically, a chimeric Ad5/48 with seven Ad5 hexon HVRs replaced with Ad48derived HVRs was constructed and produced, confirmed to be stable and infectious, and was shown to generate robust HIV-gag specific immune responses while evading pre-existing Ad5 immunity in a non-human primate model [77].

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Alternative Ad serotypes

Development of recombinant Ad vectors or vaccines, based entirely upon use of rarer alternative serotype Ads or nonhuman-derived Ads in lieu of Ad5, is a seemingly simple and straightforward alternative method for overcoming preexisting Ad5 immunity. Rare Ad serotypes can be selected based upon their minimal cross reactivity with Ad2- and Ad5-neutralizing antibodies, a feature, which by definition clinically delineated human Ad serotypes. Low seroprevalence for Ad serotypes from subgroups B (Ads 11, 35 and 50) and D (Ads 26, 48 and 49) justified their initial selection as possible alternatives to Ad5-based Ad vaccines, and testing for immunogenicity commenced in small animal models [32]. Interestingly, Ad5-based vaccines revealed the highest SIV-gag-specific T and B cell responses in Ad-naı¨ve mice, with Ad26 being the most potent of the alternative-Adserotype-derived recombinant vectors tested [32]. Subsequent studies, however, revealed that cross-reactive T cell responses between the various serotype-based vectors were still present and significant, suggesting that use of alternative-serotypederived Ad vectors will not substantially mitigate the problems associated with this form of pre-existing anti-Ad immunity [30,32,78,79]. Despite this reality, vectors based on Ad26 and Ad35, had the lowest cross-reactivity with Ad5 vectors and efficiently generated SIV-gag-specific immune responses in Ad5-pre-immune mice and non-human primates, whereas conventional Ad5 vaccines failed to generate substantial SIV-gag specific responses under the same conditions [30,78,79]. Recombinant-Ad26-based vaccines also elicited SIV-gag specific cellular immune responses with increased polyfunctionality (as measured by IFN-g/IL-2/TNF-a producing subpopulations of T cells), breadth and magnitude as well as increased humoral immune responses in Ad5 immune mice, as compared with use of an Ad5-based vaccine [30]. Importantly, utilization of priming vaccinations with an Ad26-based SIV-gag vaccine, followed by boosting immunization with the Ad5-based SIV-gag vaccine significantly improved survival of non-human primates subsequently

challenged with SIV infection, as compared with similar challenge studies utilizing Ad5 SIV-gag vaccine as both the priming and the boosting vaccine [30]. The improved capability of the Ad26/Ad5 combination also correlated with reduced SIV peak and setpoint viral loads, and a reduced rate of decline of the CD4 population in the SIV challenged animals [30]. Ad vectors derived from chimpanzee serotypes C68 and C1 expressing HIV-1 derived gag, pol, gp140 and Nef antigens were designed, produced, and tested in several animal models. AdC6-gag immunizations induced increased frequencies of HIV-gag specific CD8+ INFg + T cells in the Ad5-immune mice, as compared with the conventional Ad5-gag vaccine [33]. AdC68 was also shown to generate robust T cell responses in rhesus macaques even in the context of pre-existing Ad5 immunity, while the Ad5 vaccine was proven ineffective under these conditions [80]. These landmark studies not only confirmed that modification of Ad vectors can improve their efficacy in difficult vaccine applications (such as preventing SIV in SIV-challenge models) but also that these same maneuvers can overcome problems associated with pre-existing Ad5 immunity. 4.

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Genome modified Ad5 vectors 465

Nearly all clinical trials utilizing Ad-based vectors have made use of Ad5-based platforms. This track record for widespread use is important to consider, as regulatory agencies can utilize this accumulated knowledge to assess the safety risks of future trials utilizing similar platforms. Herein lies a very important dilemma. If an investigator chooses to utilize a capsid chimeric Ad vaccine, or a novel Ad vaccine based entirely upon use of an alternative-serotype-based Ad, that investigator must now demonstrate that the altered capsid of the novel vector does not significantly alter the biological, and/or clinical characteristics of the novel vector. Use of novel Ad capsids have been confirmed to have significantly altered biological characteristics in vivo, inclusive of significantly altered biodistribution profiles, significantly different induction of innate and pro-inflammatory immune responses, as well as heightened ability to cause toxicity relative to Ad5-based vector platforms in vivo [32,81-83]. Furthermore, lack of in vitro cross neutralization by Ad5-serotype-specific antibodies may not guarantee efficient evasion of all forms of pre-existing Ad5 immunity, especially in vivo [84]. For example, T-cell-based cross reactivity between distinct Ad serotypes has been confirmed, and may be more responsible for the lack of efficacy of Ad5-based vaccines in the Ad-immune host [35,85-91]. As but one specific example, it has been recently reported that CD8+ T cells against the E2b-encoded polymerase protein were found in Ad-immune subjects at similar frequencies as CD8+ T cells against hexon, the latter is considered a major immuno-dominant Ad protein [92]. Thus strategies that attempt to retain the Ad5 capsid, but modify portions of the Ad genome to minimize expression of Ad5 immuno-dominant


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proteins, have recently been described. E1-deleted Ad5 vectors (Ad5 [E1-]) are constructed in a manner such that a transgene replaces only the E1 region of genes; thus, 90% of the wildtype Ad5 genome is generally retained in the vector. In an effort to reduce immuno-dominant viral protein expression from Ad5 vaccines, multiply deleted Ad5 vectors, deleted for genes in addition to the E1 genes have been created [89,93-97]. Specifically, [E1-, E2b-] Ad5 vectors have the E1 region deleted, as well additional deletions in the E2b region, removing the Ad encoded DNA polymerase (pol) and the preterminal (pTP) protein genes [95]. These vectors can be grown to as high as titers as [E1-] Ads, utilizing identical procedures, and have an expanded cloning capacity, theoretically up to a 13 kb gene-carrying capacity as compared with the 8.0 kb capacity of Ad5 [E1-] vectors. A key aspect of the Ad5 [E1-, E2b-] vector is that expression of Ad5 proteins is greatly reduced as compared with [E1-] Ad5 vectors [90,95,98]. Several studies have demonstrated an improved safety of these genome-modified Ads in animal models [93]. We recently confirmed that the minimized ability of [E1-, E2b-] Ad5 vaccines to replicate or express late viral genes may result in evasion of pre-existing Ad5 cellular immunity, increasing the capability for the Ad5 [E1-, E2b-] vectors to induce beneficial immune responses in Ad5-immune animal models [89,94-97,99]. This confirmation included blunted induction of Ad-specific T cell proliferative immune responses by the [E1-, E2b-] Ad5 vaccines in the Ad5-immune hosts, results that were simultaneous with improved NK cell and antigen-specific CD8+ T cell responses to [E1-, E2b-] Ad5-expressed antigens, inclusive of HIV-gag and the tumor antigen CEA [94,97]. Upon immunization of Ad5-immune mice as well cynamalogous macaques made hyper-immune to Ad5, [E1-, E2b-] Ad5 vaccines induced HIV-gag-specific T cells producing IL-2, which can result in augmented CD4+ clonal expansion [97], and production of HIV-gag specific CD8+, IFN-g-secreting T cells. In a similar vain, ‘helper-virus-dependent’ Ads (HDAds) completely lack virus-encoded sequences, except for the viral ITRs, and allow for cloning capacities approaching 36 kb. These vectors can be grown to very high titers, and only require a helper virus in addition to specific packaging cell lines to allow for their large scale production. The absence of any Ad-encoded genes in HDAds has convincingly proven that many deleterious adaptive immune responses are directed to Ad genes [100]. In a recent study more relevant to this review, HDAds were also found to be capable of generating antigen-specific immune responses in Ad5-immune hosts [101]. Specifically, use of an HIV gp140 env-expressing HDAd5 yielded increased env-specific cellular and humoral immune responses in Ad5-pre-immune mice, and increased env specific antibody titers in Ad5 pre-immune macaques over fivefold, as compared with a traditional Ad vaccine [101]. These early studies suggests that Ad5-based vaccines can be improved upon to improve their efficacy in the face of pre-existing anti-Ad 6

immunity without requiring major alterations to their capsid 551 coat. Such vectors when used clinically will benefit from the long-term safety record of traditional Ad5-based vaccines, while potentially providing for improved efficacy in the 555 Ad5-immune host in general. 5.

Expert opinion and overall summary

The use of Ad5 vectors as a platform for vaccine development has been gaining steady momentum. Ad5-based vaccines have been repeatedly shown to outperform other vaccine platforms expressing identical antigens, including other Ad serotype vaccines [32-34], poxvirus based vectors [34,36], and DNA-based vaccines [35,36]. Additionally, the large number of patients safely treated with the Ad5 platform confirms its high likelihood of acceptance by regulatory bodies, relative to less well tested platforms, an important point when considering the considerable risks and costs involved in developing new therapeutics. Unfortunately, despite this tremendous track record for safe and potent induction of immune responses in vaccine applications, conventional, E1-deleted Ad5-based vaccines have several confirmed limitations. These include a sometimes less than optimal induction of antigen-specific adaptive immune responses, made worse in the context of pre-existing Ad immunity. These facts indicate a need for development of more potent Ad-based vaccines, vaccines that are efficacious even in the context of pre-existing Ad5 immunity. In this review, we have summarized how many groups have attempted to address the need to improve the efficacy of Ad-based vaccines in general, and/or avoid the problem of pre-existing Ad5 immunity specifically, as summarized in Figure 1. Some groups have proposed the utilization of alternative-human-Ad-serotype-based vaccines (some derived from non-human primates) as a relatively simple method to avoid pre-existing Ad5 immunity [30,82,102,103]. Use of these options has facilitated induction of improved T cell responses to Ad-expressed HIV antigens despite Ad5 immunity. These landmark studies also prove that improved induction of HIV-antigen-specific T cell responses can result in superior performance in very stringent SIV challenge studies in nonhuman primates, a feat that cannot be achieved with other vaccines, nor conventional [E1-] Ad5 vaccines [30,103-105]. Despite these achievements, use of alternative-Adserotype-based vaccines will also face neutralizing antibody induction after a single usage, and as we have detailed in the text, will probably be affected upon by crossreactive immune responses present in the Ad5-immune host [39,42-44,104]. More importantly, however, use of alternative-serotype-based Ad vaccines also poses several new serious problems including: i) lack of previous usage in humans; ii) significantly altered biodistribution profiles; iii) lack of induction of potent immune responses relative to Ad5 vaccines; and iv) induction of more deleterious inflammatory immune responses relative to Ad5, in some cases causing excessive morbidity and mortality in animal


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“HVR5 Hexon” displaying Ad5

Ad5 WT ITR E1 E2b

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ITR

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3 “Protein IX” displaying Ad

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[E1-] Ad

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[E1 , E2b ] Ad

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E1- region of Ad genome E2b- region of Ad genome Ad genome sequences Vector-encoded transgene Inverted terminal repeats

“Fully deleted” HDAd

Ad5/26 HVR5/7 chimera

2 “PEGylated” Ad vectors

Figure 1. Current approaches to design advanced Adenovirus based vaccines. Group 1. Starting with a wild-type Ad5 virus the E1 region is deleted to generate [E1-] Ad5 vectors; Group 2. Adenovirus vectors of any group can be PEGylated to improve evasion of pre-existing Ad immunity, see Section 2.1 for full descriptions; Group 3. Capsid ‘display’ of antigens at various locations on the Ad capsid where indicated, see Section 2.2.1 for full descriptions; Group 4. Chimeric Adenovirus vectors, that contain capsid proteins derived from two different Ad serotypes, see Section 2.2.2 for full descriptions; Group 5. Adenovirus vectors derived completely from alternative serotypes, see Section 3 for full descriptions; Group 6. Genome-modified Adenovirus vectors, that retain the parental serotype capsid, but are deleted for portions of the parental genome, to improve evasion of pre-existing cellular immunity, see Section 4 for full descriptions.

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models [32,81-83]. All of these caveats may prevent/hinder regulatory approval of alternative-serotype-based Ad vaccines for widespread deployment. With this in mind, we have also reviewed efforts to improve the Ad5 platform in a fashion that retains major portions of the Ad5 capsid. We have reviewed how subtle modifications (such as amino acid substitution of immuno-dominant peptides present in the Ad5 capsid, or swapping of the HVR region of the Ad5 hexon) can improve the efficacy of these modified, Ad5-based vaccines, in the Ad5-naı¨ve or Ad5-immune host. Not so subtle modifications include complete incorporation of Ad5 hexon or fiber proteins derived from alternative-serotype Ads into the Ad5 capsid. While these novel vaccine platforms also improve performance in

the context of the Ad5-immune host, they also may face the same regulatory issues facing vaccines constructed entirely from alternative Ad serotypes. Strategies that do not alter the Ad5 capsid (such as use of [E1-, E2b-] Ads or HDAds) may avoid many of the regulatory (safety) issues associated with proposed use of alternativeserotype Ad-based vaccines. These strategies may also provide an improvement that allows for efficacy in the most difficult of applications, such as for use as a HIV prophylactic vaccine in the Ad-immune host. However, these modifications may not overcome certain aspects of pre-existing Ad5 immunity as well. As a result of these considerations, it is clear that each of the modified Ad vaccines described in this manuscript may be better utilized for some clinical applications than for


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others. Despite this context-specific utility, one should also be impressed that the Ad vaccine platform appears to be highly ‘plastic’ and capable of tolerating a number of elegant molecular manipulations. In many instances, these modifications not only preserve important benefits inherent to the use of the traditional Ad5-based vaccine platform, but also provide for improved efficacy, even in the context of pre-existing Ad5 immunity. Future studies will expand upon these findings. Incorporation of some of the modifications summarized herein will probably improve the capabilities of this important vaccine platform for expanded use in a number of additional human and agricultural applications not targeted to date. One can also envision combining some of the approaches described in this review. For example, Ad5 vaccine platforms could be theoretically generated that are deleted for the E1 and E2b genes, as well having portions of the hexon HVR5 and HVR7 regions replaced with similar regions derived from Ad26.

PEGylation of such a novel vaccine could also then be con- 652 templated. Similarly, these multiple manoeuvres can be theoretically applied to any Ad serotype. However, theoretical contemplation of these multiply modified vaccines is not a 655 guarantee of reduction to practice, as nuances regarding compatibility of multiple manipulations to allow for viability and large-scale production of the resultant vaccine must be respected. We leave our readers with the view that despite these pragmatic considerations, the continued improvement 660 of Ad-based vectors is likely to yield several important vaccine platforms that will have high utility in a number of clinical and agricultural applications, even in the context of pre-existing Ad immunity. 665

Declaration of interest The authors state no conflict of interest and have received no 669 payment for preparation of this manuscript.

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Affiliation Sergey S Seregin1 & Andrea Amalfitano†1,2 † Author for correspondence 1 Michigan State University, Department of Microbiology and Molecular Genetics, 4194 Biomedical and Physical Sciences Bldg, East Lansing, MI 48823, USA Tel: +1 517 884 5324; Fax: +1 517 353 8957; E-mail: [email protected] 2 Michigan State University, Department of Pediatrics, East Lansing, MI 48824, USA

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